Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis

Microtubules in the mitotic spindles of newt lung cells were marked using local photoactivation of fluorescence. The movement of marked segments on kinetochore fibers was tracked by digital fluorescence microscopy in metaphase and anaphase and compared to the rate of chromosome movement. In metaphase, kinetochore oscillations toward and away from the poles were coupled to kinetochore fiber shortening and growth. Marked zones on the kinetochore microtubules, meanwhile, moved slowly polewards at a rate of approximately 0.5 micron/min, which identifies a slow polewards movement, or "flux," of kinetochore microtubules accompanied by depolymerization at the pole, as previously found in PtK2 cells (Mitchison, 1989b). Marks were never seen moving away from the pole, indicating that growth of the kinetochore microtubules occurs only at their kinetochore ends. In anaphase, marked zones on kinetochore microtubules also moved polewards, though at a rate slower than overall kinetochore-to-pole movement. Early in anaphase-A, microtubule depolymerization at kinetochores accounted on average for 75% of the rate of chromosome-to-pole movement, and depolymerization at the pole accounted for 25%. When chromosome-to-pole movement slowed in late anaphase, the contribution of depolymerization at the kinetochores lessened, and flux became the dominant component in some cells. Over the whole course of anaphase-A, depolymerization at kinetochores accounted on average for 63% of kinetochore fiber shortening, and flux for 37%. In some anaphase cells up to 45% of shortening resulted from the action of flux. We conclude that kinetochore microtubules change length predominantly through polymerization and depolymerization at the kinetochores during both metaphase and anaphase as the kinetochores move away from and towards the poles. Depolymerization, though not polymerization, also occurs at the pole during metaphase and anaphase, so that flux contributes to polewards chromosome movements throughout mitosis. Poleward force production for chromosome movements is thus likely to be generated by at least two distinct molecular mechanisms.     

Microinjection of mitotic cells with the 3F3/2 anti-phosphoepitope antibody delays the onset of anaphase

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.     

The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.     

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule-nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.     

Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores.We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.     

Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle

To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.     

Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle–regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NΔ253), or the conserved Spindly box (ΔSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.     

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.     

Silencing mitosin induces misaligned chromosomes, premature chromosome decondensation before anaphase onset, and mitotic cell death

Mitosin (also named CENP-F) is a large human nuclear protein transiently associated with the outer kinetochore plate in M phase. Using RNA interference and fluorescence microscopy, we showed that mitosin depletion attenuated chromosome congression and led to metaphase arrest with misaligned polar chromosomes whose kinetochores showed few cold-stable microtubules. Kinetochores of fully aligned chromosomes often failed to show orientation in the direction of the spindle long axis. Moreover, tension across their sister kinetochores was decreased by 53% on average. These phenotypes collectively imply defects in motor functions in mitosin-depleted cells and are similar to those of CENP-E depletion. Consistently, the intensities of CENP-E and cytoplasmic dynein and dynactin, which are motors controlling microtubule attachment and chromosome movement, were reduced at the kinetochore in a microtubule-dependent manner. In addition, after being arrested in pseudometaphase for approximately 2 h, mitosin-depleted cells died before anaphase initiation through apoptosis. The dying cells exhibited progressive chromosome arm decondensation, while the centromeres were still associated with spindles. Mitosin is therefore essential for full chromosome alignment, possibly by promoting proper kinetochore attachments through modulating CENP-E and dynein functions. Its depletion also prematurely triggers chromosome decondensation, a process that normally occurs from telophase for the nucleus reassembly, thus resulting in apoptosis.     

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.     

Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase

We have conducted a detailed ultrastructural analysis of the distribution of the kinesin-related centromere protein CENP-E during mitosis in cultured human, rat kangaroo and Indian muntjac cells. Using an affinity-purified polyclonal antibody and detection by 0.8 nm colloidal gold particles, CENP-E was localized primarily to the fibrous corona of the kinetochore in prometaphase and metaphase cells. Some labeling of the kinetochore outer plate was also observed. The distribution of fibrous corona-associated CENP-E did not change dramatically following the attachment of microtubules to the kinetochore. Thus, the normal disappearance of this kinetochore substructure in conventional electron micrographs of mitotic chromosomes with attached kinetochores is not due to the corona becoming stretched along the spindle microtubules as has been suggested. Examination of cells undergoing anaphase chromatid movement revealed the presence of CENP-E still associated with the outer surface of the kinetochore plate. At the same time, the majority of detectable CENP-E in these cells was associated with the bundles of antiparallel microtubules in the central spindle. CENP-E in this region of the cell is apparently associated with the stem body matrix material. The simultaneous localization of CENP-E on centromeres and the central spindle during anaphase was confirmed by both wide-field microscopy of human cells and conventional fluorescence microscopy of rat kangaroo cells. Together, the observations reported here are consistent with models in which CENP-E has a role in promoting the poleward migration of sister chromatids during anaphase A. Received: 21 July 1997 /Accepted: 19 September 1997     

MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.     

Phallacidin stains the kinetochore region in the mitotic spindle of the green algaeOedogonium spp.

Summary We found previously that in living cells ofOedogonium cardiacum andO. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (<2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore ofOedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.Abbreviations CD cytochalasin D - EM electron microscopy - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - MTs microtubules     

Micromanipulation of Chromosomes in Mitotic Vertebrate Tissue Cells: Tension Controls the State of Kinetochore Movement

In mitotic vertebrate tissue cells, chromosome congression to the spindle equator in prometaphase and segregation to the poles in anaphase depend on the movements of kinetochores at their kinetochore microtubule attachment sites. To test if kinetochores sense tension to control their states of movement poleward (P) and away from the pole (AP), we applied an external force to the spindle in preanaphase newt epithelial cells by stretching chromosome arms with microneedles. For monooriented chromosomes (only one kinetochore fiber), an abrupt stretch of an arm away from the attached pole induced the single attached kinetochore to persist in AP movement at about 2 μm/min velocity, resulting in chromosome movement away from the pole. When the stretch was reduced or the needle removed, the kinetochore switched to P movement at about 2 μm/min and pulled the chromosome back to near the premanipulation position within the spindle. For bioriented chromosomes (sister kinetochores attached to opposite poles) near the spindle equator, stretching one arm toward a pole placed the kinetochore facing away from the direction of stretch under tension and the sister facing toward the stretch under reduced tension or compression. Kinetochores under increased tension exhibited prolonged AP movement while kinetochores under reduced tension or compression exhibited prolonged P movement, moving the centromeres at about 2 μm/min velocities off the metaphase plate in the direction of stretch. Removing the needle resulted in centromere movement back to near the spindle equator at similar velocities. These results show that tension controls the direction of kinetochore movement and associated kinetochore microtubule assembly/disassembly to position centromeres within the spindle of vertebrate tissue cells. High tension induces persistent AP movement while low tension induces persistent P movement. The velocity of P and AP movement appears to be load independent and governed by the molecular mechanisms which attach kinetochores to the dynamic ends of kinetochore microtubules.     

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.     

Microtubules,chromosome movement,and reorientation after chromosomes are detached from the spindle by micromanipulation

The relationship between chromosome movement and mirotubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.     

Molecular analysis of kinetochore-microtubule attachment in budding yeast

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.     

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.     

Quantitative initiation of microtubule assembly by chromosomes from Chinese hamster ovary cells

The microtubule nucleating capacity of chromosomes was tested in vitro in lysates of Chinese hamster ovary cells. Colcemid-blocked mitotic cells were lysed with the detergent Triton X-100, incubated with exogenous porcine brain tubulin, attached to electron microscope grids and observed as whole-mounts. Under suitable conditions, greater than 98% of the chromosomes gave rise to microtubules at their kinetochore regions, thus unequivocally demonstrating that chromosomes are competent to initiate specifically microtubule formation. The average number of microtubules that polymerized onto a chromosome was 8 +/- 5, and greater than 36% of the chromosomes had between 10 and 19 microtubules per kinetochore region. We conclude that under the lysis conditions employed, virtually all the chromosomes retain their kinetochores, and that the kinetochores retain a substantial fraction of their microtubule nucleating capacity.