COMMENTARY Do anaphase chromosomes chew their way to the pole or are they pulled by actin?

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1 COMMENTARY Do anaphase chromosomes chew their way to the pole or are they pulled by actin? ARTHUR FORER Biology' Department, York University, Dotvnsvietu, Ontario, M3J 1P3, Canada Introduction In a current model of anaphase chromosome movement it is envisaged that depolymerization of microtubules at the kinetochore produces the force to move a chromosome to the pole. The chromosome itself generates the force for chromosome movement by 'chewing' its way to the pole along stationary microtubules, behaving as does 'PacMan' in the video game of the same name (reviews by Cassimeris et al. 1987; Nicklas, 1988; Nicklas, 1988a; Wolniak, 1988). [This model is a re-statement of a model presented much earlier (Gruzdev, 1972).] I describe herein data that are not consistent with the PacMan model and present an alternative view on how chromosomes move to the poles during anaphase. I first look at the evidence for the model. Evidence for the model The PacMan model derives from three kinds of experiment, all of which were interpreted as evidence that the chromosomes chew their way to the pole along stationary kinetochore microtubules. FRAP (Fluorescence Redistribution After Photobleachirig) experiments Fluorescently labelled tubulin was injected into dividing cells, a local region of the spindle between chromosomes and poles was photobleached, and the bleached region either rapidly regained fluorescence, while remaining stationary and not moving polewards (Salmon et al. 1984; Wadsworth & Salmon, 1986), or remained visible, without gaining fluorescence, as the chromosomes moved into and past the bleached region (Gorbsky et al. 1987, 1988). Biotinylated tubulin experiments Biotinylated tubulin was injected into living cells and the incorporation of tubulin monomers into kinetochore microtubules was followed by immunoelectron microscopy. Tubulin monomers appeared at the kinetochore ends of kinetochore microtubules during meta- Journnl of Cell Science 91, (1988) Printed in Great Britain The Company of Biologists Limited 1988 phase, but the same microtubules lost their labelled subunits during anaphase (Mitchison et al. 1986). Experiments on miovtubules in vitro Microtubules polymerized in vitro from microtubuleassociated protein (MAP)-free tubulin were captured by isolated chromosomes, and under appropriate conditions the captured microtubules shortened. Those captured with the same structural polarities as found in spindles;'/; vivo shortened from the kinetochore ends, as determined from microtubules labelled differently in different portions of the microtubule (Koshland et al. 1988). None of these interpretations is without flaws. With respect to the FRAP experiments, the bleached region of the spindle was not stationary in sea-urchin zygotes (Hamaguchi et al. 1987), in either metaphase or anaphase. Further, the tubulin that was bleached was not shown to be in kinetochore microtubules - it might have been primarily in non-kinetochore microtubules or in oligomeric tubulin. Until one is certain that the results pertain to kinetochore microtubules one does not know if the results from the FRAP experiments are relevant to chromosome-to-pole motion. With respect to the experiments using biotinylated tubulin, the interpretation of events in anaphase is not secure. There was variability in that after injection of labelled tubulin in metaphase some cells in anaphase still had label associated with kinetochore microtubules, and some injections in anaphase resulted in incorporation of labelled tubulin at the kinetochore. Serial-section analysis was not performed, so there is uncertainty about which microtubules were or were not labelled. Anaphase in the cells studied consists primarily of spindle elongation - chromosome-to-pole motion is a very minor component of that particular anaphase, having been described as being completed in s (Mitchison et al. 1986) - so the results, even if completely accurate, may not be relevant to the movement of chromosomes to the poles. With respect to the experiments on in vitro microtubules, there was indeed depolymerization of microtubules at their kinetochore ends for that set of microtubules studied, but it is not at all clear that the shortening in vitro of uncapped, MAP-free microtubules 449

2 is relevant to the shortening in vivo of microtubules which are capped at both ends and associated with MAPs. Even if all the experiments discussed above unambiguously showed that kinetochore microtubules shortened at the kinetochore in vivo as the chromosome moved to the pole, there is no evidence that this has anything to do with producing the force to move that chromosome to the pole of a spindle. I conclude that the evidence that suggests that kinetochore microtubules shorten at the kinetochore during anaphase chromosome-to-pole motion is only suggestive, and therefore that this interpretation of where kinetochore microtubules depolymerize during anaphase cannot be accepted without reservation. For purposes of discussion I will assume that kinetochore microtubules do depolymerize at the kinetochore in anaphase; but there is no evidence that this depolymerization of microtubules produces the force that moves a chromosome to the pole. c c CO B 8 " c<u 00 C C - Birel 1 K Distance along fibre K, 1 ; Distance along fibre \ \ P P Data that the PacMan model Is inconsistent with There are a variety of data with which the PacMan model is inconsistent. I summarize some of those that are particularly persuasive to me: (1) The birefringence along the length of an individual chromosomal spindle fibre is constant from the kinetochore half-way to the pole and then it decreases linearly from there to the pole (Forer, 1976; Salmon & Begg, 1980; Schaap & Forer, 1984a,b), as illustrated in Fig. 1A. If the microtubules between chromosome and pole remain static in anaphase as they are depolymerized at the chromosomal end, then, if the birefringence is due to microtubules (Inoue\ 1981), the PacMan model predicts that (a) as the chromosome chews its way along the microtubules the length of the region of constant birefringence should get shorter and shorter, and that (b) after the chromosome reaches the region of the fibre in which the birefringence linearly declines the birefringence at the kinetochore should get smaller and smaller as the chromosome chews its way closer and closer to the pole, as illustrated in Fig. IB. The actual events indeed satisfy the prediction up to the point at which the chromosomal spindle fibre birefringence decreases linearly to the pole; after that the predicted changes do not occur: the birefringence at the kinetochore remains constant rather than decreasing (fig. 4 of Forer (1976); fig. 5 of Salmon & Begg (1980); C. J. Schaap & A. Forer, unpublished results), as illustrated in Fig. 1C. What actually occurs in mid- to late-anaphase, then, is not consistent with the basic postulate of the PacMan model that chromosome-to-pole microtubules are depolymerized in anaphase at their kinetochore ends. (2) The force on a chromosome is proportional to the length of the chromosomal spindle fibre to which that chromosome is attached (e.g. see Wise, 1978; Hays et al. 1982; Nicklas, 19886). This is inconsistent with the PacMan model: the length of the fibre would not determine the amount of force if the driving force for motion were depolymerization of microtubules at the kinetochore. CQ Distance along fibre Fig. 1. Birefringence of an individual chromosomal spindle fibre (ordinate) during metaphase measured at different positions along the length of the fibre (abscissa), and the changes in this pattern of birefringence during anaphase as predicted by the PacMan model (B) and as actually measured (C). P, position of the pole; K, position of the kinetochore in metaphase; Kj and Kz, positions of the kinetochores at successively later stages of anaphase. Based on data from Forer (1976), Salmon & Begg (1980) and Schaap & Forer (1984a, b). (3) Various ultraviolet (u.v.) microbeam experiments are also inconsistent with the PacMan model. In order to explain the implications of these data I first summarize some of the basic results. u.v. microbeam irradiation of a single chromosomal spindle fibre can produce an area of reduced birefringence (ARB) at the site of the irradiation; the ARB is a region in which there is local depolymerization (absence) of microtubules and thus the ARB is a discontinuity in the kinetochore microtubules (Wilson & Forer, 1988). u.v. microbeam irradiation of a single chromosomal spindle fibre can also block (temporarily) the movement of the associated chromosome, but the blocking of movement is completely independent of effects on spindle fibre birefringence (Forer, 1966; Sillers, 1983): the wavelength sensitivities for producing ARBs are different from those for blocking chromosome movement (Fig. 2). Thus, by controlling wavelength and dose one can predictably produce an ARB with no effect on chromosome movement, or can block chromosome movement with no effect on spindle fibre birefringence, or simultaneously can get an ARB and block chromosome movement (Sillers & Forer, 1983). These experiments are relevant to the PacMan model as follows. Chromosome movement can be blocked by irradiations 450 A. Forer

3 0-6' 0-5-? nm Fig. 2. The energy per area (ordinate) necessary to produce either an ARB ( + ) or block chromosome movement (closed circles) at various wavelengths (abscissa), after individual chromosomal spindle fibres in crane fly spermatocytes were irradiated with a u.v. microbeam. Based on data in Sillers & Forer (1983) and Hughes et al. (1988). along the length of the chromosomal spindle fibre (e.g. Forer, 1969); this should not occur were the kinetochore the site of force production. One can produce ARBs that are immediately adjacent to the kinetochore, yet chromosome movement is normal (e.g. Forer, 1966): this should not occur were the PacMan model correct, for irradiations that destroy all microtubules at the kinetochore should block chromosome movement. Consider a cell in which an ARB is produced several micrometres from the kinetochore and chromosome movement is blocked: the ARB immediately moves poleward (while the chromosome does not), and the chromosome resumes movement before the ARB reaches the pole (e.g. Forer, 1966). In this experiment not only is movement blocked by irradiation at a site distant from the chromosome, but the poleward motion of the ARB also shows that nothing is wrong with the kinetochore microtubules - indeed, the resumption of chromosome motion before the ARB reaches the pole shows that there is no need to 'repair' the microtubules in the irradiated region in order for chromosome movement to resume. All of these observations suggest that the force for movement does not arise at the kinetochore-spindle fibre junction, and thus these observations are inconsistent with the PacMan model. (4) Observations on metaphase are inconsistent with the PacMan model. Metaphase chromosomes are subject to forces to both poles, as indicated by u.v. microbeam irradiation of spindle fibres (Sillers et al. 1983) or laser microbeam ablation of kinetochores (McNeill & Berns, 1981; Rieder et al. 1986). Oppositely directed poleward forces in metaphase can not be due to depolymerization at the kinetochore, as required by the PacMan model, because in metaphase there is incorporation of tubulin at the kinetochore, as indicated by localized release from inhibition by colcemid (Czaban & Forer, 1985a,6), by incorporation of biotinylated tubulin (Mitchison et al. Fig. 3. A model for chromosome movement in which the microtubules (straight lines) are separate from the forceproducing component(s) of the spindle fibre (zig-zag lines), and in which both components are attached to the kinetochore (bottom, dark) and extend along the length of the spindle fibre. 1986), and by poleward movement of a bleached region in spindles labelled with fluorescent tubulin (Hamaguchi et al. 1987). Unless one argues that force production at metaphase is by a mechanism completely different from that responsible for force production at anaphase, these observations are inconsistent with the PacMan model. (5) After an anaphase half-bivalent is pushed polewards using micromanipulation techniques, the chromosome sits and does not move until the other chromosomes nearly catch up to it, as if it had to wait for the pushed spindle fibre to be shortened (fig. 3 of Nicklas & Staehly, 1967). Were the force for chromosome movement to be produced by depolymerization at the kinetochore, the chromosome would continue to move as the fibre shortened. Thus this observation is not consistent with the PacMan model. These are persuasive reasons, to me, for rejecting the PacMan model. There is a simple alternative. An alternative model for force production during anaphase I propose that something other than microtubules produces the force to move a chromosome to the pole (Fig. 3). The role of the kinetochore microtubules is not specified, but microtubules might act as "governor" (Forer, 1974; Nicklas, 1975). The force-producing component is present along the length of the fibre, so irradiations along the fibre could interfere with the forceproducing component at any position. Length could matter because the force-producing component is distributed along the length of the fibre. And the model is independent of wherever kinetochore microtubules depolymerize in anaphase. What might the force-producing component(s) be? Cellular motile systems are based either on microtubules or on actin filaments (e.g. Forer, 1978), so if microtubules do not produce the force for chromosome motion one would expect that actin filaments do. There is indeed evidence implicating actin filaments. First, there are no solid data that rule out actin or myosin as producing the force to move a chromosome to the pole in anaphase (reviewed by Forer, 1985). Second, actin is present in chromosomal spindle fibres, with consistent structural Anaphase chivmosome movement 451

4 0-6H nm Fig. 4. The energy per area (ordinate) necessary to block chromosome movement (closed circles) at various wavelengths (abscissa) after individual chromosomal spindle fibres were irradiated with a u.v. microbeam, in comparison with the energies per area necessary to block myofibril contraction after u.v. microbeam irradiation of the A band (open circle) or I band (open square). Data on chromosome movement are from Sillers & Forer (1983) and on myofibrils from Wilson & Forer (1987). polarity (Forer et al. 1979; reviewed by Forer, 1985; additional evidence from staining with fluorescent phalloidin, see Seagull et al. 1987; Traas et al. 1987; Sheldon & Hawes, 1988). Third, actin is implicated experimentally, as follows. When chromosomal spindle fibres are irradiated with a u.v. microbeam the peak sensitivities for blocking chromosome movement are at wavelengths of 270 nm and 290 nm (Fig. 2); these match the peak sensitivities for blocking contraction of myofibrils but are quite different from those for blocking ciliary beating (Sillers & Forer, 1981). The peaks at 270 nm and 290 nm match absorption peaks of actin (270 nm) and heavy meromyosin (290 nm) but are quite different from the absorption peaks of tubulin or microtubules (Forer, 1985). Carrying further the comparisons of spindle fibres with muscle proteins, the energies per area required to block chromosome movement when a spindle fibre is irradiated with u.v. light of wavelength 270nm or 290 nm match exactly those needed to block myofibril contraction by irradiating the actin filaments in the I band with u.v. light of wavelength 270 nm or the A band with u.v. light of wavelength 290 nm (Wilson & Forer, 1987), as illustrated in Fig. 4. All these comparisons show a remarkable coincidence between effects on chromosome movement and effects on muscle proteins. Considering effects on birefringence, however, the peak sensitivities for producing ARBs in vivo, 260 nm and 280 nm (Fig. 2), and the doses, are the same as those for production of ARBs on microtubules in vittv (Hughes et al. 1988). Hence the effects on birefringence are indeed directly on chromosomal spindle fibre microtubules (Hughes et al. 1988); since these are independent of effects on force production (Fig. 2), this strongly argues that microtubules do not produce the force that moves the chromosome to the pole. I conclude that these data are consistent with and provide strong support for the suggestion that actin filaments are involved in force production; they are difficult to explain on any hypothesis other than that the microtubules do not produce the force for chromosome movement. While the arguments for my view of force production are not airtight, the model is testable. One test is to see if there is a correlation between local depolymerization of the putative non-microtubule force producer - actin filaments - and blocking of chromosome movement, the kind of correlation that does not exist between production of ARBs (depolymerization of microtubules) and blocking of chromosome movement. A second test is to see if agents, such as phalloidin, that selectively stabilize actin filaments will protect the chromosomal spindle fibre against u.v. microbeam irradiation, that otherwise would have blocked chromosome movement. Agents, such as taxol, that selectively stabilize microtubules, however, might protect the chromosomal spindle fibre against production of ARBs. These are the kinds of experiments that we will be doing to test this alternative view of anaphase chromosome movement. In sum, the evidence used to support the PacMan model is not compelling. I described evidence that persuades me that the site of force production is not at the kinetochore but along the chromosomal spindle fibre. In my view, even if it is substantiated that the kinetochore microtubules depolymerize at the kinetochores as the chromosomes move poleward, this does not mean that the depolymerization generates the force that moves the chromosome - microtubule depolymerization may only be the governor, the rate-limiting step that controls the velocity of the chromosomes. I argue that the force that causes the chromosome to move most likely arises from a non-microtubule component of the spindle fibre, probably actin. I thank Paula Wilson for a tough, critical reading of the manuscript. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. References CASSIMERIS, L. U., WALKER, R. A., PRYER, N. K. & SALMON, E. D. (1987). Dynamic instability of microtubules. BioEssavs 7, CZABAN, B. B. & FORER, A. (1985a). The kinetic properties of spindle microtubules in vtvo in crane-fly spermatocytes. I. Kinetochore microtubules that re-form after treatment with colcemid.7. Cell Set. 79, CZABAN, B. B. & FORER, A. (19856). The kinetic properties of spindle microtubules in vtvo in crane-fly spermatocytes. II. Kinetochore microtubules in non-treated spindles. J. Cell Sci. 79, A. Forer

5 FORER, A. (1966). Characterization of the mitotic traction system, and evidence that birefnngent spindle fibers neither produce nor transmit force for chromosome movement. Chromosoma 19, FORER, A. (1969). Chromosome movements during cell-division. In Handbook of Molecular Cytology (ed. A. Lima-de-Faria), pp Amsterdam, London: North Holland Publishing. 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 NY, London: Academic Press. FORER, A. (1976). Actin filaments and birefnngent spindle fibers during chromosome movements. In Cell Motility, Book C, Microtubules and Related Proteins (ed. R. Goldman, T. Pollard & J. Rosenbaum), pp New York: Cold Spring Harbor Press. FORER, A. (1978). Chromosome movements during cell-division: possible involvement of actin filaments. In Nuclear Division in the Fungi (ed. I. B. Heath), pp New York, London: Academic Press. FORER, A. 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Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J. Cell Biol. 103, SALMON, E. D. & BEGG, D. A. (1980). Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase. J. Cell Biol. 85, SALMON, E. D., LESLIE, R. J., SAXTON, W. M., KAROW, M. L. & MCINTOSH, J. R. (1984). Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J. Cell Biol. 99, SCHAAP, C. J. & FORER, A. (1984a). Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. I. Description of the system and general results. J. Cell Sci. 65, SCHAAP, C. J. & FORER, A. (19846). Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. II. 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