Mitosis in Tilia americana endosperm

The endosperm cells of the American basswood Tilia americana are favorable experimental material for investigating the birefringence of living plant spindles and anaphase movement of chromosomes. The behavior of the chromosomes in anaphase and the formation of the phragmoplast are unique. The numerous (3 n equals 123), small chromosomes move in precise, parallel rows until midanaphase when they bow away from the poles. Such a pattern of anaphase chromosome distribution has been described once before, but was ascribed to fusion of the chromosomes. The bowing of chromosome rows in Tilia is explainable quantitatively by the constant poleward velocity of the chromosomes during anaphase. Peripheral chromosomes are moving both relative to the spindle axis and laterally closer to the axis, whereas chromosomes lying on the spindle axis possess no lateral component in their motion, and thus at uniform velocity progress more rapidly than peripheral chromosomes relative to the spindle axis. The chromosomes are moved poleward initially by pole-to-pole elongation of the spindle, then moved farther apart by shortening of the kinetochore fibers. In contrast to other plant cells where the phragmoplast forms in telophase, the phragmoplast in Tilia endosperm is formed before midanaphase and the cell during midanaphase, while the chromosomes are still in poleward transit.     

Laser microirradiation of kinetochores in mitotic PtK2 cells: chromatid separation and micronucleus formation

Irradiation of the kinetochore region of PtK2 chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create an artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgen-stained monolayers of PtK2 cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Laser microirradiation of kinetochores in mitotic PtK2 cells

Irradiation of the kinetochore region of PtK₂ chromosomes by laser light of 532 nm was used to study the function of the kinetochore region in chromosome movement and to create artificial micronuclei in cells. When the sister kinetochores of a chromosome were irradiated at prometaphase, the affected chromosome detached from the spindle and exhibited no further directed movements for the duration of mitosis. The chromatids of the chromosome remained attached to one another until anaphase, at which point they separated. No poleward movement of the chromatids was observed, and at telophase they passively moved to one of the daughter cells and were enclosed in a micronucleus. The daughter cell containing the micronucleus was then isolated by micromanipulation and followed through subsequent mitoses. At the next mitosis, two chromosomes, each with two chromatids, condensed in the micronucleus. These chromosomes did not attach to the spindle and showed chromatid separation, but no poleward movements at anaphase. They were again enclosed in micronuclei at telophase. The third generation mitosis was similar to the second. Occasionally, both the irradiation-produced and naturally occurring micronuclei exhibited no chromosome condensation at mitosis. Feulgenstained monolayers of PtK₂ cells with naturally occurring micronuclei showed that some micronuclei stain positive for DNA and others do not. This finding raises questions about the fate of chromosomes in a micronucleus.     

Differential expression of a phosphoepitope at the kinetochores of moving chromosomes

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.     

Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.     

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.     

Backward chromosome movement in crane-fly spermatocytes after UV microbeam irradiation of the interzone and a kinetochore

Single anaphase chromosomes (in crane-fly spermatocytes) moved backwards after double irradiations with an ultraviolet light (UV) microbeam, first of the interzone and then of a kinetochore: the chromosome irradiated at the kinetochore moved backwards rapidly, across the equator and into the other half-spindle. High irradiation doses at the kinetochore were required to induce backward movement. Single irradiations of kinetochores or interzones were ineffective in inducing backward movements.     

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.     

Bipolar spindle attachments affect redistributions of ZW10, a Drosophila centromere/kinetochore component required for accurate chromosome segregation

Previous efforts have shown that mutations in the Drosophila ZW10 gene cause massive chromosome missegregation during mitotic divisions in several tissues. Here we demonstrate that mutations in ZW10 also disrupt chromosome behavior in male meiosis I and meiosis II, indicating that ZW10 function is common to both equational and reductional divisions. Divisions are apparently normal before anaphase onset, but ZW10 mutants exhibit lagging chromosomes and irregular chromosome segregation at anaphase. Chromosome missegregation during meiosis I of these mutants is not caused by precocious separation of sister chromatids, but rather the nondisjunction of homologs. ZW10 is first visible during prometaphase, where it localizes to the kinetochores of the bivalent chromosomes (during meiosis I) or to the sister kinetochores of dyads (during meiosis II). During metaphase of both divisions, ZW10 appears to move from the kinetochores and to spread toward the poles along what appear to be kinetochore microtubules. Redistributions of ZW10 at metaphase require bipolar attachments of individual chromosomes or paired bivalents to the spindle. At the onset of anaphase I or anaphase II, ZW10 rapidly relocalizes to the kinetochore regions of the separating chromosomes. In other mutant backgrounds in which chromosomes lag during anaphase, the presence or absence of ZW10 at a particular kinetochore predicts whether or not the chromosome moves appropriately to the spindle poles. We propose that ZW10 acts as part of, or immediately downstream of, a tension-sensing mechanism that regulates chromosome separation or movement at anaphase onset.     

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.     

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.     

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.     

Condensin Regulates the Stiffness of Vertebrate Centromeres

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores near the metaphase plate and delays anaphase onset.     

Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.     

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.     

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 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.     

Microtubule dynamics and chromosome motion visualized in living anaphase cells

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.     

Observations on dicentrics in living cells

Summary In previously irradiated endosperm cells of Haemanthus katherinae studied in vitro by means of micro-cinematography, two-kinetochore chromatids and dicentric chromosomes have been observed. Breaking of such dicentric chromatids and chromosomes has been analysed. Behaviour of some of the dicentric chromosomes during anaphase deserves special attention: interlocking dicentrics cut one through another and rejoin in a few minutes. In this way from a metaphase interlocking dicentric, two sister anaphase dicentrics are formed. Interlocked dicentrics can also uncoil and not break at all. In this case no activity was observed in one kinetochore of one dicentric in later stages of anaphase (two kinetochores were active in one dicentric and only one in its sister). Analysis of chromosome movements in two-kinetochore chromatids and dicentrics is also presented.