MIAMI UNIVERSITY The Graduate School. Certificate for Approving the Dissertation. We hereby approve the Dissertation. Amrita Kabi

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1 MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Amrita Kabi Candidate for the Degree: Doctor of Philosophy Director David G. Pennock, Ph.D. Reader Phyllis A. Callahan, Ph.D. Reader Jack C. Vaughn, Ph.D. Reader Joyce J. Fernandes, Ph.D. Graduate School Representative Eileen Bridge, Ph.D.

2 ABSTRACT ROLE OF INNER ARM DYNEINS AND HYDIN IN CILIARY MOTILITY IN Tetrahymena thermophila by Amrita Kabi The focuses of my doctoral research were: (a) Dynein family of motors and (b) Hydrocephalus inducing protein Hydin. Axonemal dyneins are molecular motors composed of heavy chains (HCs), intermediate and light chains. HCs comprise of the motor domain and make up most of the mass of the dynein complex. Previous studies identified eight inner arm dynein HC (DHC) in different ciliated and flagellated organisms. Recent comparative sequence analyses using completed genomes of different species resulted in identification of additional one-headed dynein HC genes in most of the organisms examined. Our proteomic studies using Tetrahymena axonemes revealed that most, if not all the dyneins are present in the axoneme and they are present in varying abundances. Functional studies with some dyneins knockouts generated suggest that dyneins are not completely redundant. Initial evidences suggest that there might be some compensation effect of other inner arm dyneins when one inner arm dynein is lost. Congenital hydrocephalus, a common birth defect, is characterized by the over accumulation of cerebrospinal fluid (CSF) within the ventricular system of the brain. Mutations in hydin result in hydrocephalus in hy3 homozygous mice. Hydin was localized to ependymal cells in the brain, but it was not known whether mutations in hydin cause hydrocephalus by impairing ciliary motility leading to disruption of CSF fluid flow, or by some other mechanisms such as signaling defects. Comparative genomics identified homologs of the hydin gene in small organisms possessing motile cilia and flagella. To determine whether hydin causes hydrocephalus by impairing ciliary motility, I knocked out hydin gene in Tetrahymena. Mutating hydin gene had a dramatic effect on cell motility. Electron microscopic studies of axonemes isolated from mutant cells showed that lack a partial central pair indicating that it is a central pair protein (Kabi et al., In prep). Hence, these results show that hydin in essential for normal ciliary motility. Ciliary movement is essential for normal CSF flow and thus, mutations in hydin can cause hydrocephalus by impairing ciliary motility.

3 ROLE OF INNER ARM DYNEINS AND HYDIN IN CILIARY MOTILITY IN Tetrahymena thermophila A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Zoology by Amrita Kabi Miami University Oxford, OH 2009 Dissertation Director: David G. Pennock, Ph.D

4 TABLE OF CONTENTS General Introduction... 1 References 6 Chapter 1: Proteomic analysis of dyneins in Tetrahymena thermophila Introduction.. 12 Materials and Methods Results.. 19 Discussion. 24 References. 30 Tables 40 Figures Chapter 2: Relative abundance and significance in ciliary motility: a different dimension of functional complexity of inner arm dyneins Introduction Materials and Methods.. 64 Results Discussion. 75 References. 81 Table Figures.. 93 Chapter 3: Disruption of hydin results in impaired ciliary motity in Tetrahymena thermophila Introduction Materials and Methods. 114 Results Discussion 127 ii

5 References 133 Table Figures Summary 161 iii

6 LIST OF TABLES Chapter 1 1. Table 1. Sample data table from a LC-MS/MS run with wild type dynein HC band ` exised from the 15% SDS-PAGE gel indicated in Figure Table 2. DHCs identified in the axonemal fraction of CU428 cells by LC-MS/MS analyses Table 3. DHCs identified in the axonemal fraction of KO3 (outer arm mutant) cells by LC-MS/MS nalyses Table 4. DHCs identified in the axonemal fraction of KO6 (two-headed inner arm mutant cells by LC-MS/MS analyses Table 5. DHCs identified in the 14S fraction of wild type cells by LC-MS/MS analyses Table 6. DHCs identified in the extracted axonemal fraction of wild type cells by LC-MS/MS analyses Chapter 2 1. Table 1. LC-MS/MS analyses of dynein heavy chains present in KO Chapter 3 1. Table 1. LC-MS/MS analysis of putative hydin protein band marked in Figure1B iv

7 LIST OF FIGURES Chapter 1 1. Fig 1. SDS-PAGE separation of axonemes Fig 2. Sucrose density gradient centrifugation analysis of dyneins extracted from wild type cells Fig 3. SDS-PAGE separation of axonemes, dyneins and extracted axonemes Fig 4. Peptide proportion analysis of one-headed inner arm dyneins of wild type, KO3 and KO6 cells Fig 5. Expression studies of representative DHCs Chapter 2 1. Fig 1. Strategy designed to create knockout of DYH15 (A) and DYH18 (B) gene Fig 2. Southern blot analyses reveal the knockout is complete in the mutants Fig 3. SDS-PAGE analysis shows that dynein heavy chain band in KO15 axonemes Fig 4. Mutants show altered swimming behavior Fig 5. KO15 mutants exhibit decreased beat frequency while KO18 mutants do not Fig 6. KO15mutants display reduced feeding rates, while KO18 mutants do not Fig 7. Loss of an inner arm may be compensated by increase in abundance levels of other one-headed inner arm dynein HCs Fig 8. Expression levels of inner arm heavy chains that showed an increase in abundance in KO15 mutants compared to wild type Chapter 3 1. Fig 1. Hydin is present in Tetrahymena thermophila Fig 2. PCR and southern blot to prove that knockout is complete in the mutants Fig 3. SDS-PAGE analysis shows that hydin protein is missing in the mutants Fig 4. Hydin mutants show altered swimming behavior Fig 5. Hydin mutants exhibit decreased beat frequency Fig 6. Hydin mutants display reduced feeding rates Fig 7. Hydin mutants respond to hyperpolarizing and depolarizing stimuli in a manner similar to wild type cells v

8 8. Fig 8. Scanning electron microscopic analyses of wild type and mutant cells. Representative images do not indicate any defect in ciliary length or density in the mutants Fig 9. Hydin mutants exhibit a defective central pair of axonemes Fig 10. Image average analyses of central pair complex of wild type and hydin mutant axonemes vi

9 Dedication I dedicate this dissertation to my husband (Dr. Kaushik Ghosal) and my advisor (Dr.David G. Pennock) whose utmost support and enthusiasm made this work possible. vii

10 Acknowledgements I sincerely extend heartfelt thanks to my advisor Dr. David G. Pennock, without whose continuous support and tremendous enthusiasm none of this work could ever have been accomplished. I consider my self extremely lucky to have found him as my advisor. I will be forever indebted to him for all the guidance and mentoring provided by him during the course of this study. Dr. Pennock not only helped me learn the ways of scientific research, but also guided me a lot in terms of professional development. Last but not least, his all time jovial nature and positive attitude have always kept us happy and going in the lab, even through the difficult phases of work and life. I would like to earnestly thank all my committee members: Drs. Phyllis Callahan, Joyce Fernandes, Jack Vaughn and Eileen Bridge for all the useful advice, guidance, support and encouragement they have provided during the course of my doctoral research. Also, I am thankful to Dr. John Hawes and Dr. Larry Sallans for sharing their expertise on mass spectrometric studies. Special thanks to my fellow students in the lab, Jingmin Zhao and Aswati Subramanian, and all the graduate students in the Department of Zoology for all their help. This work would not have been possible without the support of numerous undergraduate students involved with various phases of the work. Finally, nothing would have been possible without the all time support and blessings of my parents, Harendra Nath and Dipa Kabi, and my parents-in law, Srikumar and Gopa Ghosal. Thanks to my brother, Abhik and my sister-in law and her fiancé, Sriparna and Soumya, for their continuous encouragement. Loads of thanks to my husband, Kaushik for providing relentless enthusiasm all these years without which this work would absolutely not have been possible. His support and untold patience kept me going all throughout this long endeavor. Support for this study was provided by a NIH grant to Dr. Pennock, Graduate School and Department of Zoology at Miami University. viii

11 Introduction One of the mechanisms by which movement is brought about at the microscopic level is by hair-like organelles called cilia and flagella. Cilia and flagella are complex, microtubular organelles that are ubiquitous among eukaryotic organisms (reviewed in Gibbons, 1981). They are present in a range of different cell types- starting from unicellular organisms such as Chlamydomonas, Tetrahymena to different parts of the human body such as brain, respiratory tract and reproductive organs. Depending on their localization, they are involved in diverse physiological functions including whole cell locomotion, fluid movement across cell surface and sensory perception. Consistent with their stringent evolutionary conservation and diverse range of roles they perform, defects in cilia have been associated with an emerging class of disorders termed as ciliopathies (Badano et al., 2006). A few of the major manifestations of ciliary disorders are respiratory dysfunction, hydrocephalus and reproductive sterility which are consistent with their specific localization of cilia in the associated organs (Pan et al., 2005; Fliegauf et al., 2007). Nearly all eukaryotic cilia and flagella are amazingly alike in their organization, comprised of a proteinaceous backbone called an axoneme, surrounded by an extension of the cell membrane. Electron microscopic images of an axonemal cross-section reveal a characteristic 9+2 arrangement of microtubules. The nine peripheral doublets, consisting of a complete microtubule with 13 protofilaments (A tubule) and an incomplete microtubule with 10 protofilaments (B-tubule), form a ring around the central pair of singlet micrutubules. Three other proteinaceous structures can be observed in the axoneme. Nexin bridges (links) connect adjacent outer microtubule doublets, an inner sheath surrounds the central pair and radial spokes connect the central singlet microtubules to the A tubules of outer doublets. These axonemal substructures are an ensemble of numerous proteins, for example there are at least 25 proteins associated with the central pair (Adams et al., 1981; Dutcher et al., 1984) and 23 proteins in radial spoke of Chlamydomonas flagella (Yang et al; 2006). The other main structures found in the axoneme are the molecular motors, dyneins, which are permanently attached to the A tubule of the doublets and make transient attachment to the adjacent B tubule. 1

12 divided into two groups- motile cilia (having a 9+2 arrangement of microtubules) and primary cilia (9+0 arrangement, lacking the central pair). For a long time, it was believed that primary cilia are only involved in sensory functions while motile cilia bring about movement of fluid across cell surface. However newer studies indicate a significant number of exceptions to this categorization, leading to a refined classification of cilia into four subtypes: motile 9+2, motile 9+ 0, non-motile 9+2 and non-motile 9+0 (Ibanez- Tallon et al., 2004). Current work unravels a few cases where primary cilia (such as nodal cilia) exhibits motility (McGrath et al., 2003; Hirokawa et al., 2006); on the other hand, sensory role of motile cilia has been established in studies with airway epithelia cilia (Shah et al., 2009). The structural complexity of ciliary machinery complements the wide range of functions performed by this organelle. Proteomic analysis of human cilia revealed that there are over 200 potential proteins present in it (Ostrowski et al., 2002). With advanced genomic techniques, additional genes are being identified which are thought to encode ciliary proteins (Ibanez-Tallon et al., 2003). This suggests that the number of ciliary proteins may be significantly higher than previously estimated (Gerdes et al., 2009). The discovery of novel ciliary proteins makes it even more challenging to comprehend how the different proteins act together in efficiently performing motility and/ signaling functions within an organism. Understanding the regulation of ciliary motility has always been a fundamental question in cell biology. Regulation of ciliary beating is not well understood but involves control of activity of molecular motors- dyneins (Satir, 1998) and interaction of other substructures such as radial spokes and the central pair (Smith and Sale, 1992; Piperno, 1995). Depending on environmental conditions, the beat frequency and bending pattern of cilia change significantly (Satir et al., 1993). The ion channels and receptors on the ciliary membrane sense the various external stimuli and trigger off different signal transduction cascades, thereby resulting in altered ciliary beat pattern. The bottom-line of all these studies is that ciliary/ flagellar beating is a complex phenomenon involving interaction of dyneins motors with an array of different proteins. In order to completely understand the mechanism of ciliary movement, it will be necessary to characterize all 2

13 the different ciliary proteins and how they interact with each other to produce a characteristic beat pattern. The primary goal of this research is to understand the mechanism of ciliary motility using Tetrahymena thermophila as our model organism. Tetrahymena possesses hundreds of cilia that are involved in crucial life processes like movement, feeding etc. The ciliated protozoan is an excellent model system to understand ciliary motility for a number of different reasons. It is easy to grow and maintain in culture under laboratory conditions. The Tetrahymena macronuclear genome has been sequenced (Eisen et al., 2005) and targeted gene knockout can be carried out efficiently (Gaetrig and Gorovsky, 1995; Cassidy-Hanley et al., 1997). The effect of a mutation affecting ciliary function can be studied in this unicellular ciliate without the secondary pleitotrophic effects which hinders the interpretation of the phenotypes in higher organisms. Last but not least, Tetrahymena exhibits a wide range of swimming behavior- depending on the environmental conditions, they swim forward at different speed, reverse ciliary beat to swim backward and change directions. These different kinds of swimming behaviors can be clearly observed and scored in Tetrahymena. The proteins involved in ciliary movement can be broadly divided into two categories- molecular motors, dyneins, and the regulatory proteins, including the proteins of radial spoke and central pair. Dyneins utilize the energy derived from hydrolysis of ATP to drive the sliding of adjacent microtubules. Sliding is converted to bending by additional accessory proteins (Smith and Sale, 1992; Satir et al., 1993). As part of this project, we decided to focus on the representative members of dynein superfamily (Chapters 1and 2) and a regulatory protein, hydin, (Chapter 3) and tried to elucidate the roles they play in ciliary motility. A combination of different molecular techniques were utilized were to generate targeted gene knockouts of the genes of interest and subsequently, behavioral analyses were performed to look for the effects of mutation in ciliary motility in the mutants. Since dyneins are the actual motors powering ciliary movement, our first goal was to take a closer look at this protein family. Axonemal dyneins are molecular motors composed of heavy chains, intermediate and light chains. We are interested in the heavy chain because of two reasons- firstly because it contains the motor domain and make up 3

14 for most of the mass of the dynein, and also, because it has been shown that disruption of the motor leads to disruption of the entire dynein complex. Previous studies identified eight inner arm dynein heavy chains in different ciliated and flagellated organisms (Goodenough et al., 1987; Kagami and Kamiya, 1995; Muto et al., 1994). However, recent comparative sequence analyses using completed genomes of different species such as Chlamydomonas, Trypanosome, Tetrahymena and Homo sapiens resulted in identification of additional one-headed dynein heavy chain genes in most of the organisms examined (Wilkes et al., 2008). Tetrahymena thermophila represents an extreme case where eighteen one-headed inner arm dynein heavy chain genes were discovered. Before we moved on to elucidating the role of individual dyneins, we first wanted to see whether all the dyneins are present in the Tetrahymena axonemal proteome (Chapter 1). Using LC-MS/MS approach we detected fifteen of the predicted eighteen one-headed dynein heavy chains in ciliary axonemes. The data also indicated that the inner arm dyneins are present in varying abundances. The genomic finding combined with our proteomics data proved that the functional complexity of the inner arm dynein family was much greater than what was previously thought. The presence of multiple inner arm dyneins and their varying abundances within the axoneme prompted the questions- i) Do all inner arm dyneins perform similar roles in ciliary motility or are involved in unique functions? ii) Does relative abundance reflect functional significance of dyneins? To address these questions (Chapter 2), we knocked out two different inner arm dyneins- DYH15, the highest abundance one-headed inner arm dynein, and DYH18, present in very low abundance. Based on behavioral analyses of these two mutants, combined with previous findings, we could conclude that inner arm dyneins are involved in playing different roles in ciliary movement. Since DYH15 was the inner arm dynein of highest abundance, we hypothesized that knocking out DYH15 would result in a severe motility defect in the mutants. However, our results did not support our hypothesis. LC-MS/MS analysis provided first hand evidence of partial redundancy of some inner arm dyneins which might account for the not so pronounced phenotype exhibited by KO15. While it is true that dyneins are the major players in ciliary motility, studies have also shown the crucial role of different regulatory proteins in controlling ciliary beat 4

15 (Satir and Guerra, 2003). One such regulatory protein is hydin (Chapter 3). Mutations in hydin (hydrocephalus inducing) gene caused hydrocephalus in mice. Congenital hydrocephalus (over accumulation of cerebrospinal fluid in the ventricles of the brain) is a tremendous health problem affecting 1 in every 1000 live births (Clewell, 1988; Schurr and Polkey, 1993). Genomics and proteomic studies revealed that hydin is a ciliary protein (Pazour et al., 2005; Broadhead et al., 2006). Previous studies have shown that some ciliary proteins are involved in motility, while others are involved in signaling functions; there may be another group of proteins that play a dual role. Thus mutations in hydin could either result in a motility defect or a defect in signaling activities or an alteration in both motility and signaling. We hypothesized that mutation in hydin cause hydrocephalus by disrupting ciliary motility. In order to test our hypothesis, we knocked out the hydin gene in Tetrahymena thermophila. The hydin mutants exhibited a dramatic motility defect; however no signaling defect was observed in the mutants. Hence, our results were consistent with our hypothesis. In conclusion, these studies will help us have a better understanding of ciliary motility from the perspective of representative members of both the classes of proteinsthe actual motors (dyneins) and the regulatory players (hydin). The information generated may also be useful to understand the molecular mechanisms leading to various ciliarybased disorders. 5

16 References Adams, G.M., B. Huang, G. Piperno, and D.J. Luck Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants. J. Cell Biol. 91, Badano, J.L., Mitsuma, N., Beales, P.L., and Katsanis, N. (2006). The ciliopathies: an emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet. 7, Broadhead, R., Dawe, H.R., Farr, H, Griffiths, S., Hart, S.R., Portman, N., Shaw, M.K., Ginger, M.L., Gaskell, S.J., McKean, P.G. and Gull K. (2006). Flagellar motility is required for viability of bloodstream trypanosome. Nature. 440, Cassidy-Hanley, D., Bowen, J., Lee, J.H., Cole, E., VerPlank, L.A., Gaetrig, J., Gorovsky, M.A. and Burns, P.J Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics. 146, Clewell, W.H Congenital hydrocephalus: treatment in utero. Fetal Ther, 3(1-2), Dutcher, S.K., Huang, B and Luck, D.J Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. J Cell Biol. 98(1): Eisen, J.A., Coyne, R.S., Wu, M., Wu, D, Thiagarajan, M., Wortman, J.R., Badger, J.H., Ren, Q., Amedeo, P., Jones, K.M., TAllon, L.J., Delcher, A.L., Salzberg, S.L., Silva, J.C., Haas, B.J., Majoros, W.H., Farzad, M., Carlton, J.M., Smith, R.K., Garg, J., Pearlman, R.E., Karrer, K.M., Sun, L., Manning, G., Elde, N.C., Turkewitz, A.O., Asai, D.J., Wilkes, D. E., Wang, Y, Cai, H, Collins, K., Stewart B.W., Lee, S.R., Wilamowska, K., Weinberg, Z., Ruzzo, W.L., Wlogo, D., Gaetriog, J., Frankel, J., Tsao, C., Gorovsky, M.A., Keeling, P.J., Waller, R.F., Patron, N.J., Cherry, J.M., Stover, N.A., Kreiger, C.J., 6

17 Toro, C., Ryder, H.F., Williamson, S.C., Barbeau, R.A., Hamilton, E.P. and Orias, E Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol Fliegauf, M., Benzing, T and Omran, H When cilia go bad: clia defects and ciliopathies. Nature, 8, Gaetrig J. and Gorovsky, M.A DNA mediated transformation of Tetrahymena. Meth. Cell Biol. 47, Gerdes, J.M.; Davis, E.E. and Katsanis N The Vertebrate Primary Cilium in Development, Homeostasis and Disease. Cell 137, Gibbons, I.R Cilia and flagella of eukaryotes. J Cell Biol. 91,107s-124s. Goodenough, U.W., Gebhart, B., Mermall, V., Mitchell, D.R. and Heuser, J.E High pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner arm dynein subunits. J Mol Biol. 194, Hirokawa, N., Tanaka, Y., Okada, Y. and Takeda, S Cell 125,33 Ibanez-Tallon, I., Heintz, N and Omran, H To beat or not to beat: roles of cilia in development and disease. Human Mol Genet. 12(1). R27-R35. Kagami, O. and Kamiya, R Separation of dynein species by high-pressure liquid chromatography. Methods in Cell Biology. 47, McGrath, J., Somlo, S., Makova, S., Tian, X. and Brueckner, M Cell 114, 61 Muto, E., Edamatsu, M., Hirono, M., and Kamiya, R Immunological detection of actin in 14S ciliary dynein of Tetrahymena. FEBS lett. 343,

18 Ostrowski, L.E., Blackburn, K., Radde, K.M., Moyer, M.B., Schlatzer, D.M., Moseley, A. and Boucher, R.C A Proteomic Analysis of Human Cilia. Mol Cell Proteom. 1.6, Pan, J. Wang, Q. and Snell, W.J Cilium generated signaling and cilia related disorders. Lab Investigations. 85, Pazour, G.J., Agrin N., Leszyk J, and Witman, G.B Proteomic analysis of a eukaryotic cilium. J Cell Biol. 170(1), Piperno, G Regulation of dynein activity within Chlamydomonas flagella. Cell Motil.Cytoskelet. 32, Satir, P Mechanisms of Ciliary motility: an update. Europ. J. Protistol. 34, Satir, P. and Guerra, C Control of ciliary motility: an unifying hypothesis. Eur. J. Protistology. 39, Satir, P., Barkalow, K. and Hamasaki, T The control of ciliary beat frequency. Trends Cell Biol. 3(11), Schurr, P.H. and Polkey, C.E. eds, Hydrocephalus. 1993, Oxford Medical Publications. Oxford University Press, New York, NY. Shah, A.S.; Ben-Shahar, Y. Moninger, T.O., Kline, J.N. and Welsh, M.J Motile Cilia of Human Airway Epithelia are Chemosensory. Science 325, Smith, E.F. and Sale, W.S Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes. J Cell Biol. 117,

19 Wilkes, D.E.; Watson, H.E.; Mitchell, D.R. and Asai, D.J Twenty-five dyneins in Tetrahymena: A re-examination of the multidynein hypothesis. Cell Motil Cytoskeleton. 65, Yang, P., Diener, D.R.,Yang, C, Kohno, T.,Pazour, G.Z., Dienes, G.M, Agrin, N, King, S.M, Sale, W.M., Kamiya, R., Rosenbaum, J.L. and Witman, G.B Radial spoke proteins of Chlamydomonas flagella. J Cell Sci. 119(6),

20 Proteomic analyses of dyneins in Tetrahymena thermophila 10

21 Abstract Dyneins are motor proteins that utilize the energy from ATP hydrolysis to translocate to the negative end of the microtubules. Axonemal dyneins are molecular motors composed of heavy chains (HCs), intermediate and light chains. HCs comprise the motor domain and make up most of the mass of the dynein complex. Previous biochemical and genetic studies identified eight inner arm dynein HC in different ciliated and flagellated organisms. Electron microscopic studies confirmed the presence of a two-headed and multiple one-headed dyneins in the axoneme. In recent years the completed genomes of different organisms have been sequenced and comparative sequence analyses using completed genomes of eleven different species including Chlamydomonas, Trypanosome, Tetrahymena and Homo sapiens resulted in identification of additional dynein HC genes in most of the organisms examined. All these dynein HCs were thought to encode oneheaded dyneins. The number of additional genes discovered varied greatly across organisms. An extreme example is Tetrahymena thermophila which has eighteen oneheaded inner arm dynein HC genes. These findings raise the question: Are all predicted dyneins present in the axonemal proteome and are they all present in equal abundance? In order to answer this question, we took a LC-MS/MS approach. Proteomic analyses of axonemes and dyneins isolated from Tetrahymena resulted in detection of fifteen oneheaded dynein HCs. The LC-MS/MS data further suggested that the HCs are present in different abundances. qrt-pcr studies supported the fact that axonemal dynein HCs are expressed at different levels and hence, correlated with the LC-MS/MS findings. Key words: axonemes, dyneins, LC-MS/MS 11

22 Introduction Dyneins are a class of molecular motors which are ubiquitous among the different eukaryotic groups. Dyneins are classified as cytoplasmic or axonemal based on their intracellular location. Cytoplasmic dyneins are involved in different kinds of intracellular movement such as cargo transport, movement of chromosomes during mitotic spindle formation (Vallee et al., 2004) and slow axonal transport (Dillman et al., 1996). Axonemal dyneins drive ciliary and flagellar movement by driving the sliding of adjacent microtubule doublets. The sliding is converted to bending by additional accessory proteins (Satir et al., 1993). Each dynein is a complex composed of one, two or three heavy chains (HCs), light chains (LCs), and some dyneins also have intermediate chains (ICs) (Witman et al., 1994). Electron microscopic studies reveal a bouquet arrangement of dyneins with one, two or three globular heads (depending on the number of heavy chains) joined through flexible stalks to a common base (Johnson and Wall, 1983; Goodenough and Heuser, 1984; Goodenough and Heuser, 1989; Witman et al., 1983). Each heavy chain makes up a single head and part of the associated stalk (King and Witman, 1989; King and Witman, 1990, King, 2000a; King, 2000b; Asai and Koonce, 2001). The rest of the stalk and the globular domains at the base are composed of intermediate and/or light chains (King and Witman, 1989; King and Witman, 1990). While the heavy chain is the actual motor domain of the dynein, the intermediate and light chains are involved in regulating the motor activity (Asai and Wilkes, 2004). The heavy chains are large, approximately 4600 amino acid residues long. Structurally each dynein heavy chain consists of two major domains- the head domain making up the C-terminal two-thirds of the polypeptide, and the tail domain forming the N-terminal third of the polypeptide (Gibbons et al., 1991). The head domain contains 6 AAA (ATPase Associated with cellular Activities) modules designated as AAA1- AAA6. AAA1 is the site of ATP hydrolysis and also the catalytic site. AAA1 and the sequences around it are highly conserved across different organisms (Gibbons et al., 1991; Ogawa 1991). AAA2-4 loops are less conserved and appear to be capable of binding but not hydrolyzing ATP; evidence suggests they may help regulate dynein HC activity (Silvanovich et al., 2003; Gibbons 1995). The remaining two modules, AAA5 12

23 and AAA6, are less conserved and do not have complete nucleotide binding sites (Asai and Koonce, 2001). Between AAA4 and AAA5 is the microtubule binding site, a slender α-helical coiled coil region known as the B-link with which the dynein makes transient attachment to the adjacent B-microtubule during sliding (Asai and Brokaw, 1993; Koonce, 1997). The N-terminal tail domain region forms the base of the dynein and is much less conserved (Asai and Koonce, 2001; Asai and Wilkes, 2004). The binding of accessory proteins to this region determines the cargo binding specificity (Asai and Koonce, 2001; Asai and Wilkes, 2004). Axonemal dyneins are classified as either outer arm dyneins or inner arm dyneins depending on their position in the outer doublets of the microtubules. In most lower eukaryotes (e.g. Chlamydomonas, Paramecium and Tetrahymena) outer arms are threeheaded and contain three different HC (Pfister and Witman, 1984; Pfister, 1982). Trypanosomes and Giardia are exceptions that possess flagella with two-headed outer arm dyneins with two HCs (Wickstead and Gull, 2007). In metazoans, outer arm dyneins are two-headed with two HC (Gibbons, 1988). The outer arm dyneins, when present, repeat every 24 nm along the outer microtubule doublets. The inner arm dyneins are structurally more complex than the outer arms. In 1985, freeze fracture studies of Chlamydomonas flagella identified two structural types of inner arms - triads (three-lobed structures) and dyads (two-lobed structures) (Goodenough and Heuser, 1985). Longitudinally along the axoneme, the inner arms were organized in 96 nm repeats with each repeat displaying a triad-dyad-dyad arrangement. Further work revealed that the triad is a two-headed dynein, and the dyads are the oneheaded dyneins (Piperno et al., 1990). The 96 nm repeat is delineated into three segments, I1, I2 and I3 by the radial spokes, S1 and S2. The two-headed dynein termed as I1 is proximal to S1, while the one-headed dyneins termed as I2 and I3 are distal to spokes S1 and S2. Studies over the years with axonemes isolated from different organisms have gradually added to our understanding of the organization and complexity of inner arm dyneins in the axoneme. The most recent study performed using different Chlamydomonas mutants showed that each 96 nm repeat has one-two headed dynein and six one-headed dyneins (Huy Bui et al., 2008). These six one-headed dyneins are organized into groups of two and hence appear as dimers; thus, there are three dimers in 13

24 addition to the two-headed I1 dynein, within each 96nm repeat. Longitudinally along the entire length of the axonemes (Piperno et al., 1991; Huy Bui et al., 2009) and radially across the axoneme (Sale, 1986; Lindemann et al., 1992; King et al., 1994), distinct subsets of inner arm dyneins are localized in specific regions; thereby adding additional levels of complexity in their arrangement. Hence, the organization of the one-headed inner arm dyneins is much more complex compared to that of the outer arms and twoheaded inner arm dyneins which appear to be uniformly distributed along the entire length of the axoneme. Results of structural studies are complemented by biochemical and genetic findings. HPLC analyses of dyneins extracted from Chlamydomonas and Tetrahymena identified a single type of two headed dynein (I1 with two heavy chains) and six different one-headed I2/3 dyneins (Kamiya et al., 1991, Kagami and Kamiya, 1992; Muto et al., 1994). The biochemical studies of dynein complexity were supported by PCR-based cloning studies using degenerate primers. Two independent studies conducted with Tetrahymena led to the identification of two genes encoding the two I1 HC and seven genes encoding one-headed inner arm dynein HCs (Xu et al., 1999; Asai and Wilkes, 2004). Similar PCR based strategies showed the presence of six genes encoding 1-headed inner arm dynein HC genes in Chlamydomonas (Porter et al., 1996), eight in sea urchins and six in mammals (Gibbons et al., 1994; Chapelin et al., 1997). Thus, different approaches using various model organisms suggested that all ciliated organisms encode multiple, 1-headed inner arm dynein HC. More recently, comparative sequence analyses of genomes from different species identified additional genes encoding 1-headed inner arm dynein HC in some of the organisms examined (Wilkes et al., 2008). Although all ciliated organisms encode multiple, 1-headed inner arm dynein HC, the total number predicted from genomic studies varies greatly among different species; for example Chlamydomonas has eight genes, Trypanosome six and Tetrahymena eighteen (Wilkes et al., 2008). The observation that all ciliated organisms encode multiple, one-headed inner arm dynein HCs begs the questions: (i) Are some of the dyneins redundant? (ii) Do the different dyneins play different roles? These questions become all the more important because all the inner arm dynein mutants, missing either single or multiple dynein heavy 14

25 chains, characterized so far in various organisms (Kato et al., 1997; Liu et al., 2004; Neesen et al., 2001; Yagi et al., 2005), displayed some phenotypic defect. This indicates that at least some of the one-headed inner arm dyneins are not completely redundant; however, it does not rule out partial redundancy. A significant amount of work also suggests that different one-headed inner arm dyneins do, in fact play different roles. Analysis of Chlamydomonas mutants missing different inner arms showed that inner arms as a group determine wave form (Brokaw and Kamiya, 1987), but recent studies suggested that some inner arms may play a role in determining beat frequency (Liu et al., 2004; Neesen et al., 2001, Yamamoto et al., 2006). Tetrahymena mutants lacking one of the inner arm dynein HC, DYH8 displayed decreased beat frequency (Liu et al., 2004). Reduced beat frequency in cilia was also observed in mice with disruption in a putative inner arm HC (Neesen et al., 2001). In Chlamydomonas, some specific inner arms were shown to play a crucial role in determining beat frequency in the absence of outer arms (Yamamoto et al., 2006). All Chlamydomonas one-headed inner arm dyneins translocated microtubules in vitro, but they did so at different rates, and five of the arms rotated the microtubules as they moved. Although it seems clear that some individual one-headed inner arm dyneins are not redundant and some play different roles in ciliary motility, it is still not entirely clear how much, if any, redundancy there is among all the one-headed inner arm dyneins. To summarize, all ciliated organisms encode a large number of one-headed inner arm dynein HCs and studies to date suggest that most if not all, axonemal dynein HC encoded by the genome are expressed and are present in the axoneme. All the eighteen one-headed dynein HC genes were expressed in Tetrahymena (Wilkes et al., 2008).All but one inner arm dynein HC was detected in proteomic studies with Chlamydomonas (Pazour et al., 2005), and all six predicted dynein HC were detected in the Trypanosome flagella (Broadhead et al., 2006). In case of Tetrahymena, which encodes an exceptionally large number of one-headed inner arm dynein HC (eighteen), only nine HC were detected in the proteomic analysis performed to date (Smith et al., 2005). To address the questions of how many one-headed inner dynein HC are present in Tetrahymena axonemes and in what relative abundance, LC-MS/MS was performed on dynein HC isolated from wild type and mutant Tetrahymena cells. We were able to detect 15

26 fifteen of the eighteen one-headed dynein heavy chains genes predicted by genomic analyses. LC-MS/MS data suggested the HC are present in variable abundance. Quantitative RT-PCR analyses of the expression of different dynein HC genes correlated with the LC-MS/MS analyses. Methods Strains and cell culture Tetrahymena thermophila CU428.2 strain (kindly provided by Dr. Peter Burns, Cornell University) was used as the wild type strain. Two different Tetrahymena mutants were used in this study: an outer arm mutant (KO3) (Zhao, unpublished data) and a twoheaded inner arm mutant (KO6) (Angus et al., 2001). Mutants were maintained in a culture at a concentration of 300 μg/ml of paromomycin. All strains were grown at 28 0 C in a shaking incubator at 125 rpm in Neff s growth medium (0.25% proteose peptone, 0.25% yeast extract, 0.55% dextrose, 33 μm FeCl 3 ). Before analysis, mutants were grown for a week without paromomycin. Axoneme Isolation Whole axonemes were isolated from wild type and mutant cells as previously described (Johnson, 1986; Mobberley et al., 1999). Briefly, 1.25 l of wild type and mutant cells were pelleted by centrifugation at 900g for 3min. The supernatant was removed by aspiration to around 5 ml of medium and the cells were gently resuspended. Dibucaine was added to a concentration of 3mM to release cilia from cells. Cells were swirled gently for 1 min and 60 ml cold Neff s (medium) was immediately added. After cell bodies were pelleted by centrifugation at 900g for 10min at 4 0 C, the supernatant was transferred to fresh tubes. 25 µl of 0.1 M PMSF (phenylmethylsulphonyl fluoride), a serine protease inhibitor, was added and any cell bodies remaining were pelleted by a second round of centrifugation as described above. Cilia were then pelleted by centrifugation at 11200g in a Sorvall SS34 rotor for 10 min at 4 0 C. The pellet was resuspended in 1ml 1X Satir Buffer (SB) (30mM HEPES, ph 7.6; 5 mm MgSO4; 0.5 mm EDTA; 20mM KCl). 5 µl leupeptin (5µg/ml), another protease inhibitor, was then added. Cilia were layered onto 20 ml of 1X SB containing 1M Sucrose and 2mM EGTA 16

27 and centrifuged at 11200g in a Sorvall SS34 rotor for 20 min at 4 0 C. Pelleted cilia were resuspended in 1ml 1X SB and 5 µl leupeptin, and 25 µl 10% NP-40 was added to remove the ciliary membranes. After incubation in NP-40 for 20 minutes, axonemes were pelleted at 11200g in a Sorvall SS34 rotor for 10 min at 4 0 C and resuspended in 1ml of 1X SB. This process was repeated twice and finally the axonemes were resuspended in 1ml of 1X SB. Dynein Isolation Axonemes were isolated from 4l of cells as described above. 10 µl100mm DTT was added to axonemes and they were pelleted at 11200g for 10 min. The pellet was resuspended in 500 µl 0.6M KCl in 1XSB to extract dyneins from axoneme. 5µl leupeptin (5µg/ml) was added, and the sample was incubated on ice for 30 min. Extracted axonemes were pelleted at 39,100g in a Sorvall SS34 rotor for 20 min at 4 0 C for 20 min. The supernatant containing dynein was stored in a fresh microfuge tube at C. 14S Dynein Fraction Dyneins were loaded carefully onto a 5-25% sucrose density gradient in 1XSB. Centrifugation was carried out at 112,000g for 22.5hr at 4 C. Twenty fractions were collected, each containing 25 drops of sample. The fractions were run on 3-5% polyacrylamide/ 0-8M urea gels and fractions containing the 22S, 18S and 14S dyneins were identified. Determination of protein concentration Protein concentration was determined using Bradford Reagent (BioRad). Samples were boiled in SDS sample buffer ( M Tris, 10% SDS, 25% βme, 33% Glycerol) for 5-10 minutes and then loaded in a gel or stored at C. SDS-PAGE and Loading of proteins For LC-MS/MS analysis, 100 µg of total axonemal/ dynein proteins were electrophoresed through 15% polyacrylamide gels for ~16-20 hr at constant voltage of 50V to resolve all the dynein HCs as a compact band. All gels were stained with 17

28 Coomassie Blue R. for 3-4 hrs. Gels were destained in Destain1 (50 % Acetic Acid, 10 % Methanol) for 3-4 days and then stored in Destain 2 (5% Acetic Acid, 7% Methanol) at 4 0 C. LC-MS/MS Analysis LC-MS/MS (Liquid chromatography- Mass spectrometry/mass-spectrometry) allows efficient identification of proteins from a complex mixture of very similar proteins (Kalia and Gupta, 2005). This method has high sensitivity and has been used successfully to detect low abundance proteins (Lahm and Langen, 2000). The dynein HC band was excised from the SDS-PAGE gel and sent to the University of Cincinnati Mass Spectrometric Facility for LC-MS/MS analysis. (Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), where it was subjected to enzymatic digestion by trypsin and prepared for LC-MS/MS. Resulting peptides were separated by HPLC and subjected to two rounds of mass spectrometry. This generated a peptide molecular weight profile termed as peptide mass fingerprint for individual proteins within the mixture. Each protein has its own enzyme-specific unique molecular weight fragment profile. Since the dyneins have high molecular weight (~ 450KDa), a huge amount of data were generated in this process. The data were then condensed with appropriate software so that the collection of most intense peaks for each sample could be submitted to the search engine. To identify proteins, the condensed fingerprint data of the sample was compared, using appropriate software, with theoretical masses of all peptides of proteins subjected to cleavage with same site-specific enzymes. The software identifies potential proteins and also scores them on the basis of the probability to being a particular protein. Based on the score, the most likely protein was reported. qrt-pcr Total RNA was isolated from Tetrahymena utilizing the Qiagen RNEasy Mini Kit (Cat # 74104). RNA purity was determined using UV-spectrometry by 260/280 nm ratio. RNA was stored at C and used within one month of isolation. For each assay, 1 ug of total RNA was subjected to reverse transcription (RT) using QuantiTect Reverse Transcription Kit for RT-PCR (Qiagen, Cat # ) as per manufacturer instructions. Briefly, first 18

29 strand cdna was prepared from total RNA using oligo (dt) primers. Once prepared, the cdna was stored at C for further use. The first strand cdna was used for quantitative PCR (qpcr) using gene specific primers and QuantiFast Sybr-green PCR kit (Qiagen, Cat # ). Briefly, for each PCR reaction cdna was mixed with forward and reverse primers, Sybr-green and distilled water in the quantity as indicated in the Qiagen kit. PCR reaction was performed using a 95 0 C (10 min) step followed by 40 cycles at 95 0 C (15 sec) and 60 0 C (1 min). Data were analyzed using the 2 -ΔCt method using DYH3 as housekeeping control. ΔCts were calculated by subtracting the Ct value of DYH3 from the Ct values for each gene. Results Dynein HCs detected in wild type axonemal preparations To determine which dynein HC are present in Tetrahymena cilia, total axonemal protein was subjected to SDS-PAGE (Fig 1) and LC-MS/MS was performed on the band containing dynein HCs. Whole axonemes were used to ensure all dynein HC present in the axonemes were in the sample being analyzed. Table 1 represents an example of a data sheet containing all the proteins detected in one LC-MS/MS run of the wild type dynein HC. Each accession number was used to search the ncbi database ( to identify each dynein HC. P (pro) is the Protein Probability and is calculated by SEQUEST, a search algorithm. The smaller the value (larger negative exponent), the greater is the probability that the reported protein is correct. Scores are also a SEQUESTspecific value but calculated differently. The higher the score, the better the fit and the greater the chance that the protein identified is correct. Coverage gives the percent amino acids identified. The greater the number of peptide hits, the higher the probability that the protein is present in the mixture. Since the dynein HCs are high molecular weight proteins (~450KDa), the high degree of sequence similarity among the different dynein HCs makes it reasonable to assume that there will be at least some peptide fragments common to different dynein HCs. The peptide hits for a given protein generated by the analysis software, however, are unique to that particular dynein HC. To confirm that, we randomly picked one peptide hit for each protein and performed a BLAST search. Each time it matched the reported dynein HC with highest probability except in cases in which 19

30 the protein was identified with only one peptide. In those cases sometimes the blast search picked up more than one match. Thus, we decided to take a conservative approach and count as positives only those proteins detected with two or more peptide hits (Zhang et al., 2006). With wild type cells, we performed three axoneme isolations and processed the three samples independently. Table 2 summarizes the three different analyses. In the summary we have organized the one-headed inner arm dynein HCs into the three subgroups, IAD 3, IAD 4, IAD 5 as proposed by Wilkes et al., There was quantitative variation among the runs, but when data from all three runs were combined, all outer arm dynein HCs, both two-headed inner arm dynein HCs, and nine of the eighteen predicted one-headed inner arm dynein HCs were detected. Out of the nine oneheaded inner arm dyneins detected, eight of them were detected in all the three preparations; Dyh10p was detected by two peptide hits in a single preparation. The eight one-headed inner arm dynein HCs we detected in all the three runs were also detected by Smith et al., They also detected one dynein HC, Dyh20p, that we did not (Smith et al., 2005). Removal of background increased the number of dynein HCs detected The band subjected to LC-MS/MS is a complex mixture containing numerous proteins. It can be difficult to detect low abundance proteins in such an ensemble of multiple proteins. One way to preferentially enrich for low abundance proteins is by removing the proteins present in high abundance. Among the members of the dynein family, the outer arm dyneins are the most abundant, followed by the two-headed inner arm dyneins and then by the individual one-headed inner arm dyneins. Hence, to remove high abundant proteins from the dynein HC mixture, we used axonemes isolated from two different mutants: an outer arm mutant (KO3, a mutation in DYH3) missing the outer arm dynein HC and a two-headed inner arm dynein mutant (KO6, a mutation in DYH6) missing both two-headed inner arm HC. Table 3 is a compilation of the different dynein HCs identified in two independent axonemal preparations from a Tetrahymena mutant missing outer dynein arms (KO3). As expected we do not see significant numbers of peptide hits from the three outer arm 20

31 dynein HC (Dyh3p, Dyh4p and Dyh5p). In addition to the nine one-headed inner arm dyneins identified in preparations of wild type axonemes, three additional one-headed inner dynein HC [Dyh14p, Dyh20p and Dyh23p] were detected in both the preparations, and an additional dynein HC, Dyh8p, was detected by more than one peptide hit in one of the two preparations. Table 4 summarizes the results of the analyses of KO6 axonemes. In addition to the one-headed inner arm dynein HCs identified in wild type preparations, Dyh20p was detected in both the samples, and Dyh9p, Dyh14p and Dyh23p were detected in one sample of KO6 axonemes. Thus, five additional dynein HCs were detected in axonemes isolated from outer arm or two-headed inner arm mutants. While it is likely that removal of the high abundant outer-arm or two-headed inner arm dynein HC allowed detection of additional dynein HCs, it is possible that loss of outer arms or two-headed inner arm dyneins in the mutants resulted in induction of genes encoding one-headed inner arm dyneins, leading to increased abundance and thus, detection of additional HCs. In order to test that possibility, we took a biochemical approach to reduce the background of the highly abundant dyneins. Based on their size, shape and density, the dyneins can be separated into three different fractions by sucrose density gradient centrifugation. The 22S fraction contains the outer arm dyneins (Warner et al., 1985; Ludman et al., 1993), the 18S fraction has the two-headed inner arm dyneins (Mobberley et al., 1999), and the 14S fraction contains the one-headed inner arm dyneins (Muto et al., 1994). Dyneins were isolated from the wild type cells, subjected to sucrose density gradient centrifugation, and fractions were collected. The fractions were run on a 3-5% 0-8M Urea gradient and the 22S, 18S and14s containing fractions were identified (Fig 2). The 14S fraction was then run on a 15% SDS-PAGE. The band with the dynein HC was excised and subjected to LC-MS/MS analysis. Results are shown in Table 5. The presence of peptide hits to outer arm and two-headed inner arm in Run1 indicated that separation was not as good in the first run compared to the second one. However, in both runs, three [Dyh8p, Dyh9p and Dyh20p] out of the five additional dynein HCs detected in KO3 and KO6 axonemal preparations were identified in the CU428 14S preparations. These data support the idea that detection of at least three of the additional dynein HCs identified in the KO3 and 21

32 KO6 mutants was due to reduction of background and not due to induction of the genes. The remaining two dynein HCs that were not detected in the 14S samples may have been induced to higher levels in the KO3 and KO6 mutants enabling their detection in those preparations. Alternatively, they may not have been extracted in the dynein fraction and are left behind with the extracted axoneme. Previous studies have shown that all dyneins are not necessarily extracted under the same conditions (Avolio et al., 1986). Since 22S, 18S, and 14S dyneins do not always resolve well during centrifugation, we isolated the 14S fraction from KO3 axonemes missing outer arm dyneins and analyzed the dynein HC with LC-MS/MS. The same one-headed inner arm dynein HC were identified as were identified in 14S dyneins isolated from wild-type axonemes. Combining analyses from all the different preparations, we detected fifteen out of predicted eighteen one-headed dynein HCs. DYH15 is not extracted from axonemes There were no peptide matches against Dyh15p in either of the 14S preparations from wild type cells or from one of the two KO3-14S preparations. In the other KO3-14S fraction, Dyh15p was detected but with relatively few peptide hits. This result was surprising because Dyh15p had been consistently identified with the highest number of peptide hits of all the one-headed inner arm dynein HCs in axonemal preparations and all other one-headed dynein HCs identified in axonemal preparations were also detected in the 14S dynein fractions. To determine whether Dyh15p is extracted from the axoneme but does not sediment with other 14S dyneins or whether Dyh15p is simply not extracted from axonemes, we subjected dynein HCs remaining in axonemes after they had been extracted with high salt (extracted axonemes) (Fig 3) to LC-MS/MS analysis. In both runs Dyh15p was detected with a relatively large number of peptides (Table 6), which supports the idea that Dyh15p is not extracted readily from Tetrahymena axonemes. Dyh12p, a one-headed inner arm dynein HC, not detected in samples from whole axonemes or 14S dyneins extracted from either mutants or wild type cells, was detected in both the extracted axonemal preparations. These data suggest Dyh12p is also not readily extracted from axonemes. In contrast, some dynein HCs, e.g. Dyh11p, Dyh21p 22

33 and Dyh24p, which were detected with high numbers of peptide hits in the 14S fraction were detected with a comparatively low number of peptides in extracted axoneme fraction. Taken together, these data indicate that some dyneins are typically extracted preferentially compared to others. Relative abundance levels of the different dynein HCs In total, fifteen out of eighteen dynein HCs were detected by LC-MS/MS, and the different dyneins were detected by different numbers of peptide hits. The absolute number of peptide hits for each dynein varied from preparation to preparation and run to run. To determine whether the relative number of peptide hits was consistent from preparation to preparation, we calculated the proportion of peptide hits for each dynein HC against the total of peptide hits generated for all one-headed inner arm dyneins in that particular run. We performed the peptide proportion study on LC-MS/MS analyses of wild type, KO3, and KO6 mutant axonemes. For each dynein HC, proportion of peptides was calculated by dividing the number of peptide hits generated for that dynein HC divided by the total number of peptide hits generated for all one-headed inner arm dyneins in that particular run. This way, we could account for the disparity in peptide hits observed across runs. In Fig 4 A, B, C the peptide proportion is plotted against the dynein HC. The three graphs represent the three IAD groups- IAD3, IAD4 and IAD5 respectively. Consistently across wild type, KO3 and KO6 runs, Dyh15p was detected with highest proportion of peptides. We observed that in each IAD group, one dynein HC was detected with a high number of peptide hits [Dyh25p in IAD3, Dyh21p in IAD4 and Dyh15p in IAD5]; one or two dynein HCs were detected with intermediate number of peptide hits [Dyh11p in IAD3, Dyh16p and Dyh19p in IAD 4, Dyh22p and Dyh24p in IAD5], and some predicted dynein HCs were either detected with very few peptide hits [Dyh8p, Dyh14p in IAD3, Dyh9p, Dyh20p in IAD4 and Dyh23p in IAD5] or were not detected [Dyh13p, Dyh17p in IAD3 and Dyh18p in IAD5]. Expression pattern of representative dynein HCs at mrna levels To determine whether the number of peptide hits correlated with the relative expression level of the gene, we performed qrt-pcr studies on representative dynein HC genes. 23

34 We selected an outer arm dynein HC-DYH3, a two-headed inner arm dynein HC-DYH6 and members of IAD Group 5. Fig 5 shows the relative expression levels of the different genes. The expression levels of all other genes were normalized to the expression level of DYH3, which was the highest of the genes that were analyzed. The expression levels of two-headed inner arm dynein heavy chain DYH6 and one-headed inner arm DYH15 were approximately in the same range. DYH18 had the lowest expression level in IAD5. Discussion The vast majority of predicted one-headed inner arm dyneins are present in Tetrahymena axonemes Here, we report a detailed proteomic analysis of the dynein heavy chain family in Tetrahymena thermophila axonemes. All outer arms and two-headed inner dynein HCs and fifteen out of the predicted eighteen one-headed inner arm dynein HC were detected. Outer arm dynein HC, two-headed inner arm dynein HC, and eight one-headed inner arm dynein HC were detected in whole axonemes. When high abundant dynein HC were removed from the analysis, either by using mutants missing those HC or by biochemically removing outer and two-headed inner arm dyneins, seven additional oneheaded inner arm dynein HC could be detected. Three one-headed dynein HCs were not detected in any preparations Dyh13p and Dyh17p of subgroup IAD3 and Dyh18p of subgroup IAD5 were not detected in any of the axonemal or dynein preparations. All three of these genes are expressed in growing cells and are also induced following deciliation (Wilkes et al., 2008). Hence, it is evident that they are ciliary genes. Thus, it is possible that these three undetected dynein HCs are present in the axonemes but the relative abundance levels of the three dynein HCs are too low to be detected by this particular technique. An alternative explanation is that one or more of these three dynein HCs have an unusual molecular weight compared to the other dynein HCs and hence did not migrate with the other dynein HCs in SDS-PAGE. Although this idea seems unlikely considering the fact that the dynein HCs are over 450kDa and were electrophoresed through 15% SDS gels, Yagi et. al. (2009) recently identified three low abundant inner arm dynein 24

35 HCs in Chlamydomonas, all of which had significantly large molecular masses compared to the other inner arm dyneins. However, since the molecular weight of all these dynein HCs (Dyh13p kda, Dyh17p kda, Dyh18p kda) is in the range of other dynein HCs in Tetrahymena, this possibility was ruled out. One-headed inner arm dyneins are present in different abundances in the axoneme One of the factors determining the number of peptide hits generated for a particular protein in LC-MS/MS is the relative abundance level of the protein in the mixture (Gao et al.,2003), and analyses of our data suggest that the relative number of peptide hits does, in fact, reflect the relative abundance of each HC in the axoneme. Previous studies have indicated that outer arm dynein HCs are the most abundant HC in axonemes and that the two-headed inner arm dynein HC are second in abundance, and consistently across the three separate runs of wild type axonemes, the outer arm dynein HCs were detected with the maximum number of peptide hits followed by the two-headed inner arm dynein HCs. Thus, it seems very likely that the number of peptide hits for each one-headed inner arm dynein HC reflects its relative abundance in the axoneme. Thus, the fifteen one-headed inner arm dyneins we detected in our analysis can be classified as high abundance, medium abundance and low abundance. Three dynein HCs (Dyh15p, Dyh21p and Dyh25p) were consistently present in high abundance. Five dynein HC can be classified as medium abundance HCs, and remaining ten can be classified as low or very low abundant dynein HCs. It is interesting that each one-headed IAD subgroup contains one high abundant dynein HC, one or two medium abundant dynein HCs, and one or more low to very low abundance dynein HCs. Abundance may reflect localization pattern of dyneins The most recent model of the arrangement of dyneins within the axoneme illustrates that each 96 nm repeat comprises of six one-headed inner arm dyneins. Longitudinally as well as radially, distinct subsets of inner arm dyneins are present in different regions of the axoneme (Piperno et al., 1991). Detailed localization studies depicting the position of each dynein have not yet been carried out in Tetrahymena. However, based on varying relative abundance levels of 25

36 the inner arm dyneins and information generated from studies conducted in other organisms, we put forward a few possible theories regarding how the different dyneins can be distributed in the axoneme. As mentioned before, all inner arm dyneins present in Tetrahymena can be broadly divided into three classes based on their abundance levels high abundance, medium abundance and low to very low abundance. Each of the three high abundance dyneins are present in association with a medium/ low abundance dyneins; this dimerization concept is consistent with the demonstrated arrangement of six dyneins into three functional dimers within each 96nm repeat in Chlamydomonas (Huy Bui et al., 2008). We suggest that the three high abundance dyneins are present in each 96nm repeat along the entire length of the axoneme. In contrast, the medium and low abundance dyneins may be selectively distributed in certain regions of the axoneme. Our theory of different dimeric combinations within the axoneme complies with the observed asymmetric distribution of dyneins in different regions of the axoneme. Our proposed model is supported by localization studies carried out in Chlamydomonas which indicate that low abundance dynein HCs, DHC3, 4 and 11, are present only in the proximal region of flagella in contrast to other dynein HCs present in higher abundance, such as DHC9, which are distributed throughout the flagella (Yagi et al., 2009). The total number of inner arm dyneins varies greatly among organisms; ciliated protozoans, Tetrahymena and Paramecium, exhibiting the maximum number among organisms studied to date. Both these organisms possess hundreds of cilia which are specialized to carry out different life processes such as movement and feeding. It is possible that not all dyneins are present in every cilium. Some dyneins may be present only in somatic cilia involved in movement while others may be localized to oral cilia involved in feeding; there may be some others which are present in all types of cilia. Closer look at these distinct groups of cilia separately will be needed to validate this hypothesis. This analysis may also give us an idea of the reason behind the variation in the number of dyneins across different organisms. A significant portion of the model of arrangement of inner arm dyneins has been developed using flagella derived from different cells and organisms (Piperno et al., 1995; Huy Bui et al., 2008; Huy Bui et al., 2009). Cilia and flagella share a conserved 9+2 arrangement of microtubules; however cilia are shorter in length compared to flagella. 26

37 Although the general arrangement of inner arm dyneins is conserved across cilia and flagella, localization studies with cilia from organisms will be helpful to complement the findings from flagella. Does relative abundance and total number of dyneins reflect functional significance? The specific role(s) assayed by each of these inner arms in ciliary motility is still in the process of being assessed. It is not known whether abundance in the axoneme reflects role in ciliary beating. The presence of so many dyneins raises a possibility that some of them may be redundant. If abundance correlates with the extent of functional importance, then we would speculate the low/ very low abundance dyneins to be redundant. All mutations affecting one-headed inner arm dyneins in Tetrahymena to date have been in genes encoding low or very low abundance dynein HCs (Liu et al., 2004; Kabi et al., unpublished data). Those mutations do tell us, however, that low to very low abundance dyneins do play a role in ciliary beating as every Tetrahymena one-headed inner arm dynein mutant to date has a motility defect, albeit a relatively minor one (Liu et al., 2004). The low abundant dyneins may function more in fine tuning the ciliary beat pattern and may not be absolutely essential for motility (Wilkes et al., 2008; Yagi et al., 2009). Hence, data, so far suggest that dyneins are not completely redundant; however we cannot rule out the possibility of partial redundancy. Variation in relative abundance of the dyneins may also be attributed to the fact that different sets of dyneins may be localized in different groups of cilia. In Tetrahymena, some dyneins may be specifically localized in oral cilia and not in somatic cilia. Somatic cilia are present all over the cell in contrast to the oral cilia which is present only in the oral apparatus. Since oral cilia are responsible for feeding; mutating those dyneins can result in a pronounced phenotype in the mutants. In higher organisms, localization may vary depending on the particular tissue or organ where cilia are localized. Collectively, we can conclude that abundance may not necessarily reflect how crucial the role of the particular dynein HC is in ciliary motility. Even though cilia and flagella are similar in most respects, there are some differences. While cells typically possess one/ two flagella, the number of cilia present in 27

38 single cells/ surface of organs in higher organisms is generally a few hundred. Ciliary movement can be compared to movement of an oar with alternate power and recovery strokes, while a flagellum exhibits a more undulatory motion. In lower organisms, observations show that the number of dynein HCs in ciliated organisms is higher than in flagellated organisms; flagellated Trypanosomes, Chlamydomonas, typically have sixnine one-headed inner arm dyneins in contrast to Tetrahymena and Paramecium which possess eighteen one-headed dyneins (Wilkes et al., 2008). The ciliated protozoans use cilia for all their different life processes such as movement and feeding. It may be that cilia need more dyneins to power their movement compared to flagella. While the 9+2 axonemal backbone has been evolutionarily conserved, the wide dissimilarity in the number of dyneins across organisms may be a reflection of the variable functional importance of these dyneins. Hence, understanding the evolutionary relationship among dyneins of different organisms may provide inputs regarding their individual function as motors (Wilkes et al., 2008). Differential extraction of dyneins LC-MS/MS analyses of 14S fractions showed that Dyh15p barely got extracted from the axoneme by high salt treatment. This was surprising because dyneins generally are thought to be extracted under high salt conditions (Avolio et al., 1986) and Dyh15p appears to be the most abundant one-headed inner arm dynein in Tetrahymena axonemes. Of the dyneins detected in the 14S fractions, most were also detected to some extent in the corresponding extracted axoneme fractions, strongly suggesting that this high salt treatment does not completely extract all dyneins. Interestingly, the 14S and extracted axoneme fraction analyses complement each other; the dyneins which were highly abundant in the 14S dynein fraction were detected with relatively few peptide hits in extracted axoneme fraction and vice versa. This raises the possibility that each dynein may be interacting differently with other axonemal protein complexes such as radial spokes and those interactions may influence the extent to which each dynein is extracted. Dyh15 is an extreme example since it appears to be very resistant to extraction. The extent to which a dynein can be extracted may be determined by its location in the axoneme. Avolio et al., 1986 showed that within each 96nm repeat, dyneins localized 28

39 between S3 of a proximal spoke group and S1 of next spoke group get extracted more easily compared to others. This phenomenon is not unique to Tetrahymena. Variability in the extent of dynein extraction by high salt treatment was also reported between gill cilia and sperm flagella present in mussel Mytilus edulis and clam Spisula solidissima (Stephens and Prior, 1992). The preferential extraction of some dyneins over others reflects that variable extent of attachment of different dyneins to the microtubule backbone. Since dyneins interact with other axonemal proteins to bring about ciliary beating, the variability in attachment of the dyneins may in some way, be instrumental for the differential roles essayed by them in ciliary motility. Conclusion In summary, we have taken a proteomic approach to identify the members of the dynein superfamily in Tetrahymena thermophila. Analyses of axonemes and dyneins isolated from wild type cells and different mutants led to the detection of twenty-two out of the predicted twenty-five dynein heavy chains predicted by genomic means; confirming that most, if not all, the dyneins are present in the organism. Proteomic analyses combined with expression studies showed that one-headed inner arms are present in varying abundances in the axoneme, thereby raising the speculation if varying abundance can help us understand better the functional diversity and asymmetric localization pattern of these dyneins. In the future, extensive functional analyses combined with localization studies of these dyneins can help us better comprehend the reason for the presence of so many dyneins within an organism. This research also supports LC-MS/MS as an excellent tool not only to perform detailed proteomic studies, but also as a time saving, efficient approach to address other issues such as proving a protein is missing in mutants and identifying the proteins that are present in a complex within the organism, which have been previously answered using complex microscopy and/ biochemical analyses. 29

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50 Table 1. Sample data table from a LC-MS/MS run with wild type dynein HC band excised from the 15% SDS-PAGE gel indicated in Figure1. Reference P (pro) Score Coverage MW Accession Peptide Hits 1 ciliary outer arm dynein beta heavy chain 1.00E ( ) 2 Dynein heavy chain family protein 1.00E ( ) 3 Dynein heavy chain family protein 1.11E ( ) 4 Dynein heavy chain family protein 1.00E ( ) 5 Dynein heavy chain family protein 2.22E ( ) 6 Dynein heavy chain family protein 1.89E ( ) 7 Dynein heavy chain family protein 5.55E ( ) 8 Dynein heavy chain family protein 1.25E ( ) 9 Dynein heavy chain family protein 1.00E ( ) 10 Dynein heavy chain family protein 6.23E ( ) 11 hypothetical protein TTHERM_ E ( ) 12 AF152585_1 dynein heavy chain 1.00E ( ) 13 Dynein heavy chain family protein 1.00E ( ) 14 Dynein heavy chain family protein 2.22E ( ) 15 AF153267_1 dynein heavy chain 1.44E ( ) 16 Dynein heavy chain family protein 5.35E ( ) 17 Dynein heavy chain family protein 8.79E ( ) 18 hypothetical protein TTHERM_ E ( ) 19 AF153268_1 dynein heavy chain 5.86E ( ) 20 hypothetical protein TTHERM_ E ( ) 21 AF153702_1 dynein heavy chain 2.32E ( ) 22 Dynein heavy chain family protein 2.56E ( ) 23 AF153704_1 dynein heavy chain 7.22E ( ) 24 AF153271_1 dynein heavy chain 1.06E ( ) 25 AF153270_1 dynein heavy chain 2.19E ( ) 26 Dynein heavy chain family protein 6.70E ( ) 27 Dynein heavy chain family protein 8.79E ( ) 28 Dynein heavy chain family protein 4.18E ( ) 29 hypothetical protein TTHERM_ E ( ) 30 cyclic nucleotide-binding domain containing protein 5.55E ( ) 31 Protein kinase domain containing protein 5.16E ( ) 32 hypothetical protein TTHERM_ E ( ) 40

51 Table 2. DHCs identified in the axonemal fraction of CU428 cells by LC-MS/MS analyses. Outer Arm/Inner Arm DHC Run 1 Run 2 Run 3 Outer Arm Dyh3p Dyh4p Dyh5p headed Inner Arm Dyh6p Dyh7p headed Inner Arm IAD 3 Dyh25p Dyh11p Dyh10p Dyh8p Dyh14p Dyh12p Dyh13p Dyh17p IAD 4 Dyh21p Dyh16p Dyh19p Dyh9p Dyh20p 1-1 IAD 5 Dyh15p Dyh22p Dyh24p Dyh23p Dyh18p Three independent preparations were analyzed. Numbers under each run column indicate the peptide hits with which each DHC was detected in that run. 41

52 Table 3. DHCs identified in the axonemal fraction of KO3 (outer arm mutant) cells by LC-MS/MS analyses. Outer Arm/Inner Arm DHC Run 1 Run 2 Outer Arm Dyh3p 1 Dyh4p 1 Dyh5p 2-headed Inner Arm Dyh6p Dyh7p headed Inner Arm IAD 3 Dyh25p Dyh11p Dyh10p 2 4 Dyh8p 3 1 Dyh14p 3 5 Dyh12p - 1 Dyh13p - - Dyh17p - - IAD 4 Dyh21p Dyh16p Dyh19p 28 3 Dyh9p 8 0 Dyh20p 7 2 IAD 5 Dyh15p Dyh22p Dyh24p Dyh23p 2 4 Dyh18p - - Two independent preparations were analyzed. Numbers under each run column indicate the peptide hits with which each DHC was detected in that run. 42

53 Table 4. DHCs identified in the axonemal fraction of KO6 (two-headed inner arm mutant) cells by LC-MS/MS analyses. Outer Arm/Inner Arm DHC Run 1 Run 2 Outer Arm Dyh3p Dyh4p Dyh5p headed Inner Arm Dyh6p - - Dyh7p headed Inner Arm IAD 3 Dyh25p Dyh11p Dyh10p 2 - Dyh8p - 1 Dyh14p 5 - Dyh12p 1 - Dyh13p - - Dyh17p 1 - IAD 4 Dyh21p Dyh16p 42 6 Dyh19p Dyh9p 7 - Dyh20p 8 2 IAD 5 Dyh15p Dyh22p 39 9 Dyh24p Dyh23p 2 1 Dyh18p - - Two independent preparations were analyzed. Numbers under each run column indicate the peptide hits with which each DHC was detected in that run. 43

54 Table 5. DHCs identified in the 14S fraction of wild type cells by LC-MS/MS analyses. Outer Arm/Inner Arm DHC Run 1 Run 2 Outer Arm Dyh3p 48 - Dyh4p 8 1 Dyh5p 40-2-headed Inner Arm Dyh6p 22 - Dyh7p 21-1-headed Inner Arm IAD 3 Dyh25p Dyh11p Dyh10p 4 5 Dyh8p 1 3 Dyh14p - - Dyh12p - - Dyh13p - 1 Dyh17p - - IAD 4 Dyh21p Dyh16p Dyh19p Dyh9p 2 3 Dyh20p 2 6 IAD 5 Dyh15p - - Dyh22p Dyh24p Dyh23p 1 1 Dyh18p S dynein fractions were run on 15% SDS-PAGE. Two independent preparations were analyzed. Numbers indicate the peptide hits with which each DHC was detected in that run. 44

55 Table 6. DHCs identified in the extracted axonemal fraction of wild type cells by LC- MS/MS analyses Outer Arm/Inner Arm DHC Run 1 Run 2 Outer Arm Dyh3p Dyh4p Dyh5p headed Inner Arm Dyh6p Dyh7p headed Inner Arm IAD 3 Dyh25p Dyh11p 4 6 Dyh10p - 1 Dyh8p - 1 Dyh14p - 6 Dyh12p 3 3 Dyh13p - - Dyh17p - - IAD 4 Dyh21p Dyh16p Dyh19p 4 11 Dyh9p 3 4 Dyh20p 3 5 IAD 5 Dyh15p Dyh22p Dyh24p 7 14 Dyh23p 2 3 Dyh18p - - Extracted axoneme fractions of wild type cells were ran on 15% SDS-PAGE. Numbers under each run column indicate the peptide hits with which each DHC was detected in each run. Two independent preparations were analyzed. 45

56 Fig 1. SDS-PAGE separation of axonemes: Axonemes isolated from wild type cells were separated on a 15% SDS-PAGE. Bands were visualized by staining with Coomassie Blue. The DHCs are packed tightly in a band at the top of the gel as indicated by the arrow. 46

57 47

58 Fig 2. Sucrose density gradient centrifugation analysis of dyneins extracted from wild type cells: Dyneins were subjected to sucrose density gradient centrifugation and fractions collected were separated on a 3-5% 0-8M Urea gradient PAGE. Fractions 1-11 were run in Gel 1 (A) and in Gel 2 (B). Gels were stained by Coomassie Blue. 22S represents the three-headed dynein, 18S the two-headed dynein and 14S the one-headed dynein. 48

59 49

60 Fig 3. SDS-PAGE separation of axonemes, dyneins and extracted axonemes: Whole axonemes (WA), dyneins (D) and extracted axonemes (EA) isolated from wild type cells were run on 15% SDS-PAGE and gels were stained using Coomassie Blue. The DHC band in all the three lanes is indicated by the arrow. The presence of a DHC band in EA lane proves that dynein extraction is not complete. 50

61 51

62 Fig 4. Peptide proportion analysis of one-headed inner arm dyneins of wild type, KO3 and KO6 cells. Graph A, B and C represents the members of IAD3, IAD4 and IAD5 respectively. Error bars represents S.D. Two independent preparations of each cell type was analyzed. 52

63 53

64 Fig 5. Expression studies of representative DHCs: qrt-pcr analysis was carried out using RNA isolated from wild type cells to determine the relative expression level of DHCs. An outer arm HC (DYH3), a two-headed inner arm HC (DYH6) and all members of one-headed inner arm subgroup IAD 5 were chosen for analysis. Expression of all other genes was normalized to that of DYH3. Error bars represent S.D. 54

65 55

66 Relative abundance and significance in ciliary motility: a different dimension of functional complexity of inner arm dyneins 56

67 Abstract Axonemal dyneins utilize the energy derived from ATP hydrolysis to drive sliding of adjacent outer doublets resulting in ciliary bending. Each dynein is a macromolecular complex comprising of heavy chains (HCs), which are the actual motors, in combination with different intermediate and light chains. Depending on their position along each microtubule doublet, dyneins are either outer arm dynein or inner arm dynein. In all organisms studied to date, the number of dyneins identified biochemically is not the same as that identified genomically. For example, in Tetrahymena, biochemical studies have identified one outer arm and seven different inner arm dyneins. Recent genomic analyses suggest that dynein HC family is more complex and predict as many as twenty-two different inner arm dynein HCs. Subsequent proteomic analyses in our lab detected nineteen out of the predicted twenty-two HCs. Further, the inner arms were present in varying abundances within the axoneme. Based on sequence analysis, the one-headed inner arms are further subdivided into three groups. These findings lead to two broad questions: i) Do all dyneins within a subgroup perform similar functions? (ii) Does abundance reflect functional significance of dyneins? To address these questions, we knocked out two different inner arm dyneins -DYH15, the highest abundant one-headed inner arm dynein, and DYH18, present in very low abundance. Both DYH15 and DYH18 are members of subgroup IAD5. Both mutants displayed a 15-25% reduction in swimming speed. Beat frequency and feeding behavior was affected in KO15, but not in KO18. These data suggest that dyneins within a subgroup are involved in different roles in ciliary motility. KO15, however, did not display as dramatic a phenotype as might be expected given its abundance within the axoneme. LC-MS/MS analyses of axonemes isolated from KO15 mutants revealed an increase in levels of three one-headed DHCs. No such compensation was observed in case of KO18. This raises the possibility that some dyneins are partially redundant. Key words: Dyneins, abundance, function 57

68 Introduction Dyneins are a class of motor proteins that move along microtubules (Porter et al., 1989). Belonging to the ATPase superfamily, dyneins power their movement by utilizing the energy released by hydrolysis of ATP. Dyneins are classified as axonemal or cytoplasmic based on their intracellular location. Cytoplasmic dyneins are present in all eukaryotic cells and are involved in different intracellular activities such as formation of spindle poles during mitosis, transport of cargo for cellular activities and axonal transport in nerve cells (Vallee et al., 2004). Axonemal dyneins, on the other hand, are localized in cilia and flagella and drive sliding of adjacent outer microtubule doublets. In cilia, accessory proteins convert sliding to different beat patterns. Ciliary beating results in movement of unicellular organisms through a fluid medium or movement of fluid across the cell surface in higher organisms. In humans, defects in dyneins have been associated with diseases such as primary ciliary dyskensia, hydrocephalus and respiratory tract infection (Pan et al., 2005). A clear understanding of the role(s) of dyneins in ciliary motility will not only help us understand the mechanism of ciliary movement, but may also provide significant insights into the cause of some human disorders. The molecular masses of dynein complexes range from MDa (Burgess and Knight, 2004). Each complex consists of heavy chains (HCs), light chains (LCs) and in some cases, intermediate chains (ICs) (Witman et al., 1994). HC form the motor domain of the dynein, contain the site of ATP hydrolysis, and make up most of the mass of the dynein complex. The intermediate and light chains influence motor activity, are involved in assembly of the motor complex and play regulatory role (s) (Baron et al., 2007; Sakato and King, 2004). Each HC is comprised of approximately 4600 amino acids (aa) and can be broadly divided into two domains- the C-terminal two-thirds making up the head domain and the remaining ~ 1300aa in the N-terminus forming the tail domain. The head domain contains six AAA (ATPase associated with cellular activities) modules (Neuwald et al., 1999; Asai and Wilkes, 2004). The sequence of the first four modules is highly conserved across dyneins from different species. Each of these modules contains a nucleotide binding site (Asai and Wilkes, 2004). The first site, AAA1 is the site of ATP 58

69 hydrolysis as well as the catalytic site. The next three sites, AAA2-4, display reduced nucleotide binding properties, but nucleotide binding to those sites may be involved in regulation of dynein activity (Hirakawa et al., 2000; Kinoshita et al., 1995). AAA5 and AAA6 are less conserved and do not have complete nucleotide binding sites (Asai and Koonce, 2001). Image average analyses illustrates that the globular head resembles a homo-hexameric wheel with each AAA module forming a distinct lobe surrounding a central cavity (Asai and Koonce, 2001). A 10-12nm coiled coil emerges from the region between AAA4 and AAA5. Also known as the B-link, the tip of this stalk is the microtubule binding site (Asai and Brokaw, 1993; Koonce, 1997) that contacts the adjacent B microtubule to drive microtubule doublet sliding. The N-terminal domain forms part of the stem structure that arises from AAA1. This N-terminal domain is less conserved than the head domain and is primarily involved in interaction with other HCs, ICs and LCs (Gibbons et al., 1991; Asai and Koonce, 2001; Asai and Wilkes, 2004). Axonemal dyneins are classified as either outer arm dynein or inner arm dynein based on their position on the outer doublets. Both groups of arms play critical roles in the sliding of adjacent microtubules that results in ciliary bending (Gibbons, 1981); however structurally, functionally and also in terms of organization along the axoneme, the two rows of arms differ from each other. Outer arms contain two or three HCs, two or more ICs and multiple LCs (Witman et al., 1994). Outer arms of Trypanosome, Giardia and higher eukaryotes are two-headed and contain two HCs while outer arms in Chlamydomonas and Tetrahymena contain three heavy chains and are three-headed (Pfister and Witman, 1984; Pfister et al., 1982). Along the axoneme, outer arm dyneins repeat every 24 nm. Multiple studies using various approaches strongly suggest that inner arms are a more heterogeneous group than the outer arms. Biochemically, eight different axonemal dynein heavy chains were identified in studies done with Chlamydomonas and Tetrahymena (Gardner et al., 1994; Goodenough et al., 1987; Kagami and Kamiya, 1992; Piperno et al., 1990). High pressure liquid chromatographic (HPLC) separation of dyneins isolated from different Chlamydomonas mutants lacking outer arms and various inner arms suggested that the eight HCs are assembled into one two-headed and six oneheaded inner arm heavy chains within the axoneme (Kagami and Kamiya, 1992). Studies 59

70 by Muto and group suggest a similar assembly of dyneins in Tetrahymena (Muto et al., 1994). The organization of inner arms is more complicated than that of the outer arms and a variety of microscopic and biochemical techniques have been employed in several model systems to get a comprehensive view of the distribution of the different inner arms within the axoneme. Electron microscopic studies performed using Chlamydomonas flagella indicated that the inner arms are organized in 96 nm repeats longitudinally along the axoneme (Goodenough and Heuser, 1989). Each repeat was subdivided into three segments termed as I1, I2 and I3 by radial spokes and showed a characteristic triad-dyaddyad arrangement. Subsequent studies revealed that triad located in the I1 region was the two-headed dynein (now called I1) while the dyads located in the I2 and 3 regions were one-headed dyneins (now called I2/3 dyneins). Immunoprecipitation studies combined with other experiments on mutants resolved I2 and I3 inner arms into two classes: one associated with light chain p28 and the other associated with centrin (LeDizet and Piperno, 1995). It was proposed that within the 96nm repeat, each I2 and I3 segment contains one dynein HC associated with p28 and a second dynein HC associated with centrin (LeDizet and Piperno, 1995). Subsequent structural studies indicated that six oneheaded dyneins are present within each 96 nm repeat and suggested that these one-headed dyneins might be present as functional dimers (Huy Bui et al., 2008). Analyses of Chlamydomonas mutants with reduced flagellar lengths suggested that axonemes have distinct populations of I2/I3 dyneins in the proximal, medial and distal regions of the axoneme (Piperno and Ramanis, 1991). Recent electron tomographic experiments with different Chlamydomonas inner arm mutants provided additional evidence of the asymmetry of inner arms longitudinally along the axonemes (Huy Bui et al., 2009). Radial asymmetry in inner arm region of axoneme has also been reported in sea urchin (Sale, 1986), rat sperm (Lindemann et al., 1992) and Chlamydomonas (King et al., 1994) flagella. Taken together, structural and biochemical suggests that variation in composition of inner arms occurs longitudinally along each doublet as well as radially among the different doublets. Initial genetic studies also supported the biochemical and structural findings. PCR-based cloning studies using degenerate primers identified nine genes encoding inner 60

71 arm dynein HCs (DYH6-14) in Tetrahymena thermophila (Xu et al., 1999; Asai and Wilkes, 2004), eight inner arm dynein HC genes in Chlamydomonas (Porter et al., 1996), eight in sea urchins (Gibbons et al., 1994) and six in mammals (Chapelin et al., 1997). More recent comparative sequence analyses using completed genomes of different species such as Chlamydomonas, Trypanosome, Tetrahymena and Homo sapiens identified additional one-headed dynein HCs in several organisms (Wilkes et al., 2008) and suggested that there is great variation in the total number of one-headed inner arm dynein HC genes in different species. For example, Chlamydomonas has eight genes; Trypanosome has six, and Tetrahymena has eighteen one-headed inner arm dynein HC genes. Unpublished work shows Paramecium, another member of the same group as Tetrahymena, may also have a large number of inner arm DHC genes (Wilkes et al., 2008). Thus, genomic studies indicate that all ciliated or flagellated organisms encode multiple one-headed inner arm dynein HC and the complexity of one-headed inner arm dyneins is even greater than previously thought. Proteomic studies generally support the genomic analyses. Mass spectrometric analyses detected all six one-headed dyneins in Trypanosome (Broadhead et al., 2006), all but one in Chlamydomonas (Pazour et al., 2005), and fifteen out of the predicted eighteen one-headed dyneins in Tetrahymena (Kabi et al., unpublished observations). These studies along with biochemical analyses in Chlamydomonas (Yagi et al., 2009) suggest that the different one-headed dyneins are present in varying abundances. Based on sequence analyses, the one-headed dynein HC gene family has been further subdivided into three groups- IAD 3, IAD 4 and IAD 5 (Wilkes et al., 2008; Morris et al., 2006; Wickstead and Gull, 2007). The number of dyneins in each of these groups varies among different organisms. Further, within a group members are present in variable abundances (Yagi et al., 2009; Kabi et al., unpublished observations). To explain the presence of three evolutionarily distinct groups of one-headed dynein HC genes in all organisms studied to date, different possibilities have been suggested: a) the three groups of dyneins could correspond to one-headed inner arm dyneins in the proximal, medial and distal regions along the axoneme (Wilkes et al., 2008); b) each group could be specialized in controlling a particular aspect of ciliary motility, but members within a 61

72 particular group may play similar role(s) (Wilkes et al., 2008) c) each group may be specifically associated with specific LCs (Wickstead and Gull., 2007). The inner arms are not only more complicated than outer arms in terms of their evolution, number and organization, but functionally also, they appear to play more diverse role(s) than the outer arms. Most evidence supports the idea that outer arms determine beat frequency while inner arms determine waveform. Gibbons and Gibbons (1973) performed functional studies of outer arms using sea urchin sperm flagellum as their model. Reactivation of spermatozoa of sea urchin, following selective extraction of their outer arms using 0.5M KCl, showed that the beat frequency of flagella was reduced by approximately 50%, but wave form was unaltered (Gibbons and Gibbons, 1973). Mutants lacking outer arm dyneins in Chlamydomonas (Brokaw and Kamiya, 1987; Kamiya and Okamoto, 1985), Tetrahymena (Zhao and Pennock-unpublished observations, Attwell et al., 1992) also exhibit significantly reduced beat frequencies with normal waveforms. Initial studies with Chlamydomonas mutants missing different inner arms showed that the mutants exhibited normal beat frequency but altered wave form (Brokaw and Kamiya, 1987). Over the past few years, however, some other inner arm mutants have been characterized in organisms such as Chlamydomonas, Tetrahymena and mouse with defects in beat frequency (Yamamoto et al., 2006; Liu et al., 2004; Neesen et al., 2001) While all outer arms appear to be identical and perform the same function, variation in function has been observed among the different inner arm family members. All the one-headed inner arms isolated from Chlamydomonas flagella translocate microtubules in vitro, but they do so at different velocities (Smith and Sale, 1991; Kagami and Kamiya, 1992). In addition, five of the one-headed inner arm dyneins rotate microtubules during translocation (Kagami and Kamiya, 1992). Although inner arms as a group appear to determine waveform, some inner arm mutants exhibit defects in beat frequency. Slightly reduced beat frequency in cilia was observed in mutant mice with disruption in a putative inner arm HC (Neesen et al., 2001) and in a Tetrahymena inner arm mutant, KO8 (Liu et al., 2004). Furthermore, studies in Chlamydomonas revealed that in the absence of outer arms, some inner arm dyneins played a crucial role in determining beat frequency (Yamamoto et al., 2006). In Chlamydomonas, an inner arm 62

73 dynein, DHC9, has been shown to play crucial role in flagellar movement, predominantly under highly viscous conditions (Yagi et al., 2005); thereby indicating that some dyneins are necessary for cells to adapt to changeable micro-environments. Taken together, evidence suggests that inner arm dyneins play a variety of different roles in ciliary motility. The complicated arrangement of inner arm dyneins within each 96 nm repeat, and the presence of distinct subsets of inner arm dyneins, both radially and longitudinally along the axoneme also supports the suggestion that one-headed inner arm dyneins display functional diversity. Although no outer arm mutant has paralyzed cilia, Chlamydomonas mutants missing the two-headed inner arm dynein along with a subset of one-headed inner arm dyneins exhibit a paralyzed flagella phenotype (Kamiya et al., 1989). These data suggest that unlike outer arms, inner arms as a group may be necessary and sufficient for ciliary beating. Furthermore, proteomic and biochemical studies reflect that within an organism, they are not equally abundant (Kabi et al., unpublished observations; Yagi et al., 2009). The varying abundances of these dyneins correlate to a certain extent, with the asymmetric distribution of the dyneins along the axoneme. However, it is not known yet whether relative abundance level can help us predict how crucial a role an inner arm dynein plays in ciliary motility. The presence of so many dyneins coupled with their great variation in abundance raises a speculation that some dyneins may be redundant. Functional redundancy is a phenomenon observed in other motor superfamilies such as myosins and kinesins (Wilkes et al., 2008). However, to date, none of the dynein mutants, characterized so far, in any organism, has reflected complete functional redundancy. Some phenotype; albeit subtle in many cases; has been observed in all mutants characterized thus far. This strengthens the possibility that some inner arms may be involved in fine tuning ciliary beat patterns, rather than being major players in controlling the ciliary beat (Wilkes et al., 2008). In Chlamydomonas, most of the oneheaded inner arm mutants described are missing multiple inner arms with the exception of ida9 which has only DHC9 missing (Kamiya et al., 1991; Kagami and Kamiya, 1992; Kato et al., 1993; Yagi et al., 2005). While the multiple mutants help us address different aspects of inner arm function, they cannot be used to determine the role of individual inner arms in flagellar movement. The three one-headed inner arm mutants reported, so 63

74 far in Tetrahymena, are all knockouts of low abundance dyneins (Liu et al., 2004; Kabi et al., unpublished observations); no high abundance dynein has so far been characterized in Tetrahymena. The vast disparity in the total number of dyneins across organisms indicates that functional significance of different dyneins may differ depending on the organism. In organisms where the total number of dyneins is less, each dynein may be involved in some important role; in contrast other organisms having a larger family of dyneins might contain members displaying fine tuning function. All these factors make it evident that detailed understanding of the role of individual inner arm dyneins in different organisms is required to comprehend their functional complexity. In summary, multiple one-headed inner arm dyneins and dynein HC genes have been identified in every ciliated species studied to date. The one-headed inner arm HC genes appear to fall into three evolutionarily distinct groups, and members within a group are present in varying abundances - each subgroup contains a high abundance, one/ two medium abundance and a few low abundance dyneins. Functional analyses suggest inner arm dyneins are heterogeneous and may play different roles. These observations beg two obvious questions- i) Do all inner arm dyneins within a group perform similar functions or are they functionally different? ii) Is there any correlation between relative abundance of a dynein and its functional significance? We have attempted to address those questions by knocking out two inner arms in Tetrahymena thermophila: DYH15, present in high abundance, and DYH18, present in very low abundance, both members of IAD5. The two mutants exhibited different phenotypes, which suggest that not all inner arms within an evolutionary group perform the same function. Interestingly, the severity of the phenotype did not correlate with the normal relative abundance of the HC that was knocked out, but LC-MS/MS analysis of DYH15 mutants raise the possibility that mutant cells may have increased the abundance of other dyneins to compensate for the loss of DYH15. Methods Cell Culture CU428 cells (kindly provided by Peter Bruns, Cornell University) were used for the transformation. All cells were grown in Neff s medium (0.25% proteose peptone, 0.25% 64

75 yeast extract, 0.55% glucose, 33uM FeCl 3 ) and incubated at 28 0 C in the shaker at 125 rpm. Two control strains were used for the behavioral experiments: CU428, the strain in which transformation was carried out and UC230 (kindly provided by Aaron Turkewitz, University of Chicago), the neo-expressing strain (Haddad and Turkewitz, 1997). UC230 cells and the mutants generated were maintained at a concentration of 300µg/ml paromomycin. Before use, the cells were grown in media without paromomycin for around a week. Construction of KO Constructs The genomic sequences of DYH15 and DYH18 were downloaded from ciliate.org. A 3.0 kbp fragment around AAA1 was PCR amplified using appropriate primer pairs for each gene of interest. The region amplified had a unique restriction site cut by an enzyme that left blunt ends near the middle of the cloned region. The PCR products were ligated into pgem-t-easy Vector (Promega), and the ligated product was transformed into chemically competent E. coli cells (New England Biolabs) following the method described by the manufacturer. The plasmid containing the cloned gene was grown in a larger volume; DNA was isolated using Promega Plasmid Maxi Prep kit and digested with the appropriate restriction enzyme. The restriction enzymes used were for cloned regions of DYH15 and DYH18 respectively. The blunt ends were phosphatased to prevent self ligation. The neo2 cassette is composed of the coding region of the neomycin resistance gene flanked by the Tetrahymena histone H4-1 gene promoter on the 5 end and Tetrahymena beta-tubulin-2 gene poly A signal on the 3 end (Gaertig et al., 1994). To make the knockout construct, the neo2 cassette was removed from the plasmid vector using appropriate restriction enzymes, gel purified, and ligated into the cut, phosphatased 3kb fragment as described previously (Angus et al., 2001; Liu et al., 2004). The ligated product was transformed into chemically competent E. coli cells (New England Biolabs) and screened for the colony with insert. To prepare the DNA for biolistic delivery into Tetrahymena, DNA was isolated at a larger scale and digested with appropriate restriction enzymes to take out the cloned DYH fragment with the neo2 cassette. Finally, the digested DNA was precipitated with ethanol and resuspended in water to a concentration of 1 µg/µl prior to biolistic transformation. 65

76 Transformation of Tetrahymena with KO Construct Wild type CU428 cells were grown to the log phase (2-5 X 10 5 cells/ml) overnight at 28 0 C in a shaking incubator (at 125rpm). Cells were starved overnight in 10mM Tris (ph 7.4) at a concentration of 2 X 10 5 cells/ml. Biolistic transformation of the macronucleus was carried out as described in Bruns and Cassidy-Hanley (2000). Briefly, 1 μg DNA was used in each transformation. 0.6µM gold particles were coated with linearized plasmid. Starved cells were washed in 10mM HEPES, ph 7.5 and finally resuspended in 1 ml HEPES. 1 X 10 7 cells was used for each transformation. Cells, resuspended in HEPES, were spread on the sterile, pre-wetted filter paper (pre-wet with HEPES) and bombarded at 900 psi using the BioRad PDS-1000/He particle delivery system with DNA-covered gold particles. Following transformation, the filter paper containing cells was carefully transferred in a 500 ml flask containing 50ml Neff s medium. After 2-6 hours, paromomycin was added to the final concentration of 100ug/ml and were incubated at 28 0 C without shaking. After 4-5 days, a few micro liters of cells were removed aseptically to check for live cells under the microscope. The presence of live, swimming cells indicated that the transformation was successful. Complete phenotypic assortment Biolistic transformation is successful when the knockout constructs intergrates into the Tetrahymena genome by homologous recombination. Tetrahymena has two nuclei- a vegetative macronucleus, the nucleus targeted in our experiment, and a germline micronucleus. The macronucleus has copies of each gene. Knockout is complete only when all the wild type copies of the gene are replaced by the mutant version. To accomplish that, transformed cells were transferred every few days into medium containing higher concentrations of paromomycin. This process enables gradual replacement of wild type copies of the gene by mutated version and is termed as phenotypic assortment. At regular intervals, DNA was isolated from the cultures and PCR was carried out to check for the extent of complete replacement. At a concentration of 27,000 μg/ml and 24,000 μg/ml of paromomycin, PCR results indicated all the wild type copies of DYH15 and DYH18 have been replaced respectively. Following selection, cells were grown in medium without paromomycin, and PCR was performed on DNA 66

77 isolated from mutant cells to determine whether reversion had occurred. Once, we had confirmed that knockout was indeed complete; clones were established in hanging drops and cultured as described in Pennock (2000). Three clones were chosen; PCR was performed, and one clonal line was used for all future analyses. Southern Blot Whole genomic DNA was isolated from wild type CU428 and KO15 and KO18 mutants as described by Gaertig and Gorovsky (1992). Restriction digestion of DNA was carried out using SpeI and HincII for KO15 and KO18 respectively. The digested DNAs were electrophoresed through a 1% agarose gel and transferred from the gel to nylon membrane (Osmonics) in 10X SSC buffer (1.5 M NaCl, 0.15 M NaCitrate.2H 2 O, ph 7.0) overnight. The membrane was dried in an 80 0 C vacuum oven for 2 hours. Specific probes were designed for each gene of interest. To make the probes, PCR amplification was carried out using wild type DNA and the PCR products were purified by Wizard SV Gel and PCR Clean-Up System (Promega, WI). The probe was radio-labeled with 32 PdCTP using the Prime-a-gene Labeling System (Promega, WI).Prior to hybridization, the membrane was incubated in ExpressHyb solution (BD Biosciences) for 30min. The membrane was then hybridized with radioactively labeled probe (used at a concentration of cpm/ml) for one hour at 60 0 C with gentle shaking. Two wash solutions were used to clear the background-wash Solution 1 (2 SSC, 0.05% SDS) and Wash Solution 2 (0.1 SSC, 0.1% SDS). Rinses were carried out for 40 min each, with both the solutions. For Wash Solution 1, rinsing was carried out at room temperature while Wash Solution 2 was used at a temperature of 50 0 C. Finally, the membrane was exposed to a phosphor-imager screen and bands were visualized using the Storm Phosphorimaging System (Molecular Dynamics). Axoneme isolation, SDS-PAGE and LC-MS/MS Axoneme isolation was carried out from three cell types: wild type (CU428) and both mutants (KO15 and KO18). One liter of cells was pelleted by centrifuging at 900g for 3 minutes. Medium was removed by aspiration to approximately 5ml and the pellet was resuspended. Cilia were removed from cells by adding dibucaine to a final concentration of 3mM. Cell bodies were pelleted at 900g for 3 minutes at 4 0 C, and supernatant 67

78 containing cilia was transferred into new tubes. 25 µl of 0.1 M PMSF (phenylmethylsulphonyl fluoride), a serine protease inhibitor, was added. Remaining cell bodies were pelleted by a second round of centrifugation as described above. Cilia were pelleted at112000g in a Sorvall SS34 rotor for 10 minutes at 4 0 C). Pelleted cilia was resuspended in 1ml 1 SB buffer (30mM HEPES, PH 7.6; 5mM MgSO 4 ; 0.5mM EDTA; 20mM KCl). 5μl leupeptin (5μg/ml) was added to cilia, and cilia were layered on 20 ml of 1M Sucrose, 2mM EGTA in 1X SB and pelleted at g in Sorvall SS34 rotor for 20 minutes at 4 0 C. The cilia pellet was resuspended in 1ml 1 SB, 5μl leupeptin (5μg/ml), and 25 μl 10% NP-40 was added to disrupt ciliary membranes, and the solution was incubated on ice for 30 minutes. Axonemes were then pelleted at g in Sorvall SS34 rotor for 10 minutes at 4 0 C and this process was repeated. Axonemes were finally resuspended in 1m 1X SB. The concentration of axonemes was determined using Bradford Reagent (BioRad). One fifth volume of 5X SDS sample buffer ( M Tris, 10% SDS, 25% βme, 33% Glycerol) was added, and samples were boiled for approximately 15 minutes. 100 µg protein was loaded on a 15% SDS-PAGE gel. Gels were ran for ~16-20 hr at constant voltage of 50V to resolve all the dynein HCs as a compact band. To visualize the bands, the gels were stained by Coomassie Blue R for 3-4 hrs and then destained using Destain1 (50 % Acetic Acid, 10 % Methanol) for 3-4 days. Finally, the gels were stored in Destain 2 (5% Acetic Acid, 7% Methanol) at 4 0 C. For LC-MS/MS analysis the dynein HC band was excised from the SDS-PAGE gel and sent to the University of Cincinnati Mass Spectrometric Facility (Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati). Swimming Speed To measure swimming speed, wild type and mutant cells were grown overnight in a shaking incubator shaking at 125 rpm. Before the start of the assay, the cell concentration was determined using a hemacytometer and 1X10 6 cells were briefly pelleted at1500 rpm for 2min, and the cells were then resuspended gently in 5ml of media. The resuspended cells were transferred to 50ml flasks and were allowed to incubate for 10 min at room temperature. 0.5 μl cells were then added to 90 μl media taken on a glass slide, and the cells 68

79 were video recorded for 5sec using a DAGE-MTI CCD100 video camera at30 frames/sec. Image J program (NIH) was used to stack 30 frames at a time. This gave the path length covered by the cells in one second. Cell lengths of five random cells were measured and the average cell length was deducted to get the actual distance traversed by each cell in one second. Swimming speed was reported in mm/sec based on the distance traversed by the cells in one sec. Three independent trials were performed. A Kruskal-Wallis Test was used to determine whether the swimming speed of the mutants was statistically different from the swimming speed of the wild-type cells. Beat Frequency To measure beat frequency, cells were grown overnight at 28 0 C in a shaking incubator shaking at125 rpm. 60 μl cells at X cells/ml were pipetted onto a plain slide with a coverslip supported by double stick tape and freely swimming cells were video recorded at 500 frames/sec using a Photronics 1280 PCI FastCam attached to a Nikon Eclipse E600 microscope. Cells were visualized using a 100X oil objective. The assay was performed at room temperature. Cells whose movement was not impaired by slide and coverslip support were chosen for analysis. In each cell, individual cilia or groups of cilia were analyzed to determine the number of frames covered to complete one full beat cycle. Based on the number of frames/cycle and frames/sec, beat frequency was reported in cycles/sec (Hz). A Kruskal-Wallis Test was used to determine whether the swimming speed of the mutants was statistically different from the swimming speed of the wild-type cells. Feeding Assay Cells were grown to early log phase (~ 2 X 10 5 cells/ml) under shaking conditions at125 rpm. 1 ml of pre-warmed Neff s containing 2% India ink was added to 9ml of cells, and cells were then incubated for 20 min at 28 0 C in a gyrotory incubator at 125rpm. One ml culture was collected, and cells were fixed with formaldehyde. Cells were scored for the presence/absence of ink stained vacuoles under a bright field microscope at a magnification of 200X. One hundred randomly chosen cells were considered for each cell type. Three independent trials were conducted. A Kruskal-Wallis Test was used to determine whether 69

80 the swimming speed of the mutants was statistically different from the swimming speed of the wild-type cells. qrt-pcr Total RNA was isolated from Tetrahymena utilizing the Qiagen RNEasy Mini Kit (Cat # 74104). RNA purity was determined using UV-spectrometry by 260/280 nm ratio. RNA was stored at C and used within one month of isolation. For each assay, 1 ug of total RNA was subjected to reverse transcription (RT) using QuantiTect Reverse Transcription Kit for RT-PCR (Qiagen, Cat #205310) as per manufacturer instructions. Briefly, first strand cdna was prepared from total RNA using oligo (dt) primers. Once prepared, the cdna will be stored at C for further use. The first strand cdna was used for quantitative PCR (qpcr) using gene specific primers and Sybr-green (Qiagen). Briefly, for each PCR reaction cdna was mixed with forward and reverse primers, Sybr-green and distilled water in the quantity as indicated in the Qiagen kit. PCR reaction was performed using a 95 0 C (10 min) step followed by 40 cycles at 95 0 C (15 sec) and 60 0 C (1 min). Data was analyzed using the 2 -ΔCt method using tubulin as housekeeping control. ΔCts were calculated by subtracting the Ct value of tubulin from the Ct values for each gene. Results Complete knockout of DYH15 and DYH18 To elucidate the role(s) of one-headed inner arm dyneins in ciliary motility, we used an insertion mutation strategy to knockout the genes, DYH15 and DYH18, in Tetrahymena thermophila. We cloned a 3Kb region of each HC gene around AAA1 site, the site of ATP hydrolysis. Fig. 1A and B illustrates the knockout strategy of DYH15 and DYH18 gene respectively. With an appropriate restriction enzyme, as mentioned in details in the methods section, the neo-cassette was inserted in the cloned fragment to make the knockout construct. The construct was then transformed into the Tetrahymena macronucleus by biolistic means. Tetrahymena exhibits nuclear dimorphism with a vegetative macronucleus and germline micronucleus. The macronucleus has copies of each gene. The transformed cells were grown in increasing concentrations of 70

81 paromomycin, which brought about gradual replacement of wild type copies of the gene by the mutant version. At regular intervals, PCR was carried out with appropriate primers to check for the extent of replacement (data not shown). PCR results indicated complete replacement of all the wild type copies of the gene at a paromomycin concentration of µg/ml and 20,000µg/ml for KO15 and KO18 respectively. To confirm complete replacement, cells were grown in medium without drug for two weeks. Under these conditions, if replacement was not complete, the absence of paromomycin would result in rapid replacement of the disrupted copies by the wild type version. PCR on genomic DNA isolated from cells grown without drug indicated there was no reversion and confirmed that replacement was complete. Single cells were isolated from the KO15 and KO18 cultures to establish clonal lines, and the clones were expanded. A Southern blot of genomic DNA isolated from the wild type and mutant cultures confirmed that replacement was complete and that the knockout cassette had integrated in the correct position (Fig. 2A and B). With wild type DNA, expected band sizes were 5500 bp for the DYH 15 fragment and 3000 bp for the DYH 18 fragment. In the mutants, the bands are expected to be around 1.4 Kb larger than the wild type band because of the insertion of the 1.4 Kb neomycin cassette. The presence of bands of sizes 6900 and 4400 bp in the KO15 and KO18 lanes confirmed that the cassette had integrated in the correct position. Since there was no band of wild type size in the KO15 lane and only a faint band (containing micronuclear DNA) of wild-type size in the KO18 lane, we concluded that the knockouts were complete. Dyh15p is missing from KO15 axonemes To confirm that the Dyh15p was missing from the KO15 mutants, we isolated axonemes from KO15 cell cultures and separated the protein mixture on a 15% SDS-PAGE gel. The dynein HCs ran tightly packed in the top band as marked in Fig 3. LC-MS/MS analysis of this band did not give a single peptide hit against Dyh15p (Table 1) in contrast to wild type preparations where Dyh15 was detected with peptide hits ranging from in three independent runs. All other DHCs detected in wild type preparations were observed in KO15 axonemes. Three independent sample preparations were analyzed and the results were consistent. 71

82 LC-MS/MS could not be used to determine whether the protein is missing from KO18 axonemes because Dyh18p cannot be detected by LC-MS/MS, presumably because it is a very low abundance dynein. However, the knockout strategy used to mutate DYH18 gene has been used by our lab and other groups to successfully knockout many different genes, including other DHCs such as DYH3, DYH15 and DYH6. In our own lab, we have confirmed that the protein is missing from KO3, KO6 and KO15 mutants. Thus, it is very likely that DYH18p is missing from KO18 mutants. Both KO15 and KO18 mutants display altered swimming behavior Ciliary beating powered by dyneins moves Tetrahymena cells through a fluid medium and pulls fluid into the oral apparatus during feeding. To understand the effects of mutation of dyneins on ciliary motility, we assayed swimming speed, beat frequency and feeding rate. To measure the swimming speed of wild type and mutant cells, freely swimming cells were video recorded for 5 sec, and the videos were analyzed with Image J software. The average swimming speed for both the wild type cell types and mutants is shown in Fig. 4. Wild type strains CU428 and UC230 had average swimming speeds of 0.40 and 0.39 mm/sec respectively. KO18 cells exhibited a 15% decrease in swimming speed (average speed of 0.34 mm/sec) while KO15 mutants swam at approximately 0.30 mm/sec (~25% slower than wild type cells). The difference between swimming speed of both the mutants when compared to that of the wild type cells was statistically significant. Variation in swimming speed can be due to alteration in beat frequency or waveform of ciliary beating or a combination of both. We determined ciliary beat frequency of mutant cells and wild type control cells (Fig. 5). Wild type Cu428 and UC230 cells exhibited beat frequencies of 34.1 ± 6.0 and 34.8 ± 5.8 beats/sec respectively. Ciliary beat frequency of 18KO mutants was 32.5 ± 5.4 beats/sec while KO15 mutants exhibited a beat frequency of 27.4 ± 6.2 beat/sec. The ciliary beat frequency of KO18 mutants was approximately 5% less than that of the wild type cells. Since the swimming speed of KO18 mutants was around 15% lower than that of wild type cells, it is unlikely that the reduction of swimming speed exhibited by KO18 mutants 72

83 is primarily due to a change in the waveform of the mutants. Since waveform is not planar in Tetrahymena, we were not able to determine whether waveform was actually altered in KO18 mutants. In contrast, in the case of KO15, the extent of decrease of swimming speed (~25%) correlated with the extent decrease in beat frequency (~20%), suggesting that the alteration observed in swimming speed in KO15 mutants is explained by the decrease in beat frequency. A closer look at the videos of swimming cells captured both at lower and higher magnification indicated a subtle difference in swimming pattern of KO15 mutant and wild type cells. When the paths traversed by the cells were traced at low magnification, KO15 cells displayed a tight spiral motion in contrast to wild type cells swimming under similar conditions. Although quantitatively, the decrease in swimming speed in KO15 can be accounted by corresponding reduction in beat frequency, the swimming pattern suggests the mutation may have an effect on the waveform also. Mutating DYH15 affects feeding behavior; mutating DYH18 does not Tetrahymena feed by the process of phagocytosis using an oral apparatus located in the anterior cell cortex. The oral apparatus contains a specialized group of cilia termed as oral cilia. There is a possibility that oral cilia do not contain a subset of dynein HC present in somatic cilia. If that is the case, mutations in those dynein HC could affect somatic cilia but not oral cilia. Alternatively, if a dynein HCs are exclusively localized to oral cilia, then mutation of that particular HC will not affect the somatic cilia but could alter the activities of oral cilia. To ascertain the effects of the KO15 and 18 mutations on feeding behavior, wild type and mutant cells were incubated in medium containing 2% India Ink for 20 min, fixed and scored, as described in details in the methods section. The percent of cells with ink-stained vacuoles gave a measure of their feeding rate. Graph in Fig 6 gives the percent of cells showing ink-stained vacuoles plotted against the different cell types. We observed that there was about a 10 % decrease of cells displaying ink stained vacuoles in KO15 cultures compared to wild type; the difference was statistically significant. No such decrease was observed for KO18 cells. Thus we concluded that feeding behavior was affected due to mutations in DYH15, but not in DYH18. 73

84 Relative abundance of DHCs in mutants Previous studies with outer arm and two-headed inner arm dyneins have shown that disruption of a HC can lead to loss of other HCs that are complexed with the HC that was disrupted. Hence, the observed phenotype is actually the cumulative effect of losses of multiple proteins and not due to absence of a single HC. It is not yet known how the different one-headed dyneins are associated within the axoneme. When dyneins are extracted with high salt, 1-headed dyneins purify as individual dyneins. However, it is possible that the 1-headed dyneins form complexes in the axoneme and that loss of one DHC could affect formation of the complex. To investigate whether the absence of DYH15 or DYH18 caused loss of other HC, we isolated axonemes from the mutants, ran them on SDS-PAGE, excised the dynein band and performed LC-MS/MS. The total number of peptides generated in the different runs varied greatly, so it was not feasible to compare the absolute number of peptide hits generated for each HC across different runs. To compare relative peptide hits in different runs, we calculated the proportion of peptides with which each HC was identified against the total number of peptide hits generated by all one-headed inner arms detected in that particular run. Results from analyses of KO15 and KO18 were compared to results from analysis of CU428. Figs. 7A and B represent the relative proportion of selected DHCs in KO15 and KO18 axonemes. High and medium abundance DHCs were illustrated in the graphs; the only exception being DYH15 since it is lost in the mutants. Our analyses indicated that there was an increase in the proportion of peptides of Dyh11p, Dyh21p and Dyh25p detected in KO15 mutants, suggesting that the levels of these proteins were higher in KO15 compared to wild type. The increases were not statistically significant, but the trend in each case was consistent across runs. Slight increase was observed in proportion of peptides of Dyh11p and Dyh24p in KO18 mutants. This result prompted us to perform similar analyses with two mutants previously made in our labs, an outer arm mutant (KO3) and two-headed inner arm mutant (KO6) to determine whether they exhibited a similar phenomenon (Fig. 7C). DYH22 seems to be up-regulated slightly in KO3 mutants while DYH21 appears to be upregulated in KO6 mutants. As in the case of KO15, the trend in KO3 and KO6 mutants was consistent but the differences were not statistically significant. 74

85 Since KO15 may have increased the abundances of three different DHCs in the axoneme, we assayed expression levels at the mrna level by performing qrt-pcr studies using total RNA isolated from CU428 and KO15. We focused on DYH11, DYH21 and DYH25, the three HCs that may be present in greater abundance in KO15 mutants. In Fig 8, the relative expression levels of both wild type and KO15 are plotted against the specific HCs. Our mrna studies showed that while expression level of DYH11 was higher in KO15 compared to wild type, for DYH21 and DYH25, there was decrease in expression levels in KO15. Discussion In this paper we describe mutation of two different dynein HC in Tetrahymena thermophila. DYH15 encodes a one-headed inner arm DHC present in the axoneme in very high abundance, and DYH18 encodes a one-headed inner arm DHC present in the axonemes in very low abundance; both are members of one-headed inner arm subgroup IAD5. Mutating either gene affects ciliary motility but in different ways. Swimming speed is decreased in both the mutants, albeit somewhat more so in the case of KO15 than KO18. Beat frequency and feeding behavior are disrupted in KO15, however, no statistically significant changes in either beat frequency or feeding rate were observed in KO18. Interestingly, LC-MS/MS analysis of KO15 axonemes suggested that Tetrahymena KO15 mutants may up regulate some one-headed DHCs to compensate for the loss of DYH15. DHCs are not redundant Since both mutations affect swimming speed, both the DHCs appear to be required for full motility, and neither is completely redundant. These data are consistent with previous findings reported in different model systems. Three of the one-headed inner arm DHCs- DYH8, DYH9, DYH12, have been previously knocked out in Tetrahymena, and all three mutants displayed reduced swimming speed (Liu et al., 2004). Mutants missing a single inner arm [DHC9] (Yagi et al., 2005) or multiple inner arms [ida5 missing four inner arms] (Kato-Minoura et al., 1993) have been made in Chlamydomonas, and analyses suggested each of them may be playing some role in flagellar motility. Impaired ciliary 75

86 beating was also observed in mice lacking a single inner arm dynein, DHC7 (Neesen et al., 2001). Hence, studies on one-headed inner arms in multiple species suggest that axonemal dyneins are not completely redundant. DHCs play various roles in ciliary motility The multi dynein hypothesis (Asai and Wilkes, 1994) suggested that each dynein plays a unique role in ciliary motility. Our data support that hypothesis. Mutations in DYH15 and DYH18 affected ciliary motility in different ways. KO15 mutants swam slower than KO18 mutants. Beat frequency and feeding behavior were altered in KO15, but not in KO18. Mutations affecting other one-headed DHC in Tetrahymena are consistent with the data we report in this paper. Work carried out in other organisms such as Chlamydomonas also supports this hypothesis. While outer arms and inner arms appear to be the major players in controlling beat frequency and waveform respectively (Kamiya and Okamoto, 1985; Mitchell and Rosenbaum, 1985; Brokaw and Kamiya, 1987; Sakakibara et al., 1991; Porter et al., 1996; Kato-Minoura et al., 1993; Myster et al., 1999; Perrone et al., 2000), multiple studies in different organisms suggest some inner arms dyneins may help determine beat frequency. Beat frequency was reduced in KO15 mutants sufficiently to account for the decrease in swimming speed, but beat frequency was not affected in KO18 mutants. Since a decrease in beat frequency cannot fully account for the reduction in swimming speed in KO18, a change in waveform appears to be the main cause for the decrease in swimming speed exhibited by KO18 mutants. Beat frequency was decreased in KO8 but not in KO9 and KO12 mutants. Studies carried out in Chlamydomonas also showed that some inner arm dyneins played important role in determining beat frequencies when outer arms were absent (Yamamoto et al., 2006). Interestingly, some one-headed inner arms may affect both beat frequency and waveform. Although the decrease in beat frequency in KO15 mutants could entirely explain the quantitative decrease in swimming speed, the mutation may also have affected waveform because KO15 cells exhibit a slightly different kind of spiral motion compared to the wild type cells. Thus, some DHCs affect beat frequency, while others affect waveform and even others appear to affect both the swimming parameters. It 76

87 remains to be sorted out, however, whether the swimming behavior displayed by KO15 is entirely due to loss of DYH15p, or whether there is some effect of simultaneous increase in abundance levels of other one-headed DHCs. In higher organisms, cilia/ flagella are present in different regions of the body where they are involved in various functions e.g. movement of ependymal fluid in the brain, mucous clearance in airway epithelia and movement of sperm towards the egg. Studies in mice indicate that mutations in MDHC7, an inner arm HC, caused a defect in waveform in spermatozoa, while an altered beat frequency in tracheal cells (Neesen et al., 2001). This suggests that in higher organisms a single dynein may be performing different role(s) depending on its localization in a tissue/ organ.. Taken together, all these data strongly support the idea that different inner arm dyneins have different functions. Members within a subgroup perform diverse functions The recent discovery of additional one-headed DHCs and their subdivision into three groups, IAD 3, IAD 4 and IAD 5 based on sequence similarity prompts the question whether members within a group perform similar functions. Our studies showed that DYH15 and DYH18, both members of same subgroup IAD5, affect swimming behavior in different ways. Mutating DYH15 affected beat frequency, possibly waveform, and the feeding rate; mutating DYH18, on the other hand, affected waveform in somatic cilia but did not appear to have a significant effect on the feeding rate of cells. These results suggest that DHCs in the same one-headed inner arm subgroup may play different roles. Previous results with mutations affecting DYH8 and DYH12, both members of IAD3, also support that idea as beat frequency is affected in KO8 mutants, while waveform appears to be affected in KO12 mutants (Liu et al., 2004). In Chlamydomonas, it has been shown that members of IAD 4 and IAD 5 are only involved in producing torque to cause microtubule bending (Kikushima and Kamiya, 2008). IAD 3 members are not involved in this process. It should be noted here, that in Chlamydomonas, IAD4 and IAD5 comprise of a single member each. Hence detailed analyses of the six different members of IAD 3 in Chlamydomonas may be more 77

88 pertinent in answering the specific question of whether DHCs in the same group play similar roles. Taken together, these studies suggest that evolutionary relationships may not completely predict function of dyneins in the axoneme. However, one must interpret these data with caution as our data raise the possibility that Tetrahymena may increase expression of other DHCs to compensate when one DHC is mutated. Proteomic studies indicate that in KO15, there appears to be an increase in abundance levels of three other one-headed inner arm dyneins. qrt-pcr studies did not completely support the idea of upregulation; however, we can not fully explain this anomalous situation. Nevertheless, the important point here is that if cells do compensate for the loss of one DHC by increasing the abundance of others, some of the phenotypes observed with individual one-headed inner arm mutants could be due to altering the relative abundances of dyneins present rather than from the absence of one specific dynein. Partial redundancy may explain the subtle phenotype of KO15 Since DYH15 is the highest abundance one-headed HC, we expected the knockout to exhibit a severe phenotype. However, contrary to our expectations, KO15 did not exhibit a dramatic phenotype. There are several possible explanations. First, DYH15 may not be as abundant as we think. However, we think this is unlikely. DHY15 generated more peptide hits in LC-MS/MS than any other one-headed inner arm dynein, and the number of peptide hits is a good predictor of DHC abundance in Tetrahymena (Kabi et al., unpublished observations). In addition, q-rtpcr analyses also suggest that DYH15 is expressed at higher levels than genes encoding other one-headed inner arm dynein HC (Kabi et al., unpublished data). A second possibility is that a high abundance dynein may not necessarily play a more substantial role than a low abundance dynein. While we cannot rule out this possibility, it also does not seem likely. Mutating other high abundant dynein HCs such as outer arm mutant (KO3) or two-headed inner arm mutant (KO6) have resulted in severe motility defects in Tetrahymena. DYH15 appears to be the most abundant oneheaded inner arm dynein HC (as abundant as DYH6 and DYH7, two-headed inner arm dynein HCs). It has to be kept in mind, however, that mutating outer/ two-headed inner 78

89 arm DHCs resulted in loss of more than one heavy chain while levels of a single DHC was increased in each case. No other DHCs were lost when DYH15 was mutated. Lastly, DYH15 mutants may have compensated for the absence of DYH15 by increasing the abundance of other inner arm dyneins. This idea is supported by LC- MS/MS data which indicates that the amounts of DYH11, a medium abundance dynein, and DYH21 and DYH25, two high abundance dyneins are increased in KO15 axonemes. These data suggest that dyneins are at least partially redundant. All studies conducted previously have shown that none of the dyneins are completely redundant. Genomic and proteomic analyses combined together show that the dynein superfamily is much bigger than what was previously thought. Considering the fact that so many inner arm dyneins are involved in bringing about ciliary motility within an organism, the theory of partial redundancy seems a reasonable explanation. Some increase in relative abundance of oneheaded inner arm dyneins in the axoneme has also been observed when outer arm [KO3] or two-headed inner arm [KO6] DHCs was knocked out. In KO3 and KO6, relative abundance levels of DYH22 and DYH21 seemed to be higher compared to wild type respectively. However, since upregulation is observed in only a single inner arm DHC in both KO3 and KO6, we cannot rule out the possibility that it is an artifact. The fact that losses of outer arm/ two-headed inner arm dyneins are not compensated much by oneheaded dyneins strengthens the notion that each of these three different classes of DHCsouter arm, two-headed inner arm and one-headed inner arm perform specialized functions. Conclusions Our current findings help us put together a few important conclusions regarding the DHC superfamily. It shows that subgrouping of one-headed inner arm DHCs does not reflect functional similarity. It reiterates the idea that different dyneins play diverse roles and some dyneins may be playing more critical role(s) in ciliary motility compared to others. This work provides the first evidence that there may be partial redundancy among the one-headed dyneins. Also, the fact that loss of one high abundant dynein HC is compensated by an increase in levels of other high abundance dyneins, may suggest that dyneins of similar abundance perform similar role(s). The most recent structural view of 79

90 the arrangement of dyneins predicts that, within each 96nm repeat different combinations of one-headed dyneins may be present as functional dimers (Huy Bui et al., 2008). In future, different combinations of double/ triple knockouts of dyneins can be generated based on genomic and proteomic findings to better comprehend the functional interaction within the axoneme. Our work brings into attention a very significant point that careful judgement has to be taken when interpreting phenotype of different DHC mutants. Since the number of dyneins varies greatly across different organisms, detailed study in diverse organisms will be essential to get a clear picture of how the one-headed dyneins function in a complex and coordinate with other axonemal proteins to bring about various forms of ciliary beating. 80

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98 Neuwald, A.F., Aravind, L, Spouge, J.L. and Koonin, E.V AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9(1), Pan, J. Wang, Q. and Snell, W.J Cilium generated signaling and cilia related disorders. Lab Investigations. 85, Pazour, G.J., B.L. Dickert, Y. Vucica, E.S. Seeley, J.L. Rosenbaum, G.B. Witman, and D.G. Cole Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J. Cell Biol. 151, Pazour, G.J., Agrin N., Leszyk J, and Witman, G.B Proteomic analysis of a eukaryotic cilium. J Cell Biol. 170(1), Pfister, K.K.and Witman, G.B Subfractionation of Chlamydomonas 18 S dynein into two unique subunits containing ATPase activity. J Biol Chem. 259(19), Pfister, K.K., Fay, R.B. and Witman, G.B Purification and polypeptide composition of dynein ATPases from Chlamydomonas flagella. Cell Motil. Cytoskelet. 2(6), Piperno, G Regulation of dynein activity within Chlamydomonas flagella. Cell Motil.Cytoskelet. 32, Piperno, G., Ramanis, Z., Smith, E.F. and Sale, W.S Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and localization in the axoneme. J. Cell. Biol. 110 (2), Piperno, G. and Ramanis, Z The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms. J Cell Biol. 112,

99 Porter, M.E., Knott, J.A., Myster, S.H., and Farlow, S.J The dynein gene family in Chlamydomonas reinhardtii. Genetics 144, Pazour, G.J., Agrin N., Leszyk J, and Witman, G.B Proteomic analysis of a eukaryotic cilium. J Cell Biol. 170(1), Sakakibara, H., Mitchell, D.R., and Kamiya, R.(1991). A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain. J. Cell. Biol. 113(3): Sakato, M., and King, SM Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein. J Biol Chem. 278(44): Sale, W.S The axonemal axis and Ca2+ induced asymmetry of active microtubular sliding in sea urchin sperm tails. J Cell Biol. 98, Satir, P., Barkalow, K. and Hamasaki, T The control of ciliary beat frequency. Trends Cell Biol. 3(11), Satir, P Mechanisms of Ciliary motility: an update. Europ. J. Protistol. 34, Satir, P.; Barkalow, K. and Hamasaki, T The control of ciliary beat frequency. Trends in Cell Biology 3, Smith, E.F. and Sale, W.S Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes. J Cell Biol. 117, Smith, J. C., Northey, J. G., Garg, J., Pearlman, R. E., and Siu, K. W Robust method for proteome analysis by MS/MS using an entire translated genome: demonstration on the ciliome of Tetrahymena thermophila. J. Proteome Res. 4,

100 Smith, E.F. and Sale, W.S Regulation of Dynein-Driven Microtubule Sliding by the Radial Spokes in Flagella. Science 257, Stephens, R.E and Prior, G Dyneins from serotonin activated cilia and flagella: extraction characteristics and distinct sites for camp dependent protein phosphorylation. J Cell Sci. 103, Vallee, R.B. and Sheetz, M.P Targeting of motor proteins. Science. 271(5255): Warner, F.D., Perreault, J.G. and McIlvain, J.H Rebinding of Tetrahymena 13S and 21S dynein ATPases to extracted doublet microtubules. J Cell Sci. 107, Wickstead, B. and Gull, K Dyneins across Eukaryotes: A Comparative genomic analysis. Traffic. 8, Wilkes, D.E., Watson, H.E., Mitchell, D.R. and Asai, D.J Twenty-five dyneins in Tetrahymena: a re-examination of the multi-dynein hypothesis. Cell Motil. Cytoskelet. 65(4), Witman, G.B., Johnson, K.A., Pfister, K.K. and Wall, J.S Fine structure and molecular weight of the outer arm dyneins of Chlamydomonas. J. Submicrosc. Cytol. 15, Witman, G.B., Wilkerson, C.G. and King, S.M The biochemistry, genetics and molecular biology of flagellar dynein. In: Hyams, J.S. & Llyod, C.W. (ed.) Microtubules. Wiley Liss, New York, p Xu, W., Royalty, M.P., Zimmerman, J.R., Angus, S.P. and Pennock, D.G The dynein heavy chain gene family in Tetrahymena thermophila. J. Eukaryot. Microbiol. 46,

101 Yagi T, Minoura I, Fujiwara A, Saito R, Yasunaga T, Hirono M, Kamiya R. 2005An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9. J Biol Chem. 280(50): Yagi, T., Uematsu, K., Liu, Z. and Kamiya, R Identification of dyneins that localize exclusively to the proximal portion of Chlamydomonas flagella. J Cell Sci Yang, P., Diener, D.R.,Yang, C, Kohno, T.,Pazour, G.Z., Dienes, G.M, Agrin, N, King, S.M, Sale, W.M., Kamiya, R., Rosenbaum, J.L. and Witman, G.B Radial spoke proteins of Chlamydomonas flagella. J Cell Sci. 119(6), Yamamoto R, Yanagisawa HA, Yagi T, Kamiya R A novel subunit of axonemal dynein conserved among lower and higher eukaryotes. FEBS Lett. 27;580(27): Zhang W, Culley, D.E., Gritsenko, M.A, Moore, R.J., Nie, L., Scholten, J.C., Petritis, K., Strittmatter, E., Camp, D.G. 2 nd, Smith, R.D and Brockjaw, F. J LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris. Biochem Biophys Res Commun. 349(4),

102 Table 1. LC-MS/MS analyses of dynein heavy chains present in KO15. Outer Arm/Inner Arm DHCs Run 1 Run 2 Run 3 Outer Arm Dyh3p Dyh3p Dyh3p headed Inner Arm Dyh3p Dyh3p headed Inner Arm IAD 3 Dyh25p Dyh11p Dyh10p Dyh8p Dyh14p Dyh12p Dyh13p Dyh17p IAD 4 Dyh21p Dyh16p Dyh19p Dyh9p 2-1 Dyh120p IAD 5 Dyh15p* Dyh22p Dyh24p Dyh23p Dyh18p * No peptide hits for DYH15 found 92

103 Fig 1. Strategy designed to create knockout of DYH15 (A) and DYH18 (B) gene. The cloned regions for both the genes are marked by the solid lines. Arrowhead marks the site of insertion of neo- cassette. Arrow indicates the position of AAA1 site in both the genes. Numbers in the diagram indicate the position in bp. 93

104 94

105 Fig 2. Southern blot analyses reveal the knockout is complete in the mutants. Whole genomic DNA was isolated from both wild type (CU428) and mutant cells and digested with NdeI (in case of DYH15) and Hinc II ( in case of DYH18). With appropriate probe, bands of predicted size were obtained. Sizes of the bands (in bp) are indicated on the side of the blot. 95

106 96

107 Fig 3. SDS-PAGE analysis shows that dynein heavy chain band in KO15 axonemes. Arrowhead indicates the band which was sent for LC-MS/MS analysis. 97

108 98

109 Fig 4. Mutants show altered swimming behavior. Swimming speed in mm/sec was plotted against cell type. Swimming speed of both the mutants (KO15 and KO18) was significantly lower compared to that of the two wild type strains (CU428 and UC230). Error bars indicate S.D. *** indicates p<0.001 (ANOVA). 99

110 100

111 Fig 5. KO15 mutants exhibit decreased beat frequency while KO18 mutants do not. Beat frequency (beats/sec) plotted against the different cell types. Significant reduction in beat frequency is observed for KO15 mutants but the decrease is not significant for KO18 mutants. when compared to the wild type cells (CU428 and UC230). Error bars indicate the S.D. ** indicates p<0.01 (ANOVA). 101

112 102

113 Fig 6. KO15mutants display reduced feeding rates, while KO18 mutants do not. Feeding assays performed on wild type and mutant cells to assess the effects on oral cilia. Graph with percentage of cells containing ink stained vacuoles plotted against cell type. KO15 mutants show significant decrease in number of cells containing ink stained vacuoles. Error bars indicate S.D. * indicates p<0.05 (ANOVA) 103

114 104

115 Fig 7. Loss of an inner arm may be compensated by increase in abundance levels of other one-headed inner arm dynein HCs. A. Peptide proportion analyses reflects increase in proportion of peptide hits against three one-headed dynein HCs (DYH11p, DYH21p and DYH25p) in KO15 mutants. Three independent analyses were performed for both the cell types. B. Peptide proportion analyses reflect no increase in proportion of peptide hits against any one-headed dynein HCs in KO18 mutants. Two independent analyses were performed for both the cell types. C. Peptide proportion analyses reflect increase in proportion of peptide hits against one one-headed dynein HCs in each case of KO3 and KO6 mutants. DYH22p and DYH21p were increased relative to wild type levels in KO3 and KO6 respectively. Two independent analyses were performed for all the three cell types. 105

116 106

117 Fig 8. Expression levels of inner arm heavy chains that showed an increase in abundance in KO15 mutants compared to wild type. DYH 11, DYH 21 and DYH 25 showed an increase in the peptide proportion in KO15. qrt-pcr analysis revealed that only DYH11 showed an increase in the transcript level. Expression levels were normalized to tubulin for both the cell types. 107

118 108

119 Disruption of hydin results in impaired ciliary motility in Tetrahymena thermophila 109

120 Abstract Congenital hydrocephalus is a common birth defect with an estimated occurrence of 1 in 1000 live births in the United States. Studies so far have implicated several genes in this disorder; however the molecular mechanisms leading to hydrocephalus has not yet been fully understood. One such gene is hydin (Hydrocephalus-inducing) which when mutated causes hydrocephalus in mice. Hydin transcripts localize to tissues with 9+2 cilia in the mouse. Hydin is a component of the Chlamydomonas proteome and RNAi directed against hydin interferes with motility in trypanosomes. These results suggest the hypothesis that mutations in hydin cause hydrocephalus by interfering with ciliary movement, but they do not rule out the possibility that hydin also plays a role in signal transduction in the cilium. To determine the role hydin plays in cilia, we introduced a mutation into the Tetrahymena hydin gene by targeted gene knockout strategy. Disruption of hydin in Tetrahymena has a dramatic effect on motility. However, response to external stimuli is not affected in the mutants. Electron microscopic studies depict an ultra structural defect in the central pair of axonemes. Our data suggest that hydin is required for normal ciliary motility but is not involved in signaling functions. Further, hydin plays a role in maintaining the structural integrity of the central pair and dispruption in ciliary motility could be due to the central pair defect. The findings strongly project disruption of ciliary motility as the primary, if not only, cause of hydrocephalus when hydin gene is mutated.. Key words: hydin, axoneme, motility 110

121 Introduction Congenital hydrocephalus is a condition that affects approximately 1 in every 1000 live births, making it a very common birth defect (Clewell, 1988; Schurr and Polkey, 1993). Hydrocephalus is characterized by the over accumulation of cerebrospinal fluid (CSF) within the ventricular system of the brain. The increased intracranial pressure due to the elevated levels of CSF causes convulsions, mental disorders and progressive enlargement of the head when it occurs before the skull is completely ossified. The condition may be lethal if left untreated. The most common procedure of treating this disorder is to insert a shunt to drain off the excess fluid; however, surgical revisions can be necessary and infection can occur. Apart from the physical costs associated with hydrocephalus, there are tremendous financial costs involved in the treatment of the condition (Patwardhan and Nanda, 2005). Due to the frequency of hydrocephalus and the associated costs incurred in its treatment, it is vital to understand the root causes of this disorder. However, the molecular mechanisms leading to hydrocephalus are not yet completely understood. Hydrocephalus can be broadly divided into two types- obstructive and communicative. Obstructive hydrocephalus arises from blockage in the path of flow of CSF. Possible causes of the obstruction include scarring of a tissue or presence of a tumor. In the communicative form of hydrocephalus, there is an increase in volume of CSF, either due to an elevated production of the fluid or reduced reabsorption of CSF by the arachnoid villi of the brain. Various genetic and epigenetic factors such as injury can give rise to hydrocephalus. A significant portion of human congenital hydrocephalus is genetic in origin and various genes have been implicated in this disorder. Multiple cases of suspected autosomal recessive hydrocephalus have been reported in humans (Game et al., 1989; Moog et al., 1998; Brady et al., 1999; Lapunzina et al., 2002); however, in most cases the chromosomal location has not been mapped (Robinson et al., 2003). To understand the molecular mechanisms leading to hydrocephalus, over the years, several different mouse models have been developed, some having targeted mutations (Moyer et al., 1994; Galbreath et al., 1995, Sapiro et al., 2002; Chen et al., 1998) while others resulting from spontaneous mutations (Ibanez-Tallon et al., 2002, Gruneberg, 1943, Hollander, 1976; Bronson and Lane, 1990). 111

122 One such mouse model where lethal communicating hydrocephalus has been observed is with early onset is the hy3 (hydrocephalus 3) mouse line (Gruneberg, 1943). Mutations in the hy3 homozygous mice appear normal externally at birth, develop progressive hydrocephalus by 3-5 days of age, and die within six weeks of age (Sapiro et al., 2006; Davy and Robinson, 2003). In recent years, another mouse line, OVE459, carrying a transgene-induced insertional mutation resulting in hydrocephalus, was characterized which was found to be a new allele of hy3 (Robinson et al., 2002). Direct cdna selection, combined with other genomic techniques, mapped the mutation to a large, novel gene, hydin (hydrocephalus inducing) (Davy and Robinson, 2003). The corresponding chromosomal location in humans has also been linked to this disorder (Callen et al., 1990). Several lines of evidence indicate that hydin is a ciliary protein. In situ hybridization studies showed hydin is expressed in the ciliated ependymal cells lining ventricles of the brain, bronchi and oviducts, as well as in developing spermatocytes in the testis (Davy and Robinson, 2003). Comparative genomic studies have identified homologs of the hydin gene in small organisms possessing motile cilia and flagella (Pazour et al., 2005; Broadhead et al., 2006), and hydin is absent in organisms and cells lacking motile cilia/ flagella. Pazour et al. (2005) performed proteomic analyses of Chlamydomonas flagella where hydin was found to be tightly associated with the axonemal fraction of the organelle. Furthermore, hydin transcripts were induced in Chlamydomonas by deflagellation suggesting its specific role in ciliary/ flagellar function (Lechtreck et al., 2007). Defects in hydin could potentially cause hydrocephalus in two ways. If hydin plays a role in ciliary motility, mutations in hydin could impair ciliary motility and disrupt CSF fluid flow, leading to aqueduct stenosis and an increase in intracranial pressure and ventricular expansion (Banizs et al., 2005). Alternatively, if hydin is involved in intracellular signaling, defects in hydin could affect one or more signaling pathways, leading to overproduction / reduced reabsorption of CSF. The ciliary membrane is the site of various receptors, ion channels and signaling molecules (Flieguff et al., 2007). Information received from the surroundings by the sensors on the ciliary membrane is converted into signaling cascades within the ciliary compartment and finally 112

123 transduced to the cell body. Hence signaling defects could lead to altered ion channel functions, thereby causing an imbalance in CSF homeostasis. Several mouse models of congenital hydrocephalus have been identified which have mutations in genes such as Spag6 (Sapiro et al., 2006), polaris (Taulman et al., 2001), and Mdnah5 (Ibanez-Tallon et al., 2002) that are involved in ciliary development or function. While impaired ciliary motility has been implicated as a possible cause of this disease in some of these models such as mutations in Spag6 (Sapiro et al., 2006), Mdnah5 (Ibanez-Tallon et al., 2004), overproduction of CSF due to signaling defects has been observed in other cases such as due to mutations in polaris (Taulman et al., 2001). More recent studies using Chlamydomonas (Lechtreck et al., 2008), Trypanosome (Dawe et al., 2007) and mice (Lechtreck et al., 2008) localized hydin to the central pair of the 9+2 axonemal backbone in cilia. Knockdown of hydin by RNAi in Trypanosomes and Chlamydomonas resulted in a severe motility phenotype (Dawe et al., 2007; Lechtreck et al., 2007). A motility defect was also manifested by ependymal and airway epithelial cilia of hydin mutant mice (Lechtreck et al., 2008). While motile cilia have been primarily associated with fluid movement, their possible role as mechanosensors remains largely unexplored. Shah et al., 2009 provided the first evidence of airway epithelia being involved in some signaling pathway. This raises the possibility that motile cilia localized in other tissues such as in ependymal layers of the brain may also be involved in signaling and that some ciliary proteins may simultaneously be involved in motility and signaling. Results from Chlamydomonas, Trypanosomes and mice suggest that hydin is essential for ciliary motility. However, it is not known yet whether hydin is involved in signaling behavior. While the mouse is an excellent model system to study human genetic disorders, to clearly understand the effects of mutation of a gene on regulation of ciliary motility, small unicellular ciliated/ flagellated organisms are of great importance. This is because the effects of mutation of a gene can be observed in these organisms without secondary, pleitotrophic effects that could hinder interpretation of phenotypes. In this paper, we describe experiments to examine the role of hydin in ciliary functions by knocking out the hydin gene in Tetrahymena thermophila. The targeted gene knockout strategy employed resulted in loss of hydin protein in the mutants. Behavioral analyses showed 113

124 that while hydin played a crucial role in ciliary motility, it may not be involved in signaling activities. Electron microscopic studies revealed that hydin also plays an important role in maintaining the structural integrity of the central pair complex in the axoneme. Methods Strains used in the experiment The hydin gene mutation was targeted in wild type strain CU428, kindly provided by Dr Peter Burns from Cornell University. All cells were grown in Neff s medium (0.25% proteose peptone, 0.25% yeast extract, 0.55% dextrose, 33 μm FeCl 3 ) at 28 0 C either under stationary conditions or at shaking 125rpm depending on the requirements of the particular experiment. UC230 (kindly provided by Dr Aaron Turkewitz, University of Chicago), expressing the neo2 gene, was used as the neomycin expressing control in the behavioral experiments apart from CU428 which was the wild type control. The HYDKO mutant and UC230 cells were maintained at a concentration of 300 /ml paramomycin. Before use, they were grown around a week in medium without paromomycin to ensure that there was no effect of the drug in the behavioral responses. Construction of KO Constructs The genomic sequence of the Tetrahymena hydin gene was obtained from ciliate.org. An approximately 3Kb region was PCR amplified using appropriate primer pairs. The PCR product was checked on a 1% agarose gel and ligated into the pcr2.1 vector (lnvitrogen, Carlsbad, CA). Transformation was carried out using INVaF E.coli by the method as described in the manual. The colonies were screened for the correct clone. To obtain larger amounts of the DNA for insertion of neo-cassette, the particular clone was grown in larger volume; DNA was extracted from it using Promega Wizard Midiplus Kit. Appropriate restriction enzyme was used so that it resulted in a blunt cut at a single location around the mid region of the cloned fragment. The blunt ends were phosphatased, and the neomycinresistant cassette (neo2 gene) was ligated at that position. The neo2 cassette contains the Tetrahymena histone H4-1 gene promoter on the 5 end and contains the Tetrahymena beta-tubulin-2 (BTU2) gene poly A signal on the 3 end (Gaertig et al., 1994). 114

125 Transformation and screening of colonies was carried out and a single colony containing the cloned region with the insert was selected to be used for transformation of Tetrahymena. Transformation of Tetrahymena with KO Construct In order to transform Tetrahymena with the hydin KO construct, the KO construct was removed from the vector by restriction digestion. The digested DNA was ethanol precipitated and resuspended to a concentration of 1µg/µl. Transformation was targeted to the macronucleus of Tetrahymena by biolistic means using the BioRad PDS-1000/He particle delivery system (Bruns and Cassidy-Hanley, 1997). Briefly, 1µg DNA was used in each transformation. CU428 cells were starved overnight in 10mM Tris (ph 7.4), at a concentration of cells/ml. Cells were pelleted and washed in 10mM HEPES (ph 7.5), and resuspended in10mm HEPES (ph 7.5) to a final concentration of cells/ml. 1ml cells were spread on a sterile, pre-wetted filter paper and bombarded at 900 psi using. The carriers were 0.6μM gold particles coated with linearized plasmid. After bombardment, the filter paper containing cells was carefully transferred into 50 ml Neff s medium in a 500 ml flask. The flask was incubated at room temperature for 2-6 hours following which paromomycin was added to the medium at a final concentration of 80 μg/ ml. The cells were incubated at 28 0 C without shaking. Selection for Complete Replacement Tetrahymena has two nuclei-a germinal micronucleus and a vegetative macronucleus. The micronucleus is diploid with five pairs of chromosomes and is transcriptionally inert. The macronucleus is transcriptionally active and contains copies of each gene. Following transformation, the macronucleus contains a few disrupted gene and many copies of the wild type version of the gene. Knockout is complete when all the copies of the wild type gene in the macronucleus get replaced by the mutant version. To achieve that, transformed cells were grown in medium containing the drug paromomycin and the concentration of paromomycin in the medium was increased gradually during each transfer. Genomic DNA was isolated from the cultures at regular intervals and PCR performed to check for the extent of complete replacement. When the concentration of paromomycin in media was 115

126 70,000 μg/ ml, PCR indicated that replacement was complete. Following this, cells were grown for a few weeks in medium without drug. If a few copies of wild type gene were left in the macronucleus, then we would have observed reversion within this time frame. However, our PCR results did not show any signs of reversion. Thus, we concluded that the knockout was complete. In the last step of this selection process, single cell clones were then isolated by hanging drop method (Pennock, 2000) and slow swimming cells were isolated. Multiple rounds of single cells were done, clones were grown at a larger scale, tested by PCR (data not shown) and one clone was selected for all future analyses. Southern Blot Total genomic DNA was isolated from wild type and mutant cells as described in Gaertig and Gorovsky (1992). 10 µg of DNA was digested with EcoRV and separated on a 1% agarose gel. DNA from the gel was transferred overnight on MagnaProbe nylon membrane (Osmonics) using 10X SSC Buffer (3M NaCl, 0.3M, Na Citrate, ph-7.0). Next day, the membrane was vacuum dried at 80 0 C for around two hours. Preparation of probe- A fragment of around bp was PCR amplified using appropriate primer pairs to make the probe. The PCR product was cleaned up using the Promega PCR Clean up System Labeling of probe- The probe was labeled with radioactive P (alpha PdCTP) using the Promega Labeling Kit following the protocol given by the manufacturer. Before use, it was purified using the ProbeQuant G-50 Micro Columns and the specific activity (cpm/ml) measured in the scintillation counter. Hybridization- The hybridization was carried out using the Express Hyb Solution (ClonTech). Briefly, the membrane (pre-wet with dd water) was set up for prehybridization in the Express Hyb solution for around 30 min at 60 0 C. This was followed by hybridizing the membrane with the labeled probe, added at a concentration of 2X 10 6 cpm/ml at 60 0 C for an hour. To remove background, the membrane was washed with Wash Solution I (2X SSC, 0.05% SDS) at room temperature and Wash solution II (0.1X SSC, 0.1% SDS) at 55 0 C for around 40 min each. Finally it was set for 116

127 exposure overnight in the cassette containing phosphor-imager screen. The bands were visualized using the Storm Phosphorimaging System (Molecular Dynamics). Axoneme Isolation Whole axonemes were isolated from wild type and mutant cells as described by Johnson, (1986); Mobberley et al., (1999). Briefly, 2 l of cells were pelleted for each cell type by centrifugation at 900g for 3min. The supernatant was removed by aspiration to around 5 ml of medium and the cells were gently resuspended. Dibucaine was added to a concentration of 3mM to release cilia from cells. Cells were swirled gently for 1 min and 60 ml cold Neff s (medium) was immediately added. Centrifugation was carried out at 900g for 10min at 4 0 C, to pellet cell bodies following which the supernatant was transferred to fresh tubes. 25 µl of 0.1 M PMSF (phenylmethylsulphonyl fluoride), a serine protease inhibitor, was added and a second round of centrifugation was carried out as described above to pellet any reaming cell bodies. Cilia were then pelleted by centrifugation at 11200g in a Sorvall SS34 rotor for 10 min at 4 0 C. The pellet was resuspended in 1ml 1X Satir Buffer (SB) (30mM HEPES, ph 7.6; 5 mm MgSO4; 0.5 mm EDTA; 20mM KCl). 5 µl leupeptin (5µg/ml), another protease inhibitor, was then added. Cilia were layered onto 20 ml of 1X SB containing 1M Sucrose and 2mM EGTA and centrifuged at 11200g in a Sorvall SS34 rotor for 20 min at 4 0 C. Pelleted cilia were resuspended in 1ml 1X SB and 5 µl leupeptin, and 25 µl 10% NP-40 was added to remove the ciliary membranes. After incubation in NP-40 for 30 minutes, axonemes were pelleted at 11200g in a Sorvall SS34 rotor for 10 min at 4 0 C and resuspended in 1ml of 1X SB. This process was repeated twice and finally the axonemes were resuspended in 1ml of 1X SB. Protein concentration was determined using Bradford Reagent (BioRad). Samples were boiled in SDS sample buffer ( M Tris, 10% SDS, 25% βme, 33% Glycerol) for 5-10 minutes and then loaded in a gel or stored at C. SDS-PAGE and LC-MS/MS- 200 µg of total axonemal proteins were electrophoresed through 4-16% polyacrylamide gels for ~22 hr at constant voltage of 50V. Gels were stained with Coomassie Blue R. for 3-4 hrs. Gels were destained in Destain1 (50 % Acetic Acid, 10 % Methanol) for 3-4 days and then stored in Destain 2 (5% Acetic Acid, 117

128 7% Methanol) at 4 0 C. For LC-MS/MS analysis the band was excised from the SDS- PAGE gel and sent to the University of Cincinnati Mass Spectrometric Facility (Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati). Swimming Speed Control cells (CU428 and neo-expressing control UC230) and the mutants were grown overnight to a concentration of 2-4 X 10 5 cell/ml. Before the start of analyses, 1 ml of cells was fixed in formaldehyde and the concentration of the cells was determined using a hemacytometer. For each cell type, 1X 10 6 cells were used for the assays. Cells were briefly centrifuged at 1000g, resuspended in 5 ml of Neff s medium, transferred to 50 ml conical flasks and incubated for 10 min. 90 µl Neff s was put on a depression slide and 0.5 µl of cells were pipetted into it. Freely swimming cells were video recorded at 30 frames/second for 5 sec using the DAGE-MTI CCD100 video camera. The videos were analyzed using Image J software. Briefly, thirty frames of recording were superimposed which presented the path length traveled by a particular cell in one second. Only the paths of those cells that stayed in the same plane were measured. To get the actual path length traversed by each cell in one second, the cells lengths of five random cells were measured and the average cell length was subtracted from each measured path length. Three trials were conducted for each cell type. Finally, a Kruskal-Wallis test was performed to determine if there were any statistically significant differences among the swimming speed of the three cell types. Feeding Assay Feeding assay was performed as described by Angus et al., In brief, cells were grown to early log phase (~ 2 X 10 5 cells/ml). To 9 ml of cells, 1 ml of pre-warmed Neff s containing 2% India ink was added. Cells were incubated at 28 0 C in a gyrotory 125rpm. 1ml of culture was collected 20 min after India ink was added and fixed in formaldehyde. One hundred randomly chosen cells were scored under a bright field microscope at a magnification of 200X. Cells were scored for the presence/absence of vacuoles. Three independent trials were conducted. A Kruskal-Wallis test was performed to determine whether any differences among samples are significant. 118

129 Beat Frequency All three strains of cells were grown to early log phase (~ ) cells/ml in a shaking incubator, at 28C. A coverslip was supported by double stick tape on a plain slide to make a slide chamber. 60 ul cells were pipetted into the slide chamber. Swimming cells were observed using a100x oil immersion lens and cells were videorecorded at high speed (500 frames/sec) by a Photronics 1280 PCI FastCam on a Nikon Eclipse E600 microscope. The experiment was conducted at room temperature. Cells whose waveform wass not impaired by the slide or coverslip were chosen for analysis. Individual cilium or group of cilia was analyzed and the number of frames to complete one full beat cycle will be recorded. Knowing the number of frames/beat cycle and the number of frames/second, the beat frequency calculated and reported in beats/sec. A Kruskal-Wallis test was performed to determine whether the differences among samples was statistically significant Hyperpolarizing stimulus 1X 10 6 cells of each cell type was used for the assays. Appropriate volume of cells were briefly centrifuged at 1000g at room temperature and resuspended in 5 ml of test solution (10mM Tris-Cl, 0.5 mm MOPS, 50 mm CaCl 2, 8mM K +, ph 7.4) with 8mM K + Cells were incubated at room temperature for 10 minutes. 0.5μl cells were then added to 90μl test solution with 0mM K + and the cells will be immediately videorecorded for 5 sec at 30 frames/sec using the DAGE-MTI CCD100 video camera. To ensure that pipetting of cells did not have an effect on swimming speed, 0.5μl cells were added to 90μl test solution with 8mM K + and videorecorded for 5 sec. Approximately cells were measured in each trial. Three independent trials were performed, both for controls and the hyperpolarizing stimulus. Swimming speed of each cell type, under control and hyperpolarizing conditions, was measured as described before. A Mann-Whitney test was performed to see if swimming speed of the cells was statistically different under the effect of the stimulus from that under control conditions. Depolarizing stimulus 119

130 Wild type, neomycin expressing control and mutant cells will be counted and 1X 10 6 cells from each cell type will be used for the assays. Cells will be briefly centrifuged at 1000g at room temperature and resuspended in 5 ml of test solution (10mM Tris-Cl, 0.5 mm MOPS, 50 mm CaCl 2, 8mM K +, ph 7.4) at room temperature for 10 minutes. 0.5μl cells will be added to 90μl test solution with 0.05M BaCl 2 and the cells will be immediately videorecorded for 5 sec at 30 frames/sec using the DAGE-MTI CCD100 video camera. To ensure that pipetting the cells does not have an effect on swimming speed, as controls, 0.5μl cells will be added to 90μl test solution and will also be videorecorded for 5 sec. Approximately 20 cells will be chosen randomly in each trial, and scored for avoidance reaction. Three trials will be performed, both for controls and for depolarizing stimulus. Transmission Electron Microscopy Axonemes were isolated from 1l of cells (both CU428 and HYDKO) as described above. Fixation, dehydration, and polymer infiltration steps were carried out following the protocol earlier described by Angus et al., (2001). The axonemes were resuspended in 2% glutaraldhyde, 0.4% tannic acid and 0.1M sodium cacodylate (ph 7.0) for 1 hour at room temperature. They were then washed with 0.1M sodium cacodylate (ph 7.4), and fixed in 1% osmium tetroxide, 0.05 M sodium cacodylate (ph 7.4) for 1 hour at room temperature. To remove excess osmium tetroxide, the samples wiere washed with 0.1M sodium cacodylate (ph 7.4) and stained in 0.5% uranyl acetate overnight. The next day, samples were subjected to systematic dehydration using a graded acetone solution and finally embedded in Spurtol (vinyl cyclohexene dioxide 10g, a polyglycol diepoxide/epoxide resin 6g, nonenyl succinic anhydride 26g, 2-dimethylaminoethanol 0.4g). A Reichert Ultracut E microtome was used to cut silver-gray sections (40 ± 50 nm thick) and the sections were stained with 2% uranyl acetate and 2% lead citrate. Finally the sections were observed with JEOL 100S TEM operating at 80kV with the magnification of 40,000X. Two independent axoneme isolations were carried out for both the cell types and they were processed independently. Scanning Electron Microscopy 120

131 Cells were grown overnight in a shaker at 28 o C in Neff s growth medium to a concentration of 2-4X 10 5 cells/ml. 4 ml of cells were added to 4 ml of mixture of 2% OsO4 in 0.01 M sodium cacodylate, ph 7.1 and glutaraldehyde in 0.01 M sodium cacodylate, ph 7.1 and the cells were fixed for 10 minutes. The osmium and glutaraldehyde mixture was replaced with three changes of fresh 2.5% glutaraldehyde in buffer (25 min each), after which cells were applied to poly- L-lysine-coated coverslips. Coverslips were rinsed with water, dehydrated in ethanol, CO 2 critical point dried, sputtered with 21 nm of gold, and observed at 10 kev in JSM T-200 SEM. Approximately 20 cells of each type from duplicate coverslips were observed and representative images taken. Results Hydin is present in Tetrahymena thermophila The putative Tetrahymena hydin gene consists of 18 exons spanning less than 17 kb of genomic DNA. According to available genomic information, the Tetrahymena hydin gene appears to a singly copy locus which is supported by southern blot analysis (data not shown). Sequence comparison studies revealed that hydin mrna coding sequence in Tetrahymena encodes a putative protein of 4925 amino acids (aa) and has an overall identity of 25% and similarity of 44% with the mouse hydin protein. RT-PCR on total RNA from growing Tetrahymena indicated that hydin is expressed in Tetrahymena (Fig 1A). To confirm the presence of hydin protein in Tetrahymena cilia, we isolated axonemes from wild type cells and ran them on a 4-16% SDS-PAGE. A protein band of the predicted size (~550KDa) was observed (Fig 1B). To confirm that the protein present in the band of interest was hydin, the band was excised from the gel and LC- MS/MS analysis was performed. Hydin was detected with as many as 145 peptide hits and exhibited peptide coverage of 44.25% (Table 1). Collectively, these results provide strong evidence at genomic, mrna and protein level of the presence of hydin in Tetrahymena. Targeted gene knockout of hydin 121

132 To explore the function of hydin in ciliary motility in Tetrahymena thermophila, we carried out a targeted gene knockout of hydin in Tetrahymena. Approximately 3 Kb region (spanning exons 9, 10 and 11) of the hydin gene was cloned. The knockout construct was made by insertion of the neo-cassette within this cloned region transformed in the macronucleus of Tetrahymena by biolistic method. Fig 2B represents a schematic diagram of the hydin gene in Tetrahymena. The cloned region as well as the neo-cassette insertion site is illustrated. Transformed cells were grown in increasing concentrations of the drug paromomycin which resulted in gradual replacement of the wild type copies of the gene by the disrupted version. PCR was carried out at regular intervals to check for the extent of replacement. Primers were designed flanking the site of insertion of neocasssette and the expected band sizes were a single band of ~500 bp in the wild type lane and two bands in the mutant lane- one at 500 bp and the other at ~1900 bp. PCR on DNA isolated from cells grown at a paromomycin concentration of 70 mg/ml indicated that the knockout was complete (data not shown). To confirm that the knockout was indeed complete (i.e. that all the wild type copies in the macronucleus had been replaced with the mutated version), the cells were grown for two months without the drug. Genomic DNA was isolated from mutant cells after the first and the eighth week of growth without paromomycin, and PCR was performed Fig 2A represents the PCR results. In the lane with wild-type DNA, a single band at ~500bp was observed. The two lanes with mutant DNA had a strong signal at 1.9 Kb and a faint signal at 500bp. The strong signal was from amplification of disrupted, macronuclear copies of the hydin gene, while the faint signal was from amplification of wild type copies of the gene present in the micronucleus of the mutant cells. Since the proportion of the two bands in the lanes containing the mutant DNA proved that no reversion to wild type copies had taken place. Finally, single cell clones were established and checked by performing PCR. Although PCR results consistently showed that the knockout was complete, as a second confirmation we performed southern blot analysis. Genomic DNA isolated from wild type cells or mutants was digested with EcoRV, electrophoresed and blotted. When hybridized with the probe indicated in the schematic diagram (Fig 2b), the lane containing wild type DNA showed a signal at 5.1 Kbp as expected, and the lane with DNA from the mutant showed a strong single signal at 6.5 Kbp (Fig 2c). Since EcoRV 122

133 cuts on either side of the KO cassette insertion site, the presence of a band at 6.5 Kbp and the absence of a 5.1 Kbp band in the mutant lane prove that the knockout is complete and that the KO construct integrated into the correct site. Together, the PCR and southern blot results conclusively proved that the neo-cassette integrated in the correct position in the endogenous macronuclear hydin gene and complete knockout of the hydin gene was achieved. Hydin protein is missing in the mutants Although our results at the genomic level indicated that all macronuclear hydin genes had been disrupted, we wanted to confirm that the hydin protein is indeed missing in the mutants. To do so, axonemes were isolated from both wild type and mutant cells and subjected to SDS-PAGE electrophoresis on a 4-16% gel. The lane containing axonemes isolated from the hydin mutants lacked the band containing hydin protein (Fig 3). We therefore confirmed that the knockout of hydin gene resulted in the loss of the hydin protein in mutant cells. Hydin mutants exhibit impaired ciliary activity Cilia in Tetrahymena, can be broadly classified into two types- somatic cilia which are involved in activities like motility, and oral cilia that are involved in feeding by the process of phagocytosis. Mutations affecting a ciliary protein can have an effect on either or both the types of cilia depending upon the localization of the protein and its functional interaction with other proteins. To determine the effects of the mutation of the hydin gene on somatic ciliary behavior, we performed swimming speed and beat frequency assays. To determine the effects on oral cilia, we measured feeding rate. To measure swimming speed, freely swimming wild type and mutant cells were videorecorded as described in details in the methods section. Fig 4A gives a representative image of the path lengths traversed by wild type (CU428) and mutant cells in 1sec. The average swimming speed of wild type cells was 0.37 mm/sec (Cu428) and 0.35 mm/sec (UC230), while that of the hydin mutants was 0.05 mm/sec. Quantitatively, swimming speed was reduced by approximately 80% in hydin mutants (Fig 4B). Swimming speed is a product of beat frequency and waveform, and decreased swimming 123

134 speed could be either a result of a defect in beat frequency or waveform or a combined effect of defects in both the parameters. Video microscopic recordings revealed no gross defect in the waveform of the mutants. However, since ciliary beat is not planar in Tetrahymena, it is very difficult to detect any subtle changes in waveform. We could assay beat frequency of the mutants, and beat frequency was reduced by approximately 70% compared to that of the wild type cells (Fig 5). These two sets of data collectively suggest that decrease in swimming speed in the mutants is as a result of the decrease in their beat frequency. To investigate the effects of the mutation of hydin gene on oral cilia, we incubated both wild type and mutant cells in media containing 2% Indian ink. In the process of feeding, the cells will take up the ink containing media and store it in their food vacuoles thereby causing the vacuoles to get stained. Hence, the presence of cells with stained vacuoles can be taken as an index of their feeding activity. Fig 6A represents a wild type cell without and with ink stained vacuoles. When quantified, the percentage of cells with ink stained vacuoles was around 20% less in hydin mutants compared to that in the wild type cells (Fig 6B). The reduction in rate of formation of food vacuoles in the mutants compared to wild type cells is consistent with the malfunctioning of oral cilia. Taken together our data strongly suggests that hydin plays an essential role in motility of both somatic and oral cilia. Hydin mutants retain their hyperpolarizing and depolarizing stimuli response mechanisms Tetrahymena demonstrates an array of diverse ciliary behavior such as fast forward swimming, slow swimming, stopping, and backward swimming in response to different kinds of external stimuli. When subjected to a hyperpolarizing stimulus, wild type cells exhibit increased beat frequency and swimming speed (Sugino and Machemer, 1988; Satir, 2003). In response to a depolarizing stimulus, wild type cells exhibit three different characteristic behaviors, all of which involve reversal of ciliary beat (Saimi et al., 1983). One of the behaviors is avoidance reaction (AR). In AR, the cells, in response to a Ba 2+ stimulus, briefly jerk backwards and then twirl around its position instead of normal forward swimming. To determine the effects of the hydin mutation in response to 124

135 external stimuli, we performed two simple assays. We measured avoidance reaction in response to depolarizing stimuli and fast forward swimming in response to hyperpolarizing stimuli. To hyperpolarize the ciliary membrane, the cells were initially incubated in a test solution containing 8mM K + for 10 minute, then transferred to test solution without K +, and swimming cells were video recorded. Swimming speed was calculated from the recorded videos. Fig 7A represents the swimming speed of the cell types, both under control conditions and under the effect of hyperpolarizing stimuli. Around 25% increase in swimming speed was observed in case of both wild type strains; in case of hydin mutants the increase was around 36% compared to their basal speed. To induce depolarizing stimulus, the cells were pipetted from test solution into a solution containing 0.5 mm Ba 2+. Cells were scored for display of avoidance reaction and percentage of cells displaying AR was plotted against cell types (Fig 7B). In normal test solution approximately 10% wild type cells have a tendency to exhibit a twirling motion. Since the basal speed of hydin mutants is significantly lower compared to the wild type cells, this twirling behavior was possibly not captured for these cells under control conditions. In the presence of Ba 2+, both the mutants and wild type displayed avoidance reaction to the same extent. These two sets of experiments collectively showed that the protein hydin may not be involved in mediating signaling responses to depolarizing and hyperpolarizing stimuli. Hydin mutants exhibit no alteration in ciliary number, organization, or length Previous studies have linked aberrant ciliary length, density and arrangement with defects in beat patterns of cilia in different organisms (Roperto et al., 1994; Asai et al., 2009). Since Tetrahymena hydin mutants displayed reduced swimming speed, beat frequency and feeding rates, we performed scanning electron microscopy on fixed whole wild type and mutants cells to look for any alteration in ciliary number, length, and organization. Fig 8 shows representative images of wild type and hydin mutant cells. Analyses revealed no obvious variation in ciliary length, density or arrangement in the mutants compared to the wild type cells. 125

136 Hydin mutants have a partial central pair defect Transmission electron microscopic images of axonemes isolated from wild type or mutant cells revealed a characteristic 9+2 arrangement of microtubules. Fig 9A illustrates a representative image of cross section of axoneme isolated from wild type cells. Analyses of mutant axonemal cross sections revealed a partial central pair missing in some cross-sections. Figs 9B-D represents images of different cross sections of mutant axoneme. The three images illustrate that the extent of central pair missing is variable. Upon quantification, it was observed that a portion of one of the central pair microtubules was missing in approximately 45% of the cross-sections of mutant axonemes examined. Next, we fixed whole cells and sectioned them to take a look at their axonemal crosssections. We observed, only 10% of the axonemal sections in the mutants had a partial central pair missing. Fig 9E represents the graph where percent of sections with intact central pair has been plotted against the two different axonemal preparations of both cell types. These two sets of data combined together raised the speculation that loss of hydin might have weakened the stability of central pair structure in Tetrahymena. To take a closer look at the central pair complex, we performed image averaging analyses using digitized images of both wild type and mutant axonemal cross sections. As illustrated earlier (Sugrue et al., 1991), the central pair complex in Tetrahymena has a characteristic structure containing two identical microtubules and an ensemble of different projections. Fig 10A represents a schematic diagram depicting the central pair complex in Tetrahymena. The distance between the centres of the two microtubules is 30nm. Two short projections an 8nm long projection repeating every 16 nm and a 12nm ribbon-like structure repeating every 32 nm, emerge from one microtubule. The remaining projections emerging from this singlet microtubule as well as the other form the D- shaped central sheath. The wild type image average analyses reflect the same arrangement of the singlet microtubules and other projections as reported previously (Fig 10B). However, in case of hydin mutants, we observed that a particular projection was missing resulting in an incomplete D-shaped sheath. An important point to note here is that in case of hydin mutants, we used only those images which had a complete central pair. Based on other projections of the central pair, we could conclude that the missing 126

137 projection was part of that singlet microtubule which appeared incomplete in all the sections lacking a partial central pair. Discussion In this manuscript, we report the targeted gene knockout of hydin gene in Tetrahymena thermophila. Biochemical analysis combined with mass spectrometric results proved that the hydin protein is missing in the mutants. Loss of hydin is manifested as a severe defect in motility of cells. The response to external stimuli, however, is not affected in the mutants. Electron microscopic examination reveals an ultrastructural defect in the central pair of the axoneme. Hydin is necessary for the stability of the central pair complex Transmission electron microscopic examination of axonemal backbone of hydin mutants revealed a projection missing from the central pair of mutant axonemes. This projection is a part of the central sheath in Tetrahymena. The partial loss of this sheath might have led to destabilization of the structure resulting in loss of the partial central pair microtubule in a portion of the axonemes isolated. Axoneme isolation by itself involves a rigorous process involving removal of cilia from organisms followed by subsequent detergent induced elimination of the ciliary membrane. The notion of destabilization of the central pair due to loss of hydin is strengthened by the observation that the percent of sections missing partial central pair from whole cell fixations was considerably lower compared to the isolated axoneme sections. Our data are consistent with studies conducted in Chlamydomonas, Trypanosomes and mice. In all these organisms, loss of hydin has been associated with similar central pair defect. Although the major structural components of the axoneme are evolutionarily conserved across organisms, there are some subtle differences. The central pair of Chlamydomonas and rat sperm flagella has two asymmetric singlet microtubule- C1 with two projections that repeat at 16nm and 32 nm intervals and C2 having three projections that repeat every 16nm (Olson and Linck, 1977; Mitchell, 2003). Localizations studies have shown that hydin is present in C2b projection (one of the two projections coming out of C2); hence it is exclusively a part of C2 microtubule. However, it connects to the 127

138 C1 microtubule via a projection coming out of C1 mirotubule (Lechtreck et al., 2007). The Tetrahymena central pair complex has a slightly different structure (as described in details earlier). The loss of the particular projection observed in Tetrahymena hydin mutants also suggests that while hydin may be exclusively part of a projection coming out of one microtubule; it is attached to the other microtubule via the other projections making up the central sheath. Hence, we can conclude that despite some subtle structural dissimilarity of the central pair complex, localization of hydin is similar across different organisms and in all cases it plays an important role in maintaining the stability of the complex. Hydin plays an essential role in ciliary beating Mutating hydin in Tetrahymena resulted in approximately 80% decrease in swimming speed compared to the wild type cells. Beat frequency measurements of the cells revealed that the decrease of beat frequency could account for the quantitative reduction in swimming speed. The videos of freely swimming wild type and mutant cells demonstrated that the waveform did not appear to be affected in the mutants. Knockdown of hydin by RNAi or loss of hydin in mutants resulted in impaired ciliary motility in all organisms studied to date (Lechtrek et al., 2008, Lechtrek et al., 2007; Dawe et al., 2007). However, the nature of the motility defect is slightly variable; in Trypanosomes and Chlaymydomonas most of flagella are paralyzed. In mice the beat frequency of both ependymal and tracheal cilia is reduced but the waveform was planar; however, a delay was observed in shifting from power to recovery stroke. Ciliary beating is a complex affair. The molecular motors, dyneins, cause adjacent microtubules to slide past one another and the sliding is converted to bending by additional accessory proteins including the proteins present in radial spoke and central pair complex (reviewed in Smith and Sale, 1994). Ciliary beating can be compared to the movement of oars with alternate power and recovery strokes. The dyneins on one side of the axoneme play a major role during the power stroke, while the dyneins on the other half are the main players involved in the recovery stroke (Satir, 2003). Studies over the years have shown that central pair proteins influence the activity of these dynein motors via the radial spokes. Recent studies in Chlamydomonas have established that the central 128

139 pair influences the action of the outer arms through regulation of inner arms (Kikushima, 2009). Multiple studies have shown that the outer arms are the main players in controlling beat frequency (Satir et al., 1993). Since hydin is localized in the central pair, loss of hydin may have altered the way the central pair controls the outer arm resulting in the observed defect in beat frequency. Defects in the central pair have been associated with different kinds of motility phenotypes. Loss of the entire central pair resulted in paralysis in mutants (Witman et al., 1978). The phenotypes were variable in cases in which a singlet or a projection from the singlet microtubules was missing. Reduced beat frequency was reported in cpc1 mutants missing C1b projection in Chlamydomonas (Mitchell and Sale, 1999); waveform was altered when C1a projection was missing (Ripp et al., 2001). This proves that the different projections are biochemically and functionally distinct from one another. Detailed studies of different central pair proteins suggest that role played by each protein in motility depends on its specific localization within the complex and its unique structural features which determines its interaction with other proteins. The stalling phenotype observed in Chlamydomonas RNAi knockdowns and mice mutants led the researchers to conclude that hydin may play a role in controlling the activity of dynein arms during the switch between the power and recovery strokes. Even though the major axonemal proteins are evolutionarily conserved, the number of isoforms of some protein families varies across organisms e.g. genomic and proteomic studies show that the number of inner arm dynein isoforms ranges from seven in mammals, to nine in Chlamydomonas and twenty in Tetrahymena (Wilkes et al., 2008). The fact that the delay between power and recovery strokes is not observed in Tetrahymena mutants could be an effect of the presence of so many inner arm dyneins which may be fine tuning the beat pattern. Additionally, in some organisms the central pair is fixed (e.g in mice, Tetrahymena etc) while in others, it rotates such as in Chlamydomonas, Paramecium (Omoto et al., 1999). This may also influence how particular groups of dynein are regulated by the different central pair proteins based on specific orientation of the complex. All these factors may be collectively responsible for the variability in phenotypes of hydin mutants observed across different organisms. 129

140 Nevertheless, the studies point to the fact that the severe defect in ciliary motility in hydin mutants is due to a defect in the central pair. Hydin is not involved in signaling activities The ciliary membrane is the site of several ion channels and receptors. In response to an external stimulus, specific ion channels and/receptors get activated triggering signaling cascades that are finally manifested as a change in cilairy beat characteristics. The best known among them are the voltage gated Ca 2+ channels. The signaling molecules activate second messengers such as Ca 2+ or camp where they act on different axonemal proteins to alter ciliary beat patterns (Satir, 2003). The pathways of Ca 2+ signaling are not yet fully resolved, but it is thought that Ca 2+ acts directly or indirectly on the inner arms. Two well characterized behavioral studies in Tetrahymena are the responses to hyperpolarizing and depolarizing stimuli. Experiments suggest that in response to hyperpolarizing stimuli; activity of the outer arms is enhanced leading to increased beat frequency and greater swimming speed of the cells (Van Houten, 1979; Naitoh and Eckert, 1969; Bonini et al., 1986; Machemer, 1974; Hennessey et al., 1985). Depolarizing stimulus, on the other hand, opens Ca2+ channels to open up. The increased intraciliary concentration alters the power stroke (Machemer., 1988); the level of intraciliary concentration determines the phenotype of the cells- cells may exhibit slow swimming, stopping, or backward swimming. Studies have shown that Ca 2+ binding to calmodullin present in the central pair acts asymmetrically on the phases of stroke during ciliary reversal (Satir, 2003). Since hydin is present in the central pair and has also been implicated in regulation of dyneins at the switch point, we determined whether the motility defect caused by the loss of hydin resulted from a defect in this signaling pathway involving Ca 2+. Motility studies showed that hydin may be involved in regulating the activity of dynein in ciliary beating. As an axonemal protein, it is possible that hydin may be playing a role in regulating any of these signaling pathways. Our behavioral analyses with hydin mutants demonstrated that the mutants responded to both the stimuli in a manner similar to that of the wild type cells. Thus, we can conclude that hydin is not involved in any of these signal transduction pathways. Hence the observed defect in ciliary motility due to loss of 130

141 hydin may be attributed to the structural defect resulting in some mechanical aberration in ciliary machinery and not due a signaling defect. Hydin is not involved in ciliogenesis Ciliogenesis involves transport of ciliary components to the ciliary tip for assembly by a microtubule-based transport mechanism termed as intra-flagellar transport. The intraflagellar transport plays a key role in determining the length of cilia and a defect in this pathway would be manifested by an abnormality of ciliary length and/ density. Some central pair mutants have been previously characterized in mice (Sapiro et al., 2002) and Yorkshire boar (Sironen et al., 2006) which exhibited abnormal flagellar length. Our SEM images of mutant whole cells showed no obvious abnormality in the structure or density of cilia. RNAi studies conducted in Trypanosomes had also shown no defects in flagellar formation (Dawe et al., 2007). Altered density or length was also not observed in ependymal and tracheal cilia of hydin mutant mice (Lechtreck et al., 2008). The only exception is the case of Chlamydomonas where cells with knockdown of hydin expression mostly had shorter flagella. Overall, these results suggest that hydin does not play a role in ciliogenesis. Hydin and hydrocephalus Our studies demonstrate the crucial role played by hydin in maintaining the structural integrity of the central pair complex and in ciliary motility. It also rules out the possible involvement of hydin in signal transduction pathways. Research conducted so far not only presents hydin as an important axonemal protein but also provides important inputs in understanding how defects in hydin can cause hydrocephalus in mammals. Based on studies with hy3 mutant mice, Lechtreck et al., (2008) outlined a possible mechanism of how ciliary dysfunction caused by mutations in hydin could result in hydrocephalus. They suggested that disruption of ciliary motility causes impaired fluid flow resulting in stenosis of cerebral aqueduct and consequent enlargement of the third ventricle. The anatomical structure of the human brain suggests that while stenosis caused by impaired ciliary motility can be an important cause of hydrocephalus, it may not be the only cause (Fliegauf et al., 2007). This is because CSF homeostasis in the human brain depends on a 131

142 few factors such as directional movement of ependymal cilia, pulsations of cerebral arteries (Perez-Figaers et al., 2001) and the varying blood pressure of the brain during systole and diastole (Bradley et al., 1986). Also, defects in signaling pathways can alter the reabsorption rate of CSF or result in overproduction of CSF resulting in hydrcephalus. Very recent studies have shown that motile cilia in airway epithelia exhibit some sensory functions as well. These findings bring into light the role of different ciliary proteins in signal transduction pathways. Our findings however do not implicate hydin in any signaling function. Hence, this study along with previous findings in other organisms supports the finding that disruption of ciliary motility as the primary cause of hydrocephalus due to a mutation in hydin gene. 132

143 References Banizs, B., Pike, M.M., Millican, C.L., Ferguson, W.B., Komiosi, P., Sheetz, J., Bell, P.D., Schwiebert, E.M. and Yoder, B.K Dysfunctional cilia lead to altered ependyma and choroid plexus function and result in the formation of hydrocephalus. Dev and Disease. 132(23), Bonini, N.M., Gustin, M.C., and Nelson, D.L Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP. Cell Motil Cytoskeleton. 6(3): Bradley, W.G. Jr., Kortman, K.E. and Burgoyne, B Flowing cerebrospinal fluid in normal and hydrocephalic states: appearance of MR images. Radiology, 159(3), Brady, T.B., Kramer, R.L., Qureshi, F., Feldman, B., Kupsky, W.J., Johnson, M.P. and evans, M.I Ontogeny of recurrent hydrocephalus: presentation in three fetuses in one consanguineous family. Fetal. Diagn. Ther. 14, Broadhead, R., Dawe, H.R., Farr, H, Griffiths, S., Hart, S.R., Portman, N., Shaw, M.K., Ginger, M.L., Gaskell, S.J., McKean, P.G. and Gull K. (2006). Flagellar motility is required for viability of bloodstream trypanosome. Nature. 440, Bruns, P.J. and Cassidy-Hanley, D. 2000a. Biolistic transformation of macro- and micronuclei. Meth Cell. Biol. 62, Callen, D.F., Baker, E.G. and Lane, S.A Re-evaluation of GM2346 from a del(16)(q22) to t(4;16)(q35;q22.1). Clin Genet. 38, Clewell, W.H Congenital hydrocephalus: treatment in utero. Fetal Ther, 3(1-2),

144 Chen, J., Knowles, H.J., Hebert, J.L., and Hackett, B.P Mutations of the mouse hepatocyte nuclear factor/ forkhead homologue 4 gene results in the absence of cilia and random left-right asymmetry. J Clin. Invest. 102, Davy, B.E. and Robinson, M.L Congenital hydrocephalus in hy3 mice is caused by a frameshift mutation in Hydin, a large novel gene. Human Molecular Genetics. 12, Dawe, H.R., Shaw, M.K., Farr, H. and Gull, K The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules. BMC Biol. 5, 33. Doerder, F.P., Deak, J.C. and Lief, J.H Rate of phenotypic assortment in Tetrahymena thermophila. Dev. Genet. 13, Dutcher, S.K., Huang, B and Luck, D.J Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. J Cell Biol. 98(1): Fliegauf, M., Benzing, T and Omran, H When cilia go bad: clia defects and ciliopathies. Nature, 8, Gaetrig J. and Gorovsky, M.A Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc. Natl. Acad. Sci. 89, Gaetrig, J., Gu, L, Hai, B, and Gorovsky, M.A.1994a. Electroporation mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila. Proc. Natl. Acad. Sci. 91, Gaetrig J. and Gorovsky, M.A DNA mediated transformation of Tetrahymena. Meth. Cell Biol. 47,

145 Galbreath, E., Kim, S.J., Park, K., Brenner, M. and Messing, A. 1995, overexpression of TGF-beta 1 in the central nervous system of transgenic mice results in hydrocephalus. J Neuropathol Exp. Neurol., 54, Gruneberg, H. Two new mutant genes in the house mouse J. Genetics. 45, Hennessey, T., Machemer, H., and Nelson, DL Injected cyclic AMP increases ciliary beat frequency in conjunction with membrane hyperpolarization. Eur J Cell Biol. 36(2): Ibanez-Tallon, I., S. Gorokhova, and N. Heintz Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum. Mol. Genet. 11, Ibanez-Tallon, I., Pagenstecher, A., Fliegauf, F., Olbrich, H., Kispert., A., Ketelsen, U., North, A., Heintz, N and Omran, H Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation. Hum. Mol. Genet. 13(18), Johnson, K.A Preparation and properties of dynein from Tetrahymena cilia. Meth. Enz. 134, Kikushima, K Central pair apparatus enhances outer-arm dynein activities thorugh regulation of inner arm dyneins. Cell Motil Cytoskelet. 66, Lapunzilia, P., Delicado, A., de Torres, M.L., Mor, M.A., Perez-Pacheco, R.F. and Lopes, P.I Autosomal recessive hydrocephalus due to agueduct stenosis: report of a further family and implications for genetic counseling. J Matern Fetal Neonatal Med. 12, Lechtreck, K. and Witman, G.B Chlamydomonas reinhardtii hydin is a central 135

146 pair protein required for flagellar motility. J Cell Biol. 176(4): Lechtreck, K. Delmotte, P, Robinson, M.L., sanderson, M.J. and Witman, G.B Mutations in Hydin impair ciliary motility in mice. 180 (3), Machemer, H Frequency and directional responses of cilia to membrane potential changes in Paramecium J. comp.physiol. 92, Macherer, H Motor control of cilia. In Gortz H-D (ed): Paramecium. Berlin: Springer-Verlag Mitchell, D.R Orientation of the central pair during flagellar bend formation in Chlamydomonas. Cell Motil. Cytoskeleton. 56, Mitchell, D.R Speculation on the evolution of 9+2 organelles and the role of the central pair microtubules. Biol Cell. 96(9), Mobberly, P.S., Sullivan, J.L., Angus, S.P., Kong, X. and Pennock D.G. (1999). New axonemal dynein heavy chains from Tetrahymena thermophila. J Euk Microbiol. 46, Moog, U., Bleeker- Wagemakers, E.M., Crobach, P., Vles, J.S., and Schrander-Stumpel, C.T Sibs with Axenfeld-Rieger anamoly, hydrocephalus and leptomeningeal calcifications: a new autosomal recessive syndrome? Am J Med Genet. 78, Moyer, J.H., Lee-Tischler, M.J., Kwon, H.Y., Schrick, J.J., Avner, E.D., Sweeney, W.E., Godfrey, V.L., Cachiero, N.L., Wilkinson, J.E. and Woychik, R.P Candidate genes associated with mutation causing recessive polycystic kidney disease in mice. Science. 264,

147 Naitoh, Y., and Eckert, R Ionic mechanisms controlling behavioral responses of Paramecium: modification of ciliary movements by calcium ions. Science. 176: Omoto, C.K., Gibbons, I.R., Kamiya, R., Shingyoji, C., Takahashi, K., and Witman, G.B. Rotation of the central pair microtubules in eukaryotic flagella. Mol. Biol.Cell. 10, 1-4. Olson, L.W. and Linck, R.W Observations of the structural components of flagellar axonemes and central pair microtubules from rat sperm. J Ultrastrucl Res. 61(1), Patwardhan, R.V. and Nanda, A Implanted ventricular shunts in the United States: the billion-dollar-a-year cost of hydrocephalus treatment. Neurosurgery. 56(1), , discussion Pazour, G.J., Agrin N., Leszyk J, and Witman, G.B Proteomic analysis of a eukaryotic cilium. J Cell Biol. 170(1), Perez-Figares, J.M., Jimenez, A.J. and Rodriguez, E.M Subcommisural Organ, Cerebrospinal Fluid Circulation, and Hydrocephalus. Micros. Res. Tech. 52, Pennock, D.G Selection of motility mutants. Meth. Cell Biol. 62, Rajagopalan, V., Subramanian, A., Wilkes, D., Pennock, D.G. And Asai, D Dynein-2 affects the Regulation of Ciliary Length but is not Required for ciliogenesis in Tetrahymena thermophila. Mol Biol Cell. 20, Robinson, M.L., C.E. Allen, B.E. Davy, W.J. Durfee, F.F. Elder, C.S. Elliott, and W.R. Harrison Genetic mapping of an insertional hydrocephalus inducing mutation allelic to hy3. Mamm. Genome. 13,

148 Roperto, F., Rossacco, P, Tartaro, A. and Galati, P Abnormal length of respiratory cilia in a pig. An ultrastructural study. 26(1), Saimi, Y., Hinrichsen, R.D., Forte, M., and Kung, C Mutant analysis shows that the Ca2+-induced K+ current shuts off one type of excitation in Paramecium. Proc Natl Acad Sci U S A 80, Sale, W.S The axonemal axis and Ca2+ induced asymmetry of active microtubular sliding in sea urchin sperm tails. J Cell Biol. 98, Sapiro, R., I. Kostetskii, P. Olds-Clarke, G.L. Gerton, G.L. Radice, and I.J. Strauss Male infertility, impaired sperm motility, and hydrocephalus in mice deficient in spermassociated antigen 6. Mol. Cell. Biol. 22(17), Satir, P. and Guerra, C Control of ciliary motility: an unifying hypothesis. Eur. J. Protistology. 39, Satir, P., Barkalow, K. and Hamasaki, T The control of ciliary beat frequency. Trends Cell Biol. 3(11), Satir, P Mechanisms of Ciliary motility: an update. Europ. J. Protistol. 34, Schurr, P.H. and Polkey, C.E. eds, Hydrocephalus. 1993, Oxford Medical Publications. Oxford University Press, New York, NY. Sironen, A., Thomsen, B., Andersson, M., Ahola, V. and Vilkki, J An intronic insertion in KPL2 results in aberrant splicing and causes the immotile short tail sperm defect in the pig. Proc. Natl. Acad. Sci U S A. 103(13),

149 Smith, E.F and Sale, W.S Regulation of dynein driven microtubule sliding by the radial spokes in flagella. Science 257(5076), Shah, A.S.; Ben-Shahar, Y. Moninger, T.O., Kline, J.N. and Welsh, M.J Motile Cilia of Human Airway Epithelia are Chemosensory. Science 325, Sugino, K., and Machemer,H The ciliary cycle during hyperpolarization-induced activity: an analysis of axonemal functional parameters. Cell Motil Cytoskeleton. 11(4): Surgue, P, Avolio, J., Satir, P and Holwill, M.E.J Computer modeling of Tetrahymena axonemes at macromolecular resolution. J Cell Sci. 98, Taulman, P.D., Haycraft, C.J., Balkovetz, D.F., and Yoder, B.K Polaris, a protein involved in left-right axis patterning, localizes to basal bodies and cilia. Mol. Biol. Cell. 23(1), Van Houten, J.L Membrane potential control of chemokinesis in Paramecium. Science. 204: Witman, G.B., Plummer, J. and Sander, G Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition and function of specific axonemal components. J Cell Biol. 76, Zhang, H. and Mitchell, D.R Cpc-1, a Chlamydomonas central pair protein with an adenylate kinase domain. J Cell Sci. 117:

150 Table 1. LC-MS/MS analysis of putative hydin protein band marked in Figure1B. Reference P (pro) Score Coverage MW Accession Peptide (Hits) 1 hypothetical protein TTHERM_ E ( ) 2 Dynein heavy chain family protein 4.44E ( ) 3 ciliary outer arm dynein beta heavy chain 8.18E ( ) 4 Dynein heavy chain family protein 1.68E ( ) 5 Dynein heavy chain family protein 1.87E ( ) 6 Tubulin beta chain, putative 1.00E ( ) 7 hypothetical protein TTHERM_ E ( ) 8 Tubulin/FtsZ family, GTPase domain containing 3.00E ( ) 9 Dynein heavy chain family protein 4.22E ( ) 10 hypothetical protein TTHERM_ E ( ) 11 Dynein heavy chain family protein 8.49E ( ) 12 Dynein heavy chain family protein 6.81E ( ) 13 IQ calmodulin-binding motif family protein 2.68E ( ) 14 EF hand family protein 1.07E ( ) 15 AF152585_1 dynein heavy chain 4.22E ( ) 16 hypothetical protein TTHERM_ E ( ) 17 Dynein heavy chain family protein 6.95E ( ) 18 Dynein heavy chain family protein 2.82E ( ) 19 Dynein heavy chain family protein 1.86E ( ) 20 SerH3 immobilization antigen, putative 2.51E ( ) 21 ubiquitin-transferase, HECT-domain 2.00E ( ) 22 E1-E2 ATPase family protein 8.59E ( ) 23 AF153267_1 dynein heavy chain 5.15E ( ) 24 hypothetical protein TTHERM_ [Tetrahym 4.99E ( ) 25 JT0492 ubiquitin 2 - Tetrahymena pyriformis (fragment) 4.04E ( ) 26 ABC transporter family protein 1.46E ( ) 27 RIH domain containing protein 5.96E ( ) 28 Dynein heavy chain family protein 1.41E ( ) 29 hypothetical protein TTHERM_ E ( ) 30 Dynein heavy chain family protein 1.58E ( ) 31 AF153702_1 dynein heavy chain 1.36E ( ) 32 hypothetical protein TTHERM_ E ( ) 33 Dynein heavy chain family protein 2.05E ( ) 34 hypothetical protein TTHERM_ E ( ) 35 hypothetical protein TTHERM_ E ( ) 36 AF153270_1 dynein heavy chain 1.14E ( ) 37 Inorganic H+ pyrophosphatase 7.72E ( ) 38 inorganic pyrophosphatase 1.42E ( ) 39 Dynein heavy chain family protein 1.58E ( ) 140

151 Fig 1. Hydin is present in Tetrahymena thermophila. A. RT-PCR showing that hydin is expressed in Tetrahymena cells. RT-PCR of Tetrahymena Hydin gene. The far left lane is a 1 kb ladder. Tetrahymena genomic DNA as well as RNA samples treated with or without reverse transcriptase (RT) were amplified with primer set 1 &2 corresponding to sequences from putative exons 4 and 5 or primer set 3 & 4 corresponding to putative exons 6 and 8. Primer set 1 & 2 was predicted to amplify a band of 798 bp from genomic DNA and a band of 583 bp in correctly spliced mrna. Primer set 3 & 4 was predicted to amplify a band of 955 bp in genomic DNA and a band of 715 bp in correctly spliced mrna. B. SDS-PAGE showing a band of the size of Hydin protein in Tetrahymena. Axoneme isolated from wild type cells were separated on a 4-16% SDS-PAGE gel. Gel was stained with Coomassie Blue. The marked band at the top of the gel is of the size of hydin protein (~550 kda) 141

152 142

153 Fig 2. PCR and southern blot to prove that knockout is complete in the mutants. A. PCR results showing that phenotypic assortment is complete. Whole genomic DNA was isolated from wild type and mutant cells and PCR carried out with appropriate primer pairs. Bands of expected sizes are observed. The left most band is the marker lane. Sizes of the bands (in bp) are indicated on the side of the blot. B. Strategy designed to create knockout of hydin gene. The cloned region is marked by the double-headed arrows (bp ). Arrowhead marks the site of insertion of neo- cassette (bp 11028). The region marked by the solid lines between bp 5530 and bp is the predicted fragment generated by EcoRV digestion to be recognized by the probe for southern-blot. Numbers in the diagram indicate the position in bp. C. Whole genomic DNA was isolated from both wild type (CU428) and mutant cells and digested with EcoRV. With appropriate probe, bands of predicted size were obtained. Sizes of the bands (in bp) are indicated on the side of the blot. 143

154 144

155 Fig 3. SDS-PAGE analysis shows that hydin protein is missing in the mutants. Whole axonemes were isolated from both wild type and mutant cells and ran on a 4-16% SDS- PAGE. Arrowhead indicates the band which is missing in the mutant lane. Two different amounts of total axonemal protein (100 µg and 150 µg) was loaded as indicated below the lanes. 145

156 146

157 Fig 4. Hydin mutants show altered swimming behavior. A. Representative photomicrographs show path lengths traversed by wild type (CU428) and mutant cells in 1sec B. Swimming speed in mm/sec was plotted against cell type. Swimming speed of mutants was significantly lower compared to that of the two wild type strains (CU428 and UC230). Error bars indicate S.D. *** indicates p<0.001 (ANOVA). 147

158 148

159 Fig 5. Hydin mutants exhibit decreased beat frequency. Beat frequency (beats/sec) plotted against the different cell types show a significant reduction in beat frequency of hydin mutants compared to the two wild type strains (CU428 and UC230). Error bars indicate the S.D. *** indicates p<0.001 (ANOVA). 149

160 150

161 Fig 6. Hydin mutants display reduced feeding rates. Feeding assays performed on wild type and mutant cells to assess the effects on oral cilia. A. Representative images of wild type cell without and with ink stained vacuoles B. Graph with percentage of cells containing ink stained vacuoles plotted against cell type. Hydin mutants show significant decrease in number of cells containing ink stained vacuoles. Error bars indicate S.D. ** indicates p<0.01 (ANOVA) 151

162 152

163 Fig 7. Hydin mutants respond to hyperpolarizing and depolarizing stimuli in a manner similar to wild type cells. A. Hyperpolarizing studies carried out by transferring cells from solution containing high K + to solution without K +. Both wild type and mutant cells display a significant increase in swimming speed in response to the stimulus. Note the basal swimming speed of hydin mutants is lower compared to wild type cells. Error bars indicate S.D. * represents p<0.05 B. Depolarizing stimulus generated by putting cells in solution containing 0.5mM Ba 2+. Both wild type and mutants exhibit avoidance reaction to the same extent. 153

164 154

165 Fig 8. Scanning electron microscopic analyses of wild type and mutant cells. Representative images do not indicate any defect in ciliary length or density in the mutants. 155

166 156

167 Fig 9. Hydin mutants exhibit a defective central pair of axonemes. Transmission electron microscopic analysis of axonemes of wild type and mutant cells was performed. A-D. Representative cross sectional image of wild type (A) and mutant (B-D) axonemes. Arrows in the mutant axoneme sections indicate the varying extent of missing portion of central pair. E. Graph indicating the percent of sections with intact central pair in cross-sections from isolated axonemes as well as cross-sections from axonemes of fixed whole cells. Error bars indicate the S.D. *** p< 0.001, * p<

168 158

169 Fig 10. Image average analyses of central pair complex of wild type and hydin mutant axonemes A. Schematic diagram representing the central pair. The two singlet microtubules, D- sheath and projections are illustrated. B. Image average analyses of six cross sections of both wild type and mutant axonemes show a missing projection in the mutant axonemes indicated by arrowhead. 159

170 160