Backward chromosome movement in anaphase, after irradiation of kinetochores or kinetochore fibres

Summary We used an ultraviolet microbeam to irradiate kinetochores, or to irradiate kinetochore fibres just in front of kinetochores, in anaphase crane-fly spermatocytes. Forward movements were blocked after most irradiations, but in 7 cells the associated anaphase half-bivalents moved backward, toward their partner half-bivalents, with speeds faster than poleward movements. The occurrence of backward movement suggests that there may be mechanical connections between separating half-bivalents. We have been unable to find conditions to obtain these results reproducibly.     

A unique tubulin antiserum attenuates the rate of poleward chromosome movement in anaphase.

An antiserum against tubulin, NS20, was previously shown to specifically attenuate both fast axonal transport in vivo (Johnston, K. M. et al., Brain Res. 385, 38-45 (1986)) and in vitro (Johnston, K. M. et al., Cell Motil. Cytoskel. 7, 110-115 (1987)) and flagellar motility (Goldsmith, M. et al., Cell Motil. Cytoskel. 20, 249-262 (1991)). We hypothesized that NS20 blocked motility by binding to a multifunctional motor binding domain on the microtubules (MTs), or axonemes. Here we have examined the effect of microinjecting NS20, at metaphase, into dividing PtK2 cells. Plotting chromosome separation (CS) as a function of time, we report here that CS rates for anaphase A (chromosome-to-pole movement) were reduced by approximately 50% relative to uninjected controls. CS rates for anaphase B (spindle pole elongation) were unaffected by the NS20 antiserum. The inhibition of CS rate during anaphase A by NS20 was significantly greater than the inhibition caused by a control antitubulin serum (PC5). Two possible mechanisms underlying NS20's inhibition of CS during anaphase A were considered. NS20 could block the binding of a kinetochore-associated motor to kinetochore MTs (kMTs) or, alternatively, NS20 could stabilize kMTs against depolymerization. Our results favor the first alternative. In a cold-induced depolymerization assay, NS20 had no selective stabilizing effect on MTs. Moreover, we show that NS20 can selectively block the binding of a well characterized MT-associated motor (kinesin) to MTs, in vitro. These results suggest that NS20 may be defining a unique tubulin binding domain common to the motors underlying vesicle transport, flagellar motility, and chromosome movements during anaphase A.     

The force for poleward chromosome motion in Haemanthus cells acts along the length of the chromosome during metaphase but only at the kinetochore during anaphase

The force for poleward chromosome motion during mitosis is thought to act, in all higher organisms, exclusively through the kinetochore. We have used time-lapse. video-enhanced, differential interference contrast light microscopy to determine the behavior of kinetochore-free "acentric" chromosome fragments and "monocentric" chromosomes containing one kinetochore, created at various stages of mitosis in living higher plant (Haemanthus) cells by laser microsurgery. Acentric fragments and monocentric chromosomes generated during spindle formation and metaphase both moved towards the closest spindle pole at a rate (approximately 1.0 microm/min) similar to the poleward motion of anaphase chromosomes. This poleward transport of chromosome fragments ceased near the onset of anaphase and was replaced. near midanaphase, by another force that now transported the fragments to the spindle equator at 1.5-2.0 microm/min. These fragments then remained near the spindle midzone until phragmoplast development, at which time they were again transported randomly poleward but now at approximately 3 microm/min. This behavior of acentric chromosome fragments on anastral plant spindles differs from that reported for the astral spindles of vertebrate cells, and demonstrates that in forming plant spindles, a force for poleward chromosome motion is generated independent of the kinetochore. The data further suggest that the three stages of non- kinetochore chromosome transport we observed are all mediated by the spindle microtubules. Finally, our findings reveal that there are fundamental differences between the transport properties of forming mitotic spindles in plants and vertebrates.     

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.     

Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit-shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10^?8 joules per μm² was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time-lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half-bivalent with the partner half-bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.     

Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.     

The role of myosin phosphorylation in anaphase chromosome movement

This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production.Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin.     

The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement

At anaphase, the linkage betweeh sister chromatids is dissolved and the separated sisters move toward opposite poles of the spindle. We developed a method to purify metaphase and anaphase chromosomes from frog egg extracts and identified proteins that leave chromosomes at anaphase using a new form of expression screening. This approach identified Xkid, a Xenopus homolog of human Kid (kinesin-like DNA binding protein) as a protein that is degraded in anaphase by ubiquitin-mediated proteolysis. Immunodepleting Xkid from egg extracts prevented normal chromosome alignment on the metaphase spindle. Adding a mild excess of wild-type or nondegradable Xkid to egg extracts prevented the separated chromosomes from moving toward the poles. We propose that Xkid provides the metaphase force that pushes chromosome arms toward the equator of the spindle and that its destruction is needed for anaphase chromosome movement.     

The kinetochore is part of the metaphase chromosome scaffold

We used antisera from patients with the CREST syndrome of scleroderma (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) to show that an antigenic component of the kinetochore present in metaphase chromosomes is also present in nonhistone chromosome scaffolds isolated following extensive digestion of the DNA and extraction of the bulk of chromosomal protein. All sera from 12 scleroderma CREST patients previously shown by immunofluorescence microscopy to have circulating antikinetochore antibodies recognise a protein of Mr 77,000 (CREST-77) in an immunoblotting assay. 9 of the 12 sera also recognise an antigen of Mr 110,000 (CREST-110). These proteins are present in isolated chromosomes and nonhistone scaffolds derived from them by two different procedures. Sera of five scleroderma CREST patients who are antikinetochore negative (by immunofluorescence) bind to neither protein in immunoblots. These data suggest that CREST-77 (and possibly CREST-110) is a component of the human kinetochore, and that the kinetochore is an integral part of the mitotic chromosome scaffolding.     

MCAK facilitates chromosome movement by promoting kinetochore microtubule turnover

Mitotic centromere-associated kinesin (MCAK)/Kif2C is the most potent microtubule (MT)-destabilizing enzyme identified thus far. However, MCAK's function at the centromere has remained mechanistically elusive because of interference from cytoplasmic MCAK's global regulation of MT dynamics. In this study, we present MCAK chimeras and mutants designed to target centromere-associated MCAK for mechanistic analysis. Live imaging reveals that depletion of centromere-associated MCAK considerably decreases the directional coordination between sister kinetochores. Sister centromere directional antagonism results in decreased movement speed and increased tension. Sister centromeres appear unable to detach from kinetochore MTs efficiently in response to directional switching cues during oscillatory movement. These effects are reversed by anchoring ectopic MCAK to the centromere. We propose that MCAK increases the turnover of kinetochore MTs at all centromeres to coordinate directional switching between sister centromeres and facilitate smooth translocation. This may contribute to error correction during chromosome segregation either directly via slow MT turnover or indirectly by mechanical release of MTs during facilitated movement.     

Pac-Man does not resolve the enduring problem of anaphase chromosome movement

Summary The Pac-Man hypothesis suggests that poleward movement of chromosomes during anaphase A is brought about by: disassembly of kinetochore microtubules (MTs) at the kinetochore; generation of the poleward force exclusively at or very close to the kinetochore; and the required energy coming from coupled disassembly of these MTs. This model has become widely accepted and cited as the sole or major mechanism of anaphase A. Rarely acknowledged are several significant phenomena that refute some or all of these postulates. We summarise these anomalies as follows: poleward movement of chromosomes occurring without insertion of any MTs at the kinetochore; anaphase shortening of kinetochore fibres in spindles entirely devoid of chromosomes and, presumably, kinetochores; continued movement of chromosomes while their severed kinetochore stub elongated poleward after treatment with UV microbeams; and fluxing of tubulin subunits through kinetochore MTs during anaphase A, indicating that during anaphase, kinetochore MTs disassemble partly or solely at the poles.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation

The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.     

Mitosin／CENP-F is a conserved kinetochore protein subjected to cyto-plasmic dynein-mediated poleward transport

Mitosin/CENP-F is a human nuclear protein transiently associated with the outer kinetochore plate in M phase and is involved in M phase progression. LEK1 and CMF1, which are its murine and chicken orthologs, however, are implicated in muscle differentiation and reportedly not distributed at kinetochores.We therefore conducted several assays to clarify this issue. The typical centromere staining patterns were observed in mitotic cells from both human primary culture and murine, canine, and mink cell lines. A C-terminal portion of LEK1 also conferred centromere localization. Our analysis further suggests conserved kinetochore localization of mammalian mitosin orthologs. Moreover, mitosin was associated preferentially with kinetochores of unaligned chromosomes. It was also constantly transported from kinetochores to spindle poles by cytoplasmic dynein. These properties resemble those of other kinetochore proteins important for the spindle checkpoint, thus implying a role of mitosin in this checkpoint. Therefore, mitosin family may serve as multifunctional proteins involved in both mitosis and differentiation.     

CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase

The human autoantigen CENP-C has been demonstrated by immunoelectron microscopy to be a component of the inner kinetochore plate. Here we have used antibodies raised against various portions of CENP-C to probe its function in mitosis. We show that nuclear microinjection of anti- CENP-C antibodies during interphase causes a transient arrest at the following metaphase. Injection of the same antibodies after the initiation of prophase, however, does not disrupt mitosis. Correspondingly, indirect immunofluorescence using affinity-purified human anti-CENP-C antibodies reveals that levels of CENP-C staining are reduced at centromeres in cells that were injected during interphase, but appear unaffected in cells which were injected during mitosis. Thus, we suggest that the injected antibodies cause metaphase arrest by reducing the amount of CENP-C at centromeres. Examination of kinetochores in metaphase-arrested cells by electron microscopy reveals that the number of trilaminar structures is reduced. More surprisingly, the few remaining kinetochores in these cells retain a normal trilaminar morphology but are significantly reduced in diameter. In cells arrested for extended periods, these small kinetochores become disrupted and apparently no longer bind microtubules. These observations are consistent with an involvement of CENP-C in kinetochore assembly, and suggest that CENP-C plays a critical role in both establishing and/or maintaining proper kinetochore size and stabilizing microtubule attachments. These findings also support the idea that proper assembly of kinetochores may be monitored by the cell cycle checkpoint preceding the transition to anaphase.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.     

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.     

The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants

Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force responsible for poleward chromosome motion.     

Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase

Successful mitosis requires that anaphase chromosomes sustain a commitment to move to their assigned spindle poles. This requires stable spindle attachment of anaphase kinetochores. Prior to anaphase, stable spindle attachment depends on tension created by opposing forces on sister kinetochores [1]. Because tension is lost when kinetochores disjoin, stable attachment in anaphase must have a different basis. After expression of nondegradable cyclin B (CYC-B(S)) in Drosophila embryos, sister chromosomes disjoined normally but their anaphase behavior was abnormal [2]. Chromosomes exhibited cycles of reorientation from one pole to the other. Additionally, the unpaired kinetochores accumulated attachments to both poles (merotelic attachments), congressed (again) to a pseudometaphase plate, and reacquired associations with checkpoint proteins more characteristic of prometaphase kinetochores. Unpaired prometaphase kinetochores, which occurred in a mutant entering mitosis with unreplicated (unpaired) chromosomes, behaved just like the anaphase kinetochores at the CYC-B(S) arrest. Finally, the normal anaphase release of AuroraB/INCENP from kinetochores was blocked by CYC-B(S) expression and, reciprocally, was advanced in a CycB mutant. Given its established role in destabilizing kinetochore-microtubule interactions [3], Aurora B dissociation is likely to be key to the change in kinetochore behavior. These findings show that, in addition to loss of sister chromosome cohesion, successful anaphase requires a kinetochore behavioral transition triggered by CYC-B destruction.