BEHAVIOUR OF KINETOCHORE FIBRES IN HAEMANTHUS KATHERINAE DURING ANAPHASE MOVEMENTS OF CHROMOSOMES

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1 J. Cell Sci. 27, (1977) Printed in Great Britain Company of Biologists Limited 1977 BEHAVIOUR OF KINETOCHORE FIBRES IN HAEMANTHUS KATHERINAE DURING ANAPHASE MOVEMENTS OF CHROMOSOMES ROBERT HARD* AND ROBERT D. ALLEN Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, U.S.A. SUMMARY A laser light source along with a new method of preparing endosperm cells of Haemanthus katherinae for differential interference contrast (DIC) microscopy has led to increased visibility of kinetochore fibres. Little information is available concerning the behaviour of these fibres during anaphase in living cells. In metaphase, kinetochore fibres are seen as distinct bundles of microtubules, here referred to as ' filaments', extending from the kinetochore to the ' diffuse' pole. They possess an apparent globular substructure which corresponds to the moving 'particles or states' described previously from cine 1 films. In early anaphase, the filaments of each kinetochore fibre lose their parallel orientation characteristic of metaphase and splay out so that the more peripheral filaments intermingle with those of other kinetochore fibres. This process begins at the poles and proceeds as a wave toward the kinetochores as chromosomal movement progresses. This behaviour has been examined in relation to a number of proposed models for the mechanism of chromosome movement and has been found to place some constraints on some models but to be consistent with any model that hypothesizes that chromosomes move as a consequence of cumulative cohesive lateral interactions of microtubules. INTRODUCTION Endosperm cells of Haemanthus katherinae are ideal for studies of mitosis, and have been examined extensively using both light and electron microscopy (see Bajer & Mole-Bajer, 1972, for a review). The cells are generally prepared either flattened on an agar-air interface or by sandwiching them between 2 agar-gelatin films and supplying them with 3-6% glucose or sucrose (Mole-Bajer & Bajer, 1968). These preparations are viable for up to 16 h and have the advantage that the cells can be subjected to various chemical treatments in perfusion experiments while under continuous observation (e.g. Jensen & Bajer, 1969). This method of preparation was found to be unsatisfactory for polarized light and differential interference contrast (DIC) studies of Haemanthus spindles, because the agar films themselves were sufficiently birefringent to interfere with accurate photometric measurements of spindle retardation. Agar films also diminish the available contrast in a DIC system. An alternative preparative method was developed which has led to increased visibility of kinetochore fibres, especially with the Nomarski differential interference contrast (DIC) microscope. Current Address: Department of Biology, University of Oregon, Eugene, Oregon, U.S.A. 4-2

2 48 R. Hard and R. D. Allen METHODS AND MATERIALS Cell preparation Since perfusion was not required in the present study, the main purpose in preparing cells was to have them remain healthy and active for the longest possible period of time for observation. It was reasoned that the normal endosperm fluid should be the optimal environment. However, cells maintained between coverglasses in endosperm fluid did not appear healthy longer than 4-5 h. This period could be extended 2-3 fold by merely coating the coverglass surfaces in contact with the cells with the sucrose polymer, Ficoll (Sigma Chem. Co., St Louis, Mo.). When prepared in this way, cells could be seen to divide after more than 10 h in culture. Ficoll was dissolved in double-distilled water (o-i %-o-s %, w/v). The solution was applied to clean coverglasses which were drained, and either air- or flame-dried. A small amount of Vaseline was then placed at the corners of the coverslip to act as a spacer. A drop of endosperm suspension from approximately 3-week-old ovules was placed on the lower coverglass, and a second coated coverglass was carefully applied. Excess endosperm fluid was slowly drawn off with filter paper to produce preparations of the desired thickness and optical quality. The preparation was then sealed with 'valap' (Vaseline, lanolin, paraffin, 1:1:1) to prevent desiccation and mounted on a brass holder. Since the cells were sensitive to mechanical deformation, extreme care was taken in handling them. The only additional difficulties experienced occurred when the cells were either extensively flattened or when there were areas in the preparation where the deposition of polymer had been excessively thick. In either case, the cells deteriorated rapidly and division was completely inhibited. Microscopy Cells were examined with a Zeiss Photomicroscope I using the 514 nm line of a phaserandomized argon ion laser described previously (Hard, Zeh & Allen, 1975). A Nomarski differential interference contrast system with a strain-free 100X achromatic objective (N.A.- 125) and achromatic condenser (N.A ) had an extinction factor of 600 at small apertures but typically at the working aperture used. Cells were photographed using either Kodak Panatomic X 35-mm or Kodak New Recordak 16-mm cin6 film, and the negatives were processed in Diafine developer. The prints were exposed with a Log E Enlarger, an automatic dodging device that rendered the background density more uniform than would have been possible by manual dodging. RESULTS The combination of the absence of an agar overlay and the use of laser illumination has made it possible to observe spindle fibres with unprecedented clarity. Although kinetochore fibres could be seen clearly in any of the dividing cells used in this study, they were especially prominent in cells in which the arms of all the larger chromosomes extended into the opposite half-spindle. With these cells, the degradation of image contrast by out-of-focus detail was minimal. Such cells were examined to find the optical section within the metaphase plate where the largest number of kinetochore fibres could be seen spanning the distance Fig. 1. Optical section within the metaphase plate of a dividing Haemanthus endosperm cell showing 7 distinct kinetochore fibres extending from kinetochore to pole. Note the hemispherical kinetochores. Zeiss photomicroscope DIC 100X N.A. = 125 oil.

3 Anaphase behaviour of kinetochore fibres 49

4 50 R. Hard and R. D. Allen from the kinetochore to the spindle pole. These fibres were then oriented at azimuths of degrees relative to the polarizer in order to maximize both resolution and contrast, and they were then compensated just off extinction (Allen, David & Nomarski, 19696). As shown in the cell in metaphase (Fig. 1), the difference in the visibility of the kinetochore fibres in the 2 half-spindles is striking. The spindle fibres extend as more or less discrete bundles of smaller structures, here termed filaments, from the chromosome to the poles. Bajer & Jensen (1969) have demonstrated that similar filaments visible with DIC in fixed cells are composed of 3-5 microtubules. The kinetochore appears as a distinct hemispherical protrusion from the primary constriction of the chromosome (Fig. 1). In one case, the kinetochores are visible in both adjacent sister chromatids (Fig. 1). Although the so-called 'ball and socket' configuration has been described previously in electron micrographs of kinetochores (Bajer, 1968), this is the first confirming evidence in living material. Such a configuration has also been seen in Haemanthus chromosomes isolated under the appropriate conditions (R. Hard, unpublished results). The kinetochores in Fig. 1 are flanked by a series of regular folds or gyres in the chromosomal surface. These structures are not evident when chromosomes reorient during anaphase. All of the 7 kinetochore fibres seen in Fig. 1, regardless of their width in the halfspindle, taper in the vicinity of the chromosomes, and all visible filaments appear to insert directly into the kinetochore. The spindle fibres extend away from the kinetochore and can be followed to the 'diffuse' spindle pole. The diameters of the kinetochore fibres are not uniform along their lengths, but vary from about 1 to 5 /tm. In several cases, the filaments which make up the individual fibres appear to be twisted loosely around one another as in strands of a rope. The filaments of the kinetochore fibres also seem to possess a repeating 'globular' substructure which is probably the result of diffraction by unresolved microdomains. This apparent substructure has not yet been seen in any other type of microscope and cannot be reconciled with any presently known features of spindle ultrastructure. It has been described as 'particles or states' moving along the kinetochore fibre filaments at uniform anaphase velocity from kinetochores toward the poles, beginning in prometaphase and lasting through anaphase (Allen, Bajer & LaFountain, 1969a). The globular substructure changes appearance slightly with azimuth in a manner somewhat similar to substructure in filamentous elements in squid axoplasm (Metuzals & Izzard, 1969). In metaphase the outermost filaments of each kinetochore fibre are almost parallel and can be followed to the diffuse polar region. However, as anaphase begins, the outermost filaments of each kinetochore fibre splay out, cross and intermingle with filaments of neighbouring kinetochore fibres. This reorientation spreads in a wave from near the polar region toward the chromosomes. As anaphase proceeds, the Fig. 2. The same cell at mid-anaphase. Note the increased angle at which the outer filaments of the kinetochore fibres leave the kinetochore. Note also the crossing of filaments between the kinetochores and the pole of the spindle, x 2550.

5 Anaphase behaviour of kinetochore fibres

6 52 R. Hard and R. D. Allen outermost kinetochore filaments extend toward the lateral margin of the spindle progressively further from the poles (compare Figs. 2 and 3). The intermingling filaments from neighbouring kinetochore fibres cross in the polar regions at angles up to 20 degrees from the interpolar axis. As the chromosomes approach the poles, the Fig. 3. Tracings of cells in division from a series of photomicrographs of which Figs. 1 and 2 correspond to A and D respectively. Solid lines represent visible boundaries of kinetochore fibres. Dashed lines (short) are the projected paths of the outermost kinetochore fibre filaments toward the polar region of the spindle. Dashed lines represent the spindle outer boundary. optical clarity of the polar region diminishes, rendering it more difficult to observe the filaments. Figs. 1 and 2 are from a series of 35-mm photomicrographs taken of the same spindle from metaphase through anaphase. Fig. 3 is a set of tracings on transparent plastic placed over photographic enlargements showing the directions taken by kinetochore fibre filaments, especially the outermost ones.

7 Anaphase behaviour of kinetochore fibres 53 Continuous fibres are readily identifiable as they extend across the interzone of the spindle. Some are visible between kinetochore fibres even in metaphase (Fig. 1). The fibres in the interzone (presumably continuous and/or interzonal fibres) differ markedly in microscopic appearance from the kinetochore fibres. For the most part, they lack both filamentous and globular substructure and are fim in diameter. Unlike kinetochore fibres, the fibres observed in the interzone showed no moving globular substructure, i.e. 'particles or states'. DISCUSSION Physiological state of the cells It is clear that the extraordinary optical clarity of these cells was due in large part to the new method of preparing endosperm fluid between Ficoll-coated coverglasses. In this way, the amount of out-of-focus phase structure is minimized. The visibility of details of spindle fibre structure is attributable to selected optical components with extinction factors in the range of depending on working aperture. It is reasonable to expect further improvements in the quality of images with better corrected DIC optics possessing still higher extinction factors, for example, following the introduction of polarizing rectifiers. The total division time (3-4 h) and the time required for anaphase (25-35 mm ) were comparable to the times observed with the agar overlay technique (Mole-Bajer, & Bajer, 1968). Only uncompressed cells were examined; therefore, the 3-dimensional relationships of cellular components were as natural as possible. It appears that the 514-nm illumination used in this study had no deleterious effect on the progress of mitosis and cytokinesis (cf. Hard et al. 1975). It, therefore, seems reasonable to conclude that the anaphase behaviour described above is as close to 'normal' as is experimentally possible at this time. Image interpretation The Nomarski differential interference contrast microscope (DIC) produces directional contrast delineating refractive interfaces in cells (Allen et al ). Despite a number of studies on Haemanthus endosperm cells with light and electron microscopy, it is not yet possible to interpret all of the structures observed. The filamentous substructure probably corresponds to groups of up to half a dozen unresolved microtubules (Bajer & Allen, 1966; Bajer & Jensen, 1969). There is no explanation why the kinetochore fibres should have a filamentous substructure that is not seen in continuous and/or interzonal fibres, unless the latter are more closely packed. Similarly, it is not known what gives rise to the apparent globular substructure seen only in the filaments of the kinetochore fibres. Although electron micrographs provide no clue as to the nature or identity of the globular substructure, its reality and potential importance are indicated by the fact that the substructure begins in prometaphase to move at anaphase velocity from the kinetochore toward the poles. The movement continues during prometaphase, metaphase, and anaphase (Allen et al a). It is apparent, therefore, that present specimen preparation techniques

8 54 R- Hard and R. D. Allen for electron microscopy have not preserved all of the important ultrastructural components that play a part in the function of the mitotic spindle. Relevance of filament obliquity to models of chromosome movement The simple observations of the behaviour of kinetochore filaments have direct bearing upon some of the mechanisms that have been proposed for the movements of chromosomes. A fundamental assumption upon which much of the evidence supporting the dynamic equilibrium model of spindle formation and activity rests is that the birefringence of the spindle is a reliable indicator of the amount of polymerized tubulin present (Inoue", 1964; Inoue" & Sato, 1967; Sato, Ellis & Inoue", 1975). While this is very likely correct in those metaphase spindles in which the microtubules are known to be parallel, it is obviously not correct when the birefringent structures'are known to be not parallel. In that case, they cross one another at various angles during anaphase, partially cancelling their respective retardations due to birefringence. It is, therefore, clear from the present results that measured changes in apparent retardation during mitosis must be interpreted with great caution as they may not be a true reflexion of the amount of birefringent material present. In the context of the present results it has become easier to interpret the apparent gradient of birefringence from the kinetochore toward the pole of the metaphase spindle and progressive decreases of birefringence at anaphase as changes in orientation as well as a reflexion of possible changes in the number of microtubules (and perhaps other filamentous structures) present. With regard to the parallel sliding microtubule model of Mclntosh, Hepler & Van Wie (1969), it is now apparent that the fact that microtubules of the anaphase spindle lose their initial parallel orientation as the chromosomes move renders the parallel sliding mechanism for chromosome movement untenable, at least for Haemanthus. The only region of the kinetochore fibres in which the filaments remain parallel is the inner region of the fibres, very close to the kinetochore. This region would appear to be the most likely to explore for possible sliding interactions. However, Jensen & Bajer (1973) have shown that during anaphase up to half of the microtubules ' disappear' from the bundle of microtubules that constitutes the kinetochore fibre. It is not possible to discuss the behaviour of kinetochore fibres in relation to the possible role of contractile proteins suggested by many authors (Behnke, Forer & Emmersen, 1971; Gawadi, 1971; Forer & Behnke, 1972; Forer, 1974; Forer & Jackson, 1976; Sanger, 1975; see also LaFountain, 1975) since evidence for a functional involvement of these contractile proteins in chromosome movement does not exist at present. Contractility of the whole kinetochore fibre appears to be ruled out by the uniformity of the velocity of the movement of the 'particles or states' along the fibre. That is, if the 'particles or states' are considered to be 'moving markers' on a contracting structure, one would predict more rapid migration near the kinetochore end of the fibre. If a contractile system is assumed to play a role in mitosis, its relationship to the microtubular portion of the spindle must be more

9 Anaphase behaviour of kinetochore fibres 55 subtle. A possible role of actin (and perhaps myosin) should be looked for in the reorientation of microtubules in the spindle. It was Bajer & Mole-Bajer (1972) who first pointed out selected examples of microtubular obliquity during anaphase and suggested a formal model for mitotic movements based on a spreading cohesive lateral interaction (or 'zipper action') of microtubules (see also Bajer, 1973). This model is certainly consistent with the observed behaviour of kinetochore fibres during anaphase, but it remains to be shown whether the force produced by lateral interactions can move chromosomes. The recent experiments of Bajer, Mole- Bajer & Lambert (1975) suggest strongly that this might be the case, as they have shown that anaphase can be stopped and restarted under conditions that reversibly alter the normal pattern of lateral associations. 'Zipping' of microtubules is not a radical concept, as microtubules would have to interact laterally in order to slide. The sliding of flagellar doublet microtubules so elegantly demonstrated by Summers & Gibbons (1971), is attributable to the action of regularly spaced dynein molecules, which serve as mechanochemical transducers. The presence of dynein or a similar transducer molecule in association with microtubules of the spindle has been suspected, but not demonstrated so far. Until the molecular interactions of tubulin with associated proteins are better understood, the fact that microtubules interact laterally and reorient in some large spindles should be accepted as part of the mitotic process in those cells. The present evidence offilamentreorientation during anaphase in Haemanthus spindles is certainly consistent with Bajer's zipping model. However, it might also be consistent with other models yet to be proposed which incorporate the observed reorientation of microtubules in kinetochore fibres. This work was begun in part at SUNY-Albany and completed at Dartmouth College under NIH research grants GM and GM We thank Robert Speck and William Ruiter for photographic assistance, and Ryland Loos for artwork. REFERENCES ALLEN, R. D., BAJER, A. & LAFOUNTAIN, J. R. (1969a). Poleward migration of particles or states in spindle fibre filaments during mitosis in Haemanthus. J. Cell Biol. 43, 4a. ALLEN, R. D., DAVID, G. B. & NOMARSKI, G. (19696). The Zeiss-Nomarski differential interference equipment for transmitted light microscopy. Z. wiss. Mikrosk. 69, BAJER, A. (1968). Behaviour and fine structure of spindle fibres during mitosis in endosperm. Chromosoma 25, BAJER, A. (1973). Interaction of microtubules and the mechanism of chromosome movement (zipper hypothesis). I. General principle. Cytobios 8, BAJER, A. & ALLEN, R. D. (1966). Structure and organization of the living mitotic spindle of Haemanthus endosperm. Science, N.Y. 151, BAJER, A. & JENSEN, C. (1969). Detectability of mitotic spindle microtubules with the light and electron microscopes. J. Microscopic 8, BAJER, A. & MOLE-BAJER, J. (1972). Spindle dynamics and chromosome movements. Int. Rev. Cytol. 34, Suppl. 3, BAJER, A. S., MOLE-BAJER, J. & LAMBERT, A. M. (1975). Lateral interactions of microtubules and chromosome movements. In Microtubules and Microtubule Inhibitors (ed. M. Borgers & M. DeBrabandes), pp Amsterdam: North-Holland Publishing.

10 56 R. Hard and R. D. Allen BEHNKE, O., FORER, A. & EMMERSEN, J. (1971). Actin in sperm tails and meiotic spindles. Nature, Lond. 234, FORER, A. (1974). Possible roles of microtubules and actin-like filaments during cell division. In Cell Cycle Controls (ed. G. M. Padilla, I. L. Cameron & A. M. Zimmerman), pp New York: Academic Press. FORER, A. & BEHNKE, O. (1972). An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew). I. The spindle. Chromosoma 39, FORER, A. & JACKSON, W. (1976). Actin filaments in the endosperm mitotic spindles in a higher plant, Haemanthus Katherinae Baker. Cytobiologie 12, GAWADI, N. (1971). Actin in the mitotic spindle. Nature, Lond. 24, 410. HARD, R., ZEH, R. & ALLEN, R. D. (1976). Phase-randomized laser illumination for microscopy. J. Cell Sci. 23, INOUE, S. (1964). Organization and function of the mitotic spindle. In Primitive Motile Systems in Cell Biology (ed. R. D. Allen & N. Kamiya), pp New York: Academic Press. INOUE, S. & SATO, H. (1967). Cell motility by labile association of molecules. The nature of mitotic spindle fibres and their role in chromosome movement. J. gen. Physiol. 50, JENSEN, C. & BAJER, A. (1969). Effects of dehydration on the microtubules of the mitotic spindle. Studies in vitro and with the electron microscope. J. Ultrastruct. Res. 26, JENSEN, C. & BAJER, A. (1973). Spindle dynamics and arrangement of microtubules. Chromosoma 44, LAFOUNTAIN, J. R. (1975). What moves chromosomes, microtubules or microfilaments? BioSystems 7, MCINTOSH, J. R., HEPLER, P. K. & VAN WIE, D. G. (1969). Model for mitosis. Nature, Lond. 224, METUZALS, J. & IZZARD, C. S. (1969). Spatial patterns of thread-like elements in the axoplasm of the giant nerve fibre of the squid (Loligo pealii L.) as disclosed by differential interference microscopy and electron microscopy. J. Cell Biol. 43, MOLE-BAJER, J. & BAJER, A. (1968). Studies of selected endosperm cells with the light and electron microscope. The technique. Cellule 67, SANGER, J. W. (1975). Presence of actin during chromosomal movement. Proc. natn. Acad. Sci. U.S.A. 72, SATO, H., ELLIS, G. W. & INOUE, S. (1975). Microtubular origin of mitotic spindle form birefringence: demonstration of the applicability of Wiener's equation. J. Cell Biol. 67, SUMMERS, K. E. & GIBBONS, I. R. (1971). Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea urchin sperm. Proc. natn. Acad. Sci. U.S.A. 68, (Received 26 October Revised 12 April 1977)