Malorientation in half-bivalents at anaphase in crane fly spermatocytes following Colcemid treatment

Addition of Colcemid to the medium in which larvae of the crane fly Nephrotoma suturalis are cultivated induces a number of anomalous patterns of chromosome segregation. One of these is the anaphase lagging of autosomal half-bivalents. To investigate the cause of anaphase lagging, the orientation of sister kinetochores in Colcemidtreated spermatocytes having lagging half-bivalents was analyzed in serial sections. In contrast to nonlaggard halfbivalents that had pure syntelic orientation (sister kinetochores having all of their kinetochores microtubules (KMTs) extending to the same pole), six of the seven autosomal laggards that were selected for analysis had kinetochores with either amphitelic orientation (sister kinetochores each with a bundle of KMTs extending to opposite poles) or merotelic orientation (a single kinetochore having KMTs extending toward both poles). An additional laggard had syntelic orientation but two of the microtubules that were in its kinetochore fiber passed through the kinetochore and extended beyond it toward the equator. The bipolar malorientations observed in anaphase half-bivalents are interpreted to be a cause of the anaphase lagging induced by Colcemid treatment. Furthermore, it is hypothesized that such bipolar malorientations also may be stabilized at metaphase and thus explain the unusual tilting of metaphase bivalents commonly observed in Colcemid-treated cells.     

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.     

Influence of autosome movements and of sex-chromosome movements on sex-chromosome segregation in crane fly spermatocytes

In crane fly spermatocyte meiosis 3 autosome half-bivalents normally move to each spindle pole in anaphase while the 2 amphitelic sex-chromosome univalents remain at the equator. The sex-chromosome univalents move to opposite poles after the autosomes reach the poles. — We used micromanipulation to detach half-bivalents in anaphase. When re-attached half-bivalents were syntelically oriented to the original pole, sex-chromosome segregation was usually not altered. When re-attached half-bivalents were amphitelically oriented, sex-chromosome segregation was usually altered: usually the amphitelic autosome segregated against one sex-chromosome while the other sex-chromosome remained at the equator. When re-attached half-bivalents were syntelically oriented to the opposite pole, sex-chromosome segregation was often altered: often one sex-chromosome moved normally to the spindle pole with 2 autosomal half-bivalents, while the other sex-chromosome did not move to the spindle pole with 4 autosomal half-bivalents, but remained at the equator. — The direction of motion of a sex-chromosome could be altered even after sex-chromosome segregation had begun, by suitable micromanipulation of the other sex-chromosome. — Amphitelic chromosomes that were not on the equator at the start of anaphase segregated predominantly to the closer spindle pole. Detached half-bivalents showed no preference for the closer pole when they re-attached with syntelic orientation. — We discuss some possible hypotheses for non-independent movements, and some implications of the results.     

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.     

Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.     

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.     

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane-fly spermatocytes

We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes.     

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.     

Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes

Summary. We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles. In this study, we tested whether locust spermatocyte spindles also utilise actin and myosin, and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (latrunculin B and cytochalasin D), myosin (BDM), or myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y-27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resumed movement at normal speed after these drugs were washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB completely removes or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

Chromosome micromanipulation

The attachment of individual chromosomes to the spindle has been studied by micromanipulation in functionally normal grasshopper spermatocytes. Prometaphase to anaphase I chromosomes can be repeatedly stretched with a microneedle without much increase in the distance between the kinetochores and the poles. Individual chromosomes can, however, be displaced laterally (prometaphase-anaphase) or toward the pole (anaphase) without loss of spindle attachment and without greatly disturbing other chromosomes. It is concluded that chromosomes are firmly and individually attached to the spindle by chromosomal spindle fibers which are capable of bearing any normal mitotic load, including the stretching of dikinetic (dicentric) chromosomes in anaphase. Prolonged or severe manipulation can produce a small — three or four micron — increase in the kinetochore-to-pole distance. Anaphase motion continues normally in spite of lateral or poleward displacements or of small increases in the kinetochore-to-pole distance. In late anaphase, chromosomes can be displaced to the opposite pole. An unusual, rapid motion back toward the original pole follows such displacements, but repeated displacements eventually result in non-disjunction. No evidence for firm interzonal connections between anaphase chromosomes was obtained. Prometaphase and metaphase bivalents can be detached from the spindle by manipulations other than bivalent stretching, but half-bivalents in anaphase are never detached by these manipulations.This investigation was supported in part by research grants GM-8480 and GM-13745 from the Division of General Medical Sciences, United States Public Health Service.     

Cine-micrographic studies on dicentric chromosomes

Summary The behavior during mitosis of SR (sister reunion) bridges and other types of dicentric anaphase bridges was analysed. The endosperm of Haemanthus katharinae was used as material, and 16 mm time-lapse cine-film was analysed frame by frame. Most observations concern anaphase and telophase. The bridges break or are stretched and their variation in behavior depends on the length of the bridge and the stage when it is stretched. Breakage in anaphase is due to the pulling action of the kinetochores, while breakage in telophase is due to phragmoplast activity and a formation of a cell plate. The kinetochore movements of the dicentrics are usually, but not always, slowed down when the bridges stretch up to when the bridges have broken. Whether the movement of the kinetochores is slowed down or not depends mostly on the metaphase position of the kinetochore relative to the equator. A retarded movement of one kinetochore influences the movements of others in its close neighborhood while kinetochores situated further away are not influenced at all. In cells with many short bridges whole groups of chromosomes are retarded and the formation of a restitution nucleus follows. The formation of a restitution nucleus may also result from the existence of numerous bridges causing failure of phragmoplast formation. The contraction of the bridges during telophase pulls the two parts of the restitution nucleus together and contributes to maintain their connection.Some conclusions concerning the mechanism responsible for the movements of the chromosomes are also given.     

Cytochalasin induces abnormal anaphase in crane-fly spermatocytes and causes altered distribution of actin and centromeric antigens

Inhibition of cytokinesis by cytochalasins without an effect on karyokinesis has been demonstrated in several types of cells. We report here that treating crane-fly spermatocytes with cytochalasins at concentrations (10 M CE, 100 M CD, and 200 CB) in excess of that needed to inhibit cell division induces one or more half-bivalents to lag at anaphase during the first meiotic division. The behavior of the laggards is similar to that of maloriented half-bivalents. Following treatment at these concentrations, probing with rhodamine-phalloidin or bodipy-phallacidin reveals loss of filamentous actin from the poles and its appearance in the spindle, predominantly in regions where centromeres and kinetochores are normally found. When either N350 anti-actin monoclonal antibody or rhodamine DNase I was used to probe for actin in cytochalasin-treated cells, a similar redistribution of actin was observed. CD and CE treatments alter the pattern of fluorescence at centromere/kinetochore regions after staining with scleroderma CREST serum: CREST-positive structures become broader, with spikes extending from them toward the pole; in addition, some strands of CREST fluorescence appear that are apparently extraneous, and not associated with chromosomes. Probes for actin yield staining patterns in centromere/kinetochore regions that match closely the cytochalasin-altered pattern of CREST staining. Our finding of actin in the vicinity of kinetochores under conditions that result in abnormal chromosome behavior raises numerous questions about the possible role(s) of actin in meiosis, particularly in chromosome orientation.Abbreviations CREST calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia by W.C. Earnshaw     

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.     

Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.     

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.     

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.     

Chromosome micromanipulation

The relationship of kinetochore orientation and reorientation to orderly chromosome distribution in anaphase has been studied experimentally by micromanipulation of living grasshopper spermatocytes. Bivalents or the X chromosome at prometaphase or metaphase I can be detached from the spindle with a microneedle and moved to any desired location within the cell. Following a pause of variable duration the detached chromosome invariably moved, kinetochores foremost, back to the spindle, reassumed its characteristic metaphase position, and, with one exception, segregated normally at anaphase I. Detachment from the spindle is demonstrated unequivocally (1) by manipulation evidence for the absence of the firm spindle connections seen both before detachment and after reattachment and (2) by a functional criterion: a given kinetochore, oriented to one pole before detachment, often orients to the opposite pole after detachment. The segregation in anaphase was always as expected from the final, post-operation, orientation. Reorientation and prometaphase and anaphase movement after detachment cannot be distinguished from their counterparts in control cells. Kinetochore position after detachment is the primary determinant of the pole to which that kinetochore will orient. Therefore, since the experimenter determines kinetochore position, he can cause any given half-bivalent to segregate to a predetermined pole at anaphase I. Similarly, orientation of both half-bivalents to the same pole can be induced. These mal-oriented bivalents invariably reorient and normal anaphase segregation ensues. Non-disjunction can, however, be produced directly in late anaphase. These experiments are based upon current views of orderly chromosome distribution; their success confirms our understanding of the fundamental orientation process.