Sucrose-induced spindle elongation in mitotic PtK-1 cells

Brief treatment of mitotic metaphase and anaphase PtK-1 cells with tissue culture medium containing 0.5 M sucrose resulted in spindle elongation without chromosome motion. Spindle birefringence also changed from a uniform appearance to one of highly birefringent bundles. Electron microscopic analysis indicated these birefringent bundles were composed of tightly packed arrays of spindle microtubules. No kinetochores could be seen following a 10 min sucrose treatment. Upon removal of sucrose, metaphase spindles returned to pretreatment lengths and the normal birefringence pattern returned. Reduction in spindle length could be temporally coupled with the reappearance of kinetochores and the reassociation of microtubules with these structures. In contrast to treated and released metaphase cells, anaphase spindles did not return to pretreatment lengths. Replacement of sucrose with medium showed the resumption of chromosome-to-pole motion within 2 min of sucrose removal. Chromosome motion could be correlated with the reappearance of kinetochores and kinetochore microtubules. These data have led us to postulate the existence of two microtubule continuums in the spindle and to discuss their roles in spindle organization and chromosome motion.     

Structural implications of kinetochore function in sucrose-treated PtK1 cells

Treatment of PtK1 cells during metaphase with solutions containing hyperosmotic concentrations of sucrose resulted in an alteration of kinetochore structure and function in a concentration-dependent manner. This alteration in kinetochore morphology was shown to be rapidly reversible upon removal of the sucrose-containing tissue culture medium. A 10-min treatment with both 0.2 M and 0.4 M sucrose resulted in a concentration-dependent aggregation of spindle fibers into bundles, loss of trilaminar kinetochore morphology as judged by electron microscopy, and induction of anaphase B-like spindle elongation as previously described. Electron microscopy showed that a 10-min treatment of metaphase cells with hyperosmotic concentrations of sucrose changed the trilaminar kinetochore structure to one of a single lamina, with an amorphous, lightly staining material distally associated with it. Sucrose-induced bundles of microtubules could usually be seen embedded or tangentially associated with this material. Rate and extent of spindle elongation in sucrose-treated metaphase cells were greater in the higher concentrations of sucrose employed. The degree of microtubule bundling was also concentration dependent, with reduced bundling occurring at lower sucrose concentrations. Within 2 min after sucrose removal kinetochores returned to a bi- or trilaminar morphology with reduction in the amount of amorphous material. Reformation of the kinetochore trilaminar structure resembled that of the normal maturation process which occurs from prophase through anaphase. These rapid changes in kinetochore morphology following release from sucrose treatment were temporally associated with restoration of spindle function and suggested that kinetochore integrity was necessary for the expression of spindle forces responsible for spindle shortening. These forces are probably generated or transduced by the continuum formed between the two spindle poles, the kinetochore microtubules, and the sister chromatids.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Role of non-kinetochore microtubules in spindle elongation in mitotic PtK1 cells

PtK1 metaphase cells were treated with varying concentrations of nocodazole to reduce spindle microtubule number and spindle length. The range of concentrations employed reduced spindle length from approximately 47% to 82% of the original pole-pole distance. Electron microscopy of cells treated with the lowest concentration of nocodazole employed (0.01 microgram/ml) showed a small decrease in the number of non-kinetochore microtubules (nkMTs), particularly evident in the astral region, with no significant effect on kinetochore microtubule number. Metaphase cells treated with 1 microgram/ml nocodazole for 2 min demonstrated a reduction in spindle length and loss of most non-kinetochore microtubules with little effect on the number and arrangement of the kinetochore class of microtubules. Following nocodazole treatment, the cells were perfused with 0.5 M sucrose dissolved in tissue culture medium, a treatment which has previously been shown to induce spindle elongation in metaphase cells. In cells where nocodazole effected a large decrease in non-kinetochore microtubule number with a concomitant decrease in spindle length, sucrose treatment had a reduced effect in inducing spindle elongation. In cells treated with lower concentrations of nocodazole, where numerous non-kinetochore microtubules remained, sucrose had a greater effect in inducing spindle elongation. These data suggest that the non-kinetochore population of microtubules is responsible for the extent of sucrose-induced spindle elongation. An explanation of these data is provided which suggests that the role of non-kinetochore microtubules is to trap energy in the developing spindle, such that it can be used to separate spindle poles during anaphase B.     

Asynchronous entry into anaphase induced by okadaic acid: spindle microtubule organization and microtubule/kinetochore attachments

Summary We have found that a brief treatment of either PtK₂ cells or stamen hair cells ofTradescantia virginiana during metaphase with okadaic acid, a potent protein phosphatase inhibitor, results in asynchronous entry into anaphase. After this treatment, the interval for the separation of sister chromatids can be expanded from a few seconds to approximately 5 min. We have performed a series of immunolocalizations of cells with anti-tubulin antibodies and CREST serum, asking whether okadaic acid induces asynchronous entry into anaphase through changes in the organization of the spindle microtubules or through a loss in the attachment of spindle microtubules to the kinetochores. Our experiments clearly indicate that asynchronous entry into anaphase after phosphatase inhibitor treatment is not the result of either altered spindle microtubule organization or the long-term loss of microtubule attachment to kinetochores. The kinetochore fiber bundles for all of the separating chromosomes are normally of uniform length throughout anaphase, but after asynchronous entry into anaphase, different groups of kinetochore fiber bundles have distinctly different lengths. The reason for this difference in length is that once split apart, the daughter chromosomes begin their movement toward the spindle poles, with normal shortening of the kinetochore fiber bundle microtubules. Thus, okadaic acid treatment during metaphase does not affect anaphase chromosome movement once it has begun. Our results suggest that one or more protein phosphatases appear to play an important role during metaphase in the regulatory cascade that culminates in synchronous sister chromatid separation.     

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.     

Effect of metabolic inhibitors on sucrose-induced metaphase spindle elongation and spindle recovery

Hyperosmotic sucrose treatment of metaphase PtK-1 cells has been shown to produce a reversible concentration-dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366-372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62-69, 1984 and 39:373-379, 1985). Addition of metabolic inhibitors to Ham's F-12 salts with deoxyglucose (D/F-12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose-induced spindle elongation; rates and extents are 60-75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F-12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose-treated cells released into D/F-12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP-containing D/F-12 medium and released into DNP-containing D/F-12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose-induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.     

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.     

Cytokinesis in cells undergoing mitosis without genome replication

Chinese hamster ovary cells can be forced to enter mitosis without prior DNA replication by treatment with hydroxyurea and caffeine. Cells treated in this way assemble a spindle that functions normally except that it does not accomplish anaphase spindle elongation (anaphase B). The chromatin detaches from the unreplicated kinetochores, which fragment, but establish microtubule attachments and migrate to the metaphase plate. Partitioning of the kinetochore fragments ensues on the normal schedule. Typical midbodies and cleavage furrows are established and daughter cells of equal size are produced. These results imply that intact chromosomes are not necessary for correct cleavage furrow placement but that kinetochores might be. Further, it is clear that cleavage furrow placement does not depend on anaphase spindle elongation.     

Merotelic kinetochores in mammalian tissue cells

Merotelic kinetochore attachment is a major source of aneuploidy in mammalian tissue cells in culture. Mammalian kinetochores typically have binding sites for about 20-25 kinetochore microtubules. In prometaphase, kinetochores become merotelic if they attach to microtubules from opposite poles rather than to just one pole as normally occurs. Merotelic attachments support chromosome bi-orientation and alignment near the metaphase plate and they are not detected by the mitotic spindle checkpoint. At anaphase onset, sister chromatids separate, but a chromatid with a merotelic kinetochore may not be segregated correctly, and may lag near the spindle equator because of pulling forces toward opposite poles, or move in the direction of the wrong pole. Correction mechanisms are important for preventing segregation errors. There are probably more than 100 times as many PtK1 tissue cells with merotelic kinetochores in early mitosis, and about 16 times as many entering anaphase as the 1% of cells with lagging chromosomes seen in late anaphase. The role of spindle mechanics and potential functions of the Ndc80/Nuf2 protein complex at the kinetochore/microtubule interface is discussed for two correction mechanisms: one that functions before anaphase to reduce the number of kinetochore microtubules to the wrong pole, and one that functions after anaphase onset to move merotelic kinetochores based on the ratio of kinetochore microtubules to the correct versus incorrect pole.     

Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 microM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2--5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.     

M phase-specific kinetochore proteins in fission yeast: microtubule-associating Dis1 and Mtc1 display rapid separation and segregation during anaphase

BACKGROUND: Kinetochore microtubules are made early in mitosis and link chromosomal kinetochores to the spindle poles. They are required later to move the separated sister chromatids toward the opposite poles upon the onset of anaphase. Very little is known about proteins that are responsible for the connection between kinetochores and mitotic microtubules. RESULTS: We here show that fission yeast Dis1 and the related protein Mtc1/Alp14 are both able to bind microtubules in vitro and share an essential function for viability in vivo. The deletion of mtc1+ results in an instability of cytoplasmic microtubules that can be suppressed by the ectopic expression of dis1+. Dis1 and Mtc1 are localized along interphase cytoplasmic microtubules and are mobilized onto the spindle upon mitotic commitment. In chromatin immunoprecipitation (CHIP) experiments Dis1 coprecipitated with the central centromeric DNA in an M phase-specific manner. Consistently, observations of both living cells in which the native, genomic copy of dis1+ tagged with GFP and cells fixed by immunostaining established that Dis1 behaves as a kinetochore protein during the progression from metaphase to anaphase. The central and C-terminal regions of Dis1 are sufficient for interactions with microtubules and the kinetochore, respectively. In anaphase, the GFP signals of both Dis1 and Mtc1 suddenly separate and move quickly toward opposite spindle poles. CONCLUSIONS: Fission yeast Dis1 and Mtc1 are members of an evolutionarily conserved microtubule binding protein family that includes frog XMAP215. Dis1 and Mtc1 are implicated in stabilizing kinetochore microtubules in metaphase and so counteract the action of microtubule destabilizing factors that dominate in anaphase. Dis1 may play a dual role by becoming a part of the kinetochores in an M phase-specific manner, and it may possibly generate connections between kinetochores and microtubules.     

Bi-orienting chromosomes: acrobatics on the mitotic spindle

To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to bi-orientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.     

EB1 targets to kinetochores with attached,polymerizing microtubules

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end-kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.     

Origin of kinetochore microtubules in Chinese hamster ovary cells

We have attempted to determine whether chromosomal microtubules arise by kinetochore nucleation or by attachment of pre-existing microtubules. The appearance of new microtubules was investigated in vivo on kinetochores to which microtubules had not previously been attached. The mitotic apparatus of Chinese hamster ovary cells was reconstructed in three dimensions from 0.25 m thick serial sections, and the location of chromosomes, kinetochore outer disks, centrioles, virus-like particles and microtubules determined. Central to the interpretation of these data is a synchronization scheme in which cells entered Colcemid arrest without forming mitotic microtubules. Cells were synchronized by the excess thymidine method and exposed to 0.3 g/ml Colcemid for 8 h. Electron microscopic examination showed that this Colcemid concentration eliminated all microtubules. Mitotic cells were collected by shaking off, and cell counts showed that over 95% of the cells were in interphase when treatment began and thus were arrested without the kinetochores having been previously attached to microtubules. Cells were then incubated in fresh medium and fixed for high voltage electron microscopy at intervals during recovery. — In early stages of recovery, short microtubules were observed near and in contact with kinetochores and surrounding centrioles. Microtubules were associated with kinetochores facing away from centrosomes and far from any centrosomal microtubules, and thus were not of centrosomal origin. At a later stage of recovery, long parallel bundles of microtubules, terminating in the kinetochore outer disk, extended from kinetochores both toward and away from centrosomes. Because microtubules had never been attached to kinetochores, the possibility that kinetochore microtubles were initiated by microtubule stubs resistant to Colcemid was eliminated. Therefore we conclude that mammalian kinetochores can initiate microtubules in vivo, thus serving as microtubule organizing centers for the mitotic spindle, and that formation of kinetochore-microtubule bundles is not dependent on centrosomal activity.     

Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes

Summary. We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the “kinetochore stubs”, the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.