Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes

Exposing crane fly larvae to 6 degrees C or returning them to 22 degrees C after exposure to 6, 2, or 0.2 degrees C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J. R. LaFountain, 1982, Chromosoma, 85:619-631). We report here documentation that lagging autosomes in cold-treated and cold- recovering cells are maloriented. During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles: one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and non-lagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold- recovering cells was similar to that observed in untreated anaphase cells. Giemsa-stained centromeric dots of sister chromatids were contiguous in non-laggards and separated in laggards at anaphase. We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.     

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.     

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.     

Functional redundancy in the maize meiotic kinetochore

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, approximately 6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.     

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.     

Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.     

Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other

To test the "traction fiber" model for metaphase positioning of bivalents during meiosis, kinetochore fibers of maloriented bivalents, induced during recovery from cold arrest, were analyzed with a liquid crystal polarizing microscope. The measured birefringence retardation of kinetochore fibers is proportional to the number of microtubules in a fiber. Five of the 11 maloriented bivalents analyzed exhibited bipolar malorientations that had at least four times more kinetochore microtubules to one pole than to the other pole, and two had microtubules directed to only one pole. Yet all maloriented bivalents had positions at or near the spindle equator. The traction fiber model predicts such maloriented bivalents should be positioned closer to the pole with more kinetochore microtubules. A metaphase position at the spindle equator, according to the model, requires equal numbers of kinetochore microtubules to both poles. Data from polarizing microscope images were not in accord with those predictions, leading to the conclusion that other factors, in addition to traction forces, must be involved in metaphase positioning in crane-fly spermatocytes. Although the identity of additional factors has not been established, one possibility is that polar ejection forces operate to exert away-from-the-pole forces that could counteract pole-directed traction forces. Another is that kinetochores are "smart," meaning they embody a position-sensitive mechanism that controls their activity.     

Malorientation and abnormal segregation of chromosomes during recovery from colcemid and nocodazole

Reversal of meiotic arrest in crane-fly spermatocytes by U. V. irradiation of Colcemid-arrested cells or by rinsing Nocodazole-arrested cells in fresh buffer results in the induction of chromosome malorientation. Malorientations observed among Colcemid-recovering and Nocodazole-recovering spermatocytes at frequencies higher than normally observed in untreated cells included associations of sister kinetochores of half-bivalents with both spindle poles (amphitely), in contrast with associations of sisters with only one pole (syntely) as is usually found during the first meiotic division. In several cases, prior to anaphase onset, maloriented bivalents appeared unusually tilted with respect to the spindle axis, and during anaphase they gave rise to laggard half-bivalents that did not segregate during anaphase along with half-bivalents having proper syntelic orientation. The results parallel previous findings obtained during cold recovery, and the properties of the drugs used here suggest that their action on microtubules, although reversible, induces malorientation during recovery from meiotic arrest.     

The aurora B kinase AIR-2 regulates kinetochores during mitosis and is required for separation of homologous Chromosomes during meiosis

BACKGROUND: Mitotic chromosome segregation depends on bi-orientation and capture of sister kinetochores by microtubules emanating from opposite spindle poles and the near synchronous loss of sister chromatid cohesion. During meiosis I, in contrast, sister kinetochores orient to the same pole, and homologous kinetochores are captured by microtubules emanating from opposite spindle poles. Additionally, mechanisms exist that prevent complete loss of cohesion during meiosis I. These features ensure that homologs separate during meiosis I and sister chromatids remain together until meiosis II. The mechanisms responsible for orienting kinetochores in mitosis and for causing asynchronous loss of cohesion during meiosis are not well understood. RESULTS: During mitosis in C. elegans, aurora B kinase, AIR-2, is not required for sister chromatid separation, but it is required for chromosome segregation. Condensin recruitment during metaphase requires AIR-2; however, condensin functions during prometaphase, independent of AIR-2. During metaphase, AIR-2 promotes chromosome congression to the metaphase plate, perhaps by inhibiting attachment of chromatids to both spindle poles. During meiosis in AIR-2-depleted oocytes, congression of bivalents appears normal, but segregation fails. Localization of AIR-2 on meiotic bivalents suggests this kinase promotes separation of homologs by promoting the loss of cohesion distal to the single chiasma. Inactivation of the phosphatase that antagonizes AIR-2 causes premature separation of chromatids during meiosis I, in a separase-dependent reaction. CONCLUSIONS: Aurora B functions to resolve chiasmata during meiosis I and to regulate kinetochore function during mitosis. Condensin mediates chromosome condensation during prophase, and condensin-independent pathways contribute to chromosome condensation during metaphase.     

Merotelic kinetochore attachment: causes and effects

Accurate chromosome segregation depends on the proper attachment of sister kinetochores to microtubules emanating from opposite spindle poles. Merotelic kinetochore orientation is an error in which a single kinetochore is attached to microtubules emanating from both spindle poles. Despite correction mechanisms, merotelically attached kinetochores can persist until anaphase, causing chromatids to lag on the mitotic spindle and hindering their timely segregation. Recent studies showing that merotelic kinetochore attachment represents a major mechanism of aneuploidy in mitotic cells and is the primary mechanism of chromosomal instability in cancer cells have underlined the importance of studying merotely. Here, we highlight recent progress in our understanding of how cells prevent and correct merotelic kinetochore attachments.     

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.     

Polarity of spindle microtubules in Haemanthus endosperm

Structural polarities of mitotic spindle microtubules in the plant Haemanthus katherinae have been studied by lysing endosperm cells in solutions of neurotubulin under conditions that will decorate cellular microtubules with curved sheets of tubulin protofilaments. Microtubule polarity was observed at several positions in each cell by cutting serial thin sections perpendicular to the spindle axis. The majority of the microtubules present in a metaphase or anaphase half-spindle are oriented with their fast-growing or "plus" ends distal to the polar area. Near the polar ends of the spindle and up to about halfway between the kinetichores and the poles, the number of microtubules with opposite polarity is low: 8-20% in metaphase and 2-15% in anaphase cells. Direct examination of 10 kinetochore fibers shows that the majority of these microtubules, too, are oriented with their plus ends distal to the poles, as had been previously shown in animal cells. Sections from the region near the spindle equator reveal an increased fraction of microtubules with opposite polarity. Graphs of polarity vs. position along the spindle axis display a smooth transition from microtubules of one orientation near the first pole, through a region containing equal numbers of the two orientations, to a zone near the second pole where the opposite polarity predominates. We conclude that the spindle of endosperm cells is constructed from two sets of microtubules with opposite polarity that interdigitate near the spindle equator. The length of the zone of interdigitation shortens from metaphase through telophase, consistent with a model that states that during anaphase spindle elongation in Haemanthus, the interdigitating sets of microtubules are moved apart. We found no major changes in the distribution of microtubule polarity in the spindle interzone from anaphase to telophase when cells are engaged in phragmoplast formation. Therefore, the initiation and organization of new microtubules, thought to take place during phragmoplast assembly, must occur without significant alteration of the microtubule polarity distribution.     

Spoke: a 120-kD protein associated with a novel filamentous structure on or near kinetochore microtubules in the mitotic spindle

We have characterized an antiserum that recognizes a single 120-kD protein in CHO cells which is soluble and cytoplasmically localized in interphase, but which is associated with a novel filamentous structure localized on or near kinetochore microtubules in mid-mitosis. These filaments, one per sister chromatid, run from near the mitotic spindle pole to within approximately 0.3 microns of each kinetochore. In metaphase, the staining pattern shows considerable substructure at light microscopy resolution, appearing as bright nodes or striations, often with a kinked or helical appearance. This overall localization pattern is retained throughout anaphase, with the filaments shortening as the chromosomes move toward the mitotic spindle poles. Also in anaphase, a separate ring-like structure lacking a tubulin-staining component appears near the spindle poles. As cells exit mitosis, the amount of this antigen in the cell decreases seven- to tenfold. The unusual staining pattern and the specific localization of this antigen on or near kinetochore microtubules in mid-mitosis indicate that the 120-kD protein defines or is associated with an important and previously unrecognized structural element of the mitotic spindle.     

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.     

Geometry and force behind kinetochore orientation: lessons from meiosis

During mitosis, replicated chromosomes (sister chromatids) become attached at the kinetochore by spindle microtubules emanating from opposite poles and segregate equationally. In the first division of meiosis, however, sister chromatids become attached from the same pole and co-segregate, whereas homologous chromosomes connected by chiasmata segregate to opposite poles. Disorder in this specialized chromosome attachment in meiosis is the leading cause of miscarriage in humans. Recent studies have elucidated the molecular mechanisms determining chromosome orientation, and consequently segregation, in meiosis. Comparative studies of meiosis and mitosis have led to the general principle that kinetochore geometry and tension exerted by microtubules synergistically generate chromosome orientation.     

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.     

The Drosophila Mei-S332 Gene Promotes Sister-Chromatid Cohesion in Meiosis following Kinetochore Differentiation

The Drosophila mei-S332 gene acts to maintain sister-chromatid cohesion before anaphase II of meiosis in both males and females. By isolating and analyzing seven new alleles and a deficiency uncovering the mei-S332 gene we have demonstrated that the onset of the requirement for mei-S332 is not until late anaphase I. All of our alleles result primarily in equational (meiosis II) nondisjunction with low amounts of reductional (meiosis I) nondisjunction. Cytological analysis revealed that sister chromatids frequently separate in late anaphase I in these mutants. Since the sister chromatids remain associated until late in the first division, chromosomes segregate normally during meiosis I, and the genetic consequences of premature sister-chromatid dissociation are seen as nondisjunction in meiosis II. The late onset of mei-S332 action demonstrated by the mutations was not a consequence of residual gene function because two strong, and possibly null, alleles give predominantly equational nondisjunction both as homozygotes and in trans to a deficiency. mei-S332 is not required until after metaphase I, when the kinetochore differentiates from a single hemispherical kinetochore jointly organized by the sister chromatids into two distinct sister kinetochores. Therefore, we propose that the mei-S322 product acts to hold the doubled kinetochore together until anaphase II. All of the alleles are fully viable when in trans to a deficiency, thus mei-S332 is not essential for mitosis. Four of the alleles show an unexpected sex specificity.     

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.     

Light and electron microscopy of rat kangaroo cells in mitosis

Chromosome orientation and behavior during prometaphase of mitosis in PtK₁ rat kangaroo cells were investigated by cinémicrography and electron microscopy. The first chromosome movements occur soon after the nuclear envelope begins to break down in the region near each pole. Initial chromosome behavior is primarily determined by the distance from the kinetochore region to the spindle poles. The predominant pattern is a movement to and/or association with the proximal pole. Movement to and association with the more distant pole, or direct alignment at or near the spindle equator (direct congression) are less frequent patterns. Except for rare cases, pole-associated chromosomes congress sooner or later and most congressed chromosomes oscillate about the equator. — Ultrastructural observations suggest that pole-associated chromosomes are oriented only to the proximal pole (monotelic or syntelic orientation) and they demonstrate that the sister-kinetochores of congressing or oscillating chromosomes are oriented to opposite poles (amphitelic orientation). — Based on the structure of the early prometaphase spindle and four assumptions concerning the formation of kinetochore fibers and their force-producing interaction with complementary elements, the different patterns of chromosome behavior observed can be explained as a result of synchronous or asynchronous formation of sister-kinetochore fibers. The few chromosomes whose kinetochore region is approximately equidistant from the poles amphi-orient immediately because their sister-kinetochores form fibers synchronously and they congress directly because of the bidirectional forces to which they are subjected. The kinetochore region of most chromosomes is not equidistant from the poles. Therefore, they form a functional fiber first to the nearer pole and move to, or associate with, it because of the unidirectional force. Eventually, however, these chromosomes achieve amphitelic orientation and congress. Once established, amphitelic orientation is stable. Re-orientations do not occur during congression or oscillatory movements.     

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.