Molecular Cell Biology (Bio 5068) Microtubules and their func<ons in cells. Moe Mahjoub. September 17, 2015

Transcription

1 Molecular Cell Biology (Bio 5068) Microtubules and their func<ons in cells. Moe Mahjoub September 17, 2015

2 About me From Vancouver, Canada PhD Simon Fraser University, Canada Dr. Lynne Quarmby Postdoc Stanford University Dr. Tim Stearns Washington University From September 2012 Medicine (Division of Renal Diseases) Department of Cell Biology and Physiology DBBS Programs: Molecular Cell Biology Developmental, Regenera<ve and Stem Cell Biology Biochemistry

3 Microtubules and their Func<ons Intro what is a microtubule? What do microtubules do in cells? Microtubule Organizing Center (MTOC) Mitosis Cilia

4 Microtubule Structure Cross- sec<on Hollow tube 24 nm wide ~13 protofilaments Helical structure Polar Plus ends generally distal Minus ends generally proximal (at MTOC) Composed of Tubulin α/β Heterodimer

5 Microtubule Structure & Assembly

6 Microtubule Dynamic Instability

7 The Master Nucleator γ- TuRC and microtubule nuclea<on

8 Microtubules are everywhere Q: How are heterogeneity and specificity of microtubule func<on regulated?

9 The Tubulin Superfamily

10 Microtubule Modifica<ons Poseransla<onal modifica<ons (PTM s)

11 Microtubule Motors Defini<on Microtubule- s<mulated ATPase Mo<lity along MT s Dynein Moves towards Minus End of MT s Kinesin Moves to Plus End of MT s

12 Kinesin Movement and Processivity Iden<fied in search for motors involved in axonal transport Movement required ATP

13 Kinesin Superfamily Structures

14 Kinesin Superfamily Phylogene<c Tree

15 Cytoplasmic Dynein Discovered biochemically Minus end motor for vesicle transport, MT mo<lity Requires Dynac<n complex (or other accessory proteins) for func<on

16 Model for interac<ons between dynein, dynac<n complex, microtubules, and cargo

17 Microtubule binding proteins Nucleators (e.g. γ- tubulin, Ninein): Provide the basis for microtubule nuclea<on and assembly Stabilizers (e.g. CLIP170, EB1, Tau): Promote microtubule growth through increased polymeriza<on and reduced catastrophe Destabilizers (e.g. Stathmin, KinI): Reduce average microtubule length by promo<ng catastrophe and suppressing rescue. Severers (e.g. Katanin, Spas<n): Cause breaks within the microtubule lakce, generally reducing average length of microtubules Bundlers (e.g. PRC1): Bind two (or more) microtubules and align them to form parallel or an<- parallel bundles Chaperones (e.g. Prefoldins): Promote folding and assembly of tubulin protein complexes facilita<ng polymeriza<on.

18 Microtubules are everywhere Q: How are heterogeneity and specificity of microtubule func<on regulated? Different tubulin isoforms A large number of tubulin PTMs Different motors and adaptors Diverse microtubule binding proteins

19 Organelle Trafficking - ER and Golgi Posi<oning ER & Golgi: Golgi near MTOC Minus Ends are at MTOC Golgi Posi<on Requires Dynein ER Tubular network spread about the cell Kinesin moves the tubules peripherally

20 Organelle Trafficking - ER at cell boundaries Microtubules (Red) and ER (Green)"

21 Organelle Trafficking - Melanophores Xenopus Melanophore Pigment Granule Movement Vesicles move along microtubules Vesicles carry dynein, kinesin & myosin- V Regula<on of the motors accounts for the dispersion / aggrega<on Inward Mo<on (Movie Loops)

22 Organelle Trafficking - Melanophores Xenopus Melanophore Pigment Granule Movement Vesicles move along microtubules Vesicles carry dynein, kinesin & myosin- V Regula<on of the motors accounts for the dispersion / aggrega<on Outward Mo<on (Movie Loops)

23 Microtubule- based Trafficking

24 Microtubules and their Func<ons Intro what is a microtubule? What do microtubules do in cells? Microtubule Organizing Center (MTOC) Mitosis Cilia

25 Boveri: The Centrosome as MTOC Theodore Boveri ( )" Boveri s predic<ons about centrosomes: (i) The centrosome is a cyclical structure a permanent reproducing organ of the cell. (ii) Centrosomes are part of a central apparatus or microcentrum in cells. (iii) They are dynamic centers' of cells and the true division organs of the cell. (iv) Centrioles (one or two) are contained within centrosomes (v) Centrosomes are the sole microtubule organizing centers (MTOC) in asters and spindles.

26 Boveri: The Centrosome as MTOC Theodore Boveri ( )" Boveri s predic<ons about centrosomes: (i) The centrosome is a cyclical structure a permanent reproducing organ of the cell. (ii) Centrosomes are part of a central apparatus or microcentrum in cells. (iii) They are dynamic centers' of cells and the true division organs of the cell. (iv) Centrioles (one or two) are contained within centrosomes (v) Centrosomes are the sole microtubule organizing centers (MTOC) in asters and spindles.

27 The centrosome as MTOC 1 centrosome per cell" " 2 centrioles per centrosome (in interphase)" " Centriole size is highly conserved throughout evolution:" ~250nm diameter, ~500nm length"

28 Centrosome structure higher order MT organiza<on

29 Centrosome assembly iden<fica<on of components Bornens and colleagues ( )" " Purified centrosomes on sucrose " gradients" " Very clean preps, as seen by EM and IF" Purified centrosomes are " functional:" " Incubated with purified cow " tubulin, starts nucleating MT"

30 Centrosome assembly iden<fica<on of components 2D-gel of isolated centrosomes. Authors surprised to find ~ unique proteins make up a centrosome"

31 Centrosome assembly iden<fica<on of components At least 500 unique proteins " present in centrosomes" " Appear in distinct modules or " complexes, based on subcellular " localization and function"

32 Mechanism of centrosome duplica<on Q: How can a cell make an exact copy of such an elaborate structure?

33 Centrosome duplica<on and the cell cycle G1/S" S" S" S/G2" G2/M" M/G1" Kuriyama and Borisy, 1981

34 Centrosome duplica<on and the cell cycle

35 Centriole structure establishing 9- fold symmetry Q: How is the 9- fold rota<onal symmetry of microtubules established during centriole duplica<on?

36 Centriole structure establishing 9- fold symmetry

37 Centriole structure establishing 9- fold symmetry Structure of purified, recombinant Sas6 " from zebrafish (Science 2011)" Structure of purified, recombinant Sas6 " from Chlamydomonas (Cell 2011)"

38 Centriole structure establishing 9- fold symmetry Sas6 has intrinsic symmetry when forming oligomers"

39 Centriole structure establishing 9- fold symmetry Q: But how come Sas6 doesn t just form a bunch of rings non- specifically in the cytoplasm? A: - Expression restricted to G1/S phase (temporal regula<on) - Recruited to parental centrioles (spa<al regula<on) how?!

40 Centriole structure establishing 9- fold symmetry

41 Pericentriolar material (PCM) and microtubule nuclea<on +! Amorphous cloud " +! +! -! -! -! Lots of γ-turc! -! -! + +! Lots of big, coiled-coil proteins in PCM" +!

42 Pericentriolar material (PCM) organiza<on Amorphous cloud " Amorphous no more!" Super-resolution microscopy to the rescue"

43 Pericentriolar material (PCM) organiza<on The PCM is organized into layers"

44 PCM size and ac<vity increase in mitosis Almost all of the PCM proteins increase in abundance in mitosis"

45 Microtubules and their Func<ons Intro what is a microtubule? What do microtubules do in cells? Microtubule Organizing Center (MTOC) Mitosis Cilia

46 Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNA Prometaphase Metaphase Early Anaphase Late Anaphase Cytokinesis Onset Late Cytokinesis

47 Mito<c Spindle Assembly Centrosome duplicates and is segragated to each side of the nucleus Nuclear envelope breakdown in prophase MT s rearrange via dynamic instability

48 Spindle microtubules Chromosomes, Kinetochores and Microtubules. Super- resolu<on Microscopy (SIM).

49 Chromosome Congression to Metaphase Plate Kinetochores capture MT s Chromosomes pulled and pushed to and from poles Forces on arms Force at kinetochore

50 Metaphase to Anaphase transi<on GFP- labeled Centromeres

51 Types of microtubule- kinetochore aeachments Amphitelic Monotelic Syntelic Merotelic

52 Metaphase/Anaphase Lagging

53 Models for Chromosomes Moving to the Pole Treadmilling? Depolymeriza<on at pole (MTOC) Depolymeriza<on at Kinetochore? How to remain bound while end shrinks? Motors at Kinetochore or Pole?

54 Poleward Tubulin Flux in Anaphase Movement towards pole... Blue: Photobleach Mark, 0.7 µm/min Yellow: Edge of Chromosome, 1.2 µm/min

55 Microtubule dynamics and motors help move chromosomes in mitosis The MTOC plays a critical role in " organizing the microtubule arrays" " "

56 Abnormal regula<on of centrosome number Q: What happens if MTOC (centrosome) assembly and duplication is defective?"

57 Abnormal regula<on of centrosome number Boveri, 1914: extra centrosomes are prevalent in tumors drive genome instability?

58 Abnormal regula<on of centrosome number Ring, Hubble, Kirschner, 1982! Ganem et al. (2009) Nature"

59 Causes and consequences of centrosome amplifica<on

60 Microtubules and their Func<ons Intro what is a microtubule? What do microtubules do in cells? Microtubule Organizing Center (MTOC) Mitosis Cilia

61 The cilium in the 17 th century Drawings from van Leeuwenhoek's letter of March 18, 1678."

62 Mo<le cilia in ac<on Euplotes " Stentor"

63 Mo<le cilia in ac<on Chlamydomonas cilia Sperm Flagellum

64 Cilia on surface of epithelial cells Mo<le mul<ciliated epithelial cells are found in: Airways (lung and trachea): help clear mucus (muco- ciliary escalator) Brain (lining the ventricles): help move cerebrospinal fluid (CSF) Fallopian tube: involved in tubular fluid flow, and thus movement of sperm and ova

65 Structure of mo<le cilium: Cross- sec<on

66 Conversion of sliding to bending

67 Conversion of sliding to bending Q: If all dyneins pull together, how does the axoneme " bend back and forth?"

68 Rota<on of central pair microtubules Whole Chlamydomonas cell " with 2 cilia" Axonemes isolated from Chlamydomonas cell "

69 Mo<le cilia defects in humans Sterility" " Respiratory defects" " Hydrocephalus" " Most of the mutations are in " dyneins and assembly factors"

70 Mo<le ciliary dysfunc<on and L- R asymmetry X!

71 Mo<le ciliary dysfunc<on and L- R asymmetry Heart-looping defects and congenital heart disease"

72 Ciliary assembly - Intraflagellar Transport (IFT)

73 Ciliary assembly - Intraflagellar Transport (IFT) 20 years" later"

74 The primary cilium: non- mo<le sensory organelle Found in almost every cell type in your body:" " Epithelia" Fibroblasts" Neurons" Stem cells" we have hypothesized that these (primary) cilia may be ves9gial organelles Webber and Lee (1975)

75 The primary cilium: non- mo<le sensory organelle

76 Signaling pathways that rely on cilia Primary cilium function is required for sonic hedgehog signaling. Huangfu, et al. Nature (2003)" Shh" PKD! Shh! PDGF! Wnt! TOR! Smoothened" Smoothened"

77 Fundamental processes in cell biology X" Cell division" Extracellular" signal" X! X" Cell differentiation" X" Cell migration" X" Cell polarity and" organization"

78 Human ciliopathies due to defec<ve primary cilia we have hypothesized that these (primary) cilia may be ves9gial organelles absolutely essen<al!!! Webber and Lee (1975)

79 Microtubules and their func<ons in cells. Thank you!" " Contact: