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The distinctive behaviors of chromosomes in mitosis and meiosis depend upon differences in kinetochore position. Kinetochore position is well established except for a critical transition between meiosis I and meiosis II. We examined kinetochore position during the transition and compared it with the position of kinetochores in mitosis. Immunofluorescence staining using the 3F3/2 antibody showed that in mitosis in grasshopper cells, as in other organisms, kinetochores are positioned on opposite sides of the two sister chromatids. In meiosis I, sister kinetochores are positioned side by side. At nuclear envelope breakdown in meiosis II, sister kinetochores are still side by side, but are separated by the time all chromosomes have fully attached in metaphase II. Micromanipulation experiments reveal that this switch from side-by-side to separated sister kinetochores requires attachment to the spindle. Moreover, it is irreversible, as chromosomes detached from a metaphase II spindle retain separate kinetochores. How this critical separation of sister kinetochores occurs in meiosis is uncertain, but clearly it is not built into the chromosome before nuclear envelope breakdown, as it is in mitosis.

In mitosis, cohesion appears to be present along the entire length of the chromosome, between centromeres and along chromosome arms. By metaphase, sister chromatids appear as two adjacent but visibly distinct rods. Sister chromatids separate from one another in anaphase by releasing all chromosome cohesion. This is different from meiosis I, in which pairs of sister chromatids separate from one another, moving to each spindle pole by releasing cohesion only between sister chromatid arms. Then, in anaphase II, sister chromatids separate by releasing centromere cohesion. Our objective was to find where cohesion is present or absent on chromosomes in mitosis and meiosis and when and how it is released. We determined cohesion directly by pulling on chromosomes with two micromanipulation needles. Thus, we could distinguish for the first time between apparent doubleness as seen in the microscope and physical separability. We found that apparent doubleness can be deceiving: Visibly distinct sister chromatids often cannot be separated. We also demonstrated that cohesion is released gradually in anaphase, with chromosomes looking as if they were unzipped or pulled apart. This implied that tension from spindle forces was required, but we showed directly that no tension was necessary to pull chromatids apart.

A reduction of chromosome number in meiosis is essential for genome transmission in diploid organisms. Reduction depends on a change in kinetochore configuration.1 A recent study2 connects changes in kinetochores with other changes in chromosome structure and raises the intriguing possibility that topoisomerase II, the DNA untangling enzyme, is involved., (Copyright 2003 Wiley Periodicals, Inc.)

The spindle checkpoint detects errors in kinetochore attachment to microtubules and delays anaphase if attachment is improper. The checkpoint is activated by attachment-sensitive components including Mad2 and certain phosphorylated proteins detected by the 3F3/2 antibody. We have studied Mad2 and 3F3/2 immunofluorescence in grasshopper spermatocytes. As in other cells, unattached kinetochores are loaded with Mad2 and are highly phosphorylated, whereas after proper attachment, Mad2 is lost and kinetochores are dephosphorylated. What is it about proper attachment that produces these changes--is it microtubule attachment itself or is it the tension from mitotic forces that follows proper attachment? Using micromanipulation, we created an intermediate state, weak attachment, that provides an answer. Weakly attached kinetochores are not under tension and have few kinetochore microtubules. Despite the absence of tension, many weakly attached kinetochores lose their Mad2 and become dephosphorylated. Therefore we conclude that microtubule attachment determines both Mad2 binding and phosphorylation. Nevertheless, tension plays an absolutely essential role. Tension elevates the number of kinetochore microtubules to the level necessary for the complete loss of Mad2 and dephosphorylation from all kinetochores. This gives a reliable 'all clear' signal to the checkpoint, allowing the cell to progress to anaphase.

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

When chromosomes attach properly to a mitotic spindle, their kinetochores generate force in opposite directions, creating tension. Tension is presumed to increase kinetochore microtubule number, but there has been no direct evidence this is true. We micromanipulated grasshopper spermatocyte chromosomes to test this assumption and found that tension does indeed affect the number of kinetochore microtubules. Releasing tension at kinetochores causes a drop to less than half the original number of kinetochore microtubules. Restoring tension onto these depleted kinetochores restores the microtubules to their original number. However, the effects of tension are limited. Prometaphase kinetochores, when under normal tension from mitotic forces, have about half as many microtubules as they will in late metaphase. We imposed a tension force of 6 x 10(-5) dynes, three times the normal tension, on prometaphase kinetochores. The elevated tension did not drive kinetochore microtubule number above normal prometaphase values. Tension probably increases the number of kinetochore microtubules by slowing their turnover rate. The limited effect of tension at prometaphase kinetochores suggests that they have fewer microtubule binding sites than at late metaphase. The relatively few sites available in prometaphase may be the decisive sites whose binding of microtubules regulates the dynamics of transient kinetochore constituents, including checkpoint components.

In meiosis I, two chromatids move to each spindle pole. Then, in meiosis II, the two are distributed, one to each future gamete. This requires that meiosis I chromosomes attach to the spindle differently than meiosis II chromosomes and that they regulate chromosome cohesion differently. We investigated whether the information that dictates the division type of the chromosome comes from the whole cell, the spindle, or the chromosome itself. Also, we determined when chromosomes can switch from meiosis I behavior to meiosis II behavior. We used a micromanipulation needle to fuse grasshopper spermatocytes in meiosis I to spermatocytes in meiosis II, and to move chromosomes from one spindle to the other. Chromosomes placed on spindles of a different meiotic division always behaved as they would have on their native spindle; e.g., a meiosis I chromosome attached to a meiosis II spindle in its normal fashion and sister chromatids moved together to the same spindle pole. We also showed that meiosis I chromosomes become competent meiosis II chromosomes in anaphase of meiosis I, but not before. The patterns for attachment to the spindle and regulation of cohesion are built into the chromosome itself. These results suggest that regulation of chromosome cohesion may be linked to differences in the arrangement of kinetochores in the two meiotic divisions.

The spindle checkpoint prevents errors in mitosis. Cells respond to the presence of kinetochores that are improperly attached to the mitotic spindle by delaying anaphase onset. Evidence suggests that phosphorylations recognized by the 3F3/2 anti-phosphoepitope antibody may be involved in the kinetochore signaling of the spindle checkpoint. Mitotic cells lysed in detergent in the absence of phosphatase inhibitors rapidly lose expression of the 3F3/2 phosphoepitope. However, when ATP is added to lysed and rinsed mitotic cytoskeletons, kinetochores become rephosphorylated by an endogenous, bound kinase. Kinetochore rephosphorylation in vitro produced the same differential phosphorylation seen in appropriately fixed living cells. In chromosomes not yet aligned at the metaphase plate, kinetochores undergo rapid rephosphorylation, while those of fully congressed chromosomes are under-phosphorylated. However, latent 3F3/2 kinase activity is retained at kinetochores of cells at all stages of mitosis including anaphase. This latent activity is revealed when rephosphorylation reactions are carried out for extended times. The endogenous, kinetochore-bound kinase can be chemically inactivated. Remarkably, a soluble kinase activity extracted from mitotic cells also caused differential rephosphorylation of kinetochores whose endogenous kinase had been chemically inactivated. We suggest that, in vivo, microtubule attachment alters the kinetochore 3F3/2 phosphoprotein, causing it to resist phosphorylation. This kinetochore modification is retained after cell lysis, producing a "memory" of the in vivo phosphorylation state., (Copyright 2000 Wiley-Liss, Inc.)

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.

Many cells have a checkpoint that detects a single misattached chromosome and delays anaphase, allowing time for error correction. Detection probably depends on tension-sensitive kinetochore protein phosphorylation. Somehow, mechanical tension, or some consequence of tension, produces a chemical change, dephosphorylation. The mechanism of tension-mediated dephosphorylation can be approached using an in vitro system. Earlier work showed that the kinetochores of washed chromosomes from a mammalian cell line can be phosphorylated in vitro simply by incubation with ATP and a phosphatase inhibitor. We confirm this for chromosomes from insect meiotic cells. Thus, kinetochores of washed chromosomes from diverse sources contain a complete phosphorylation system: a kinase, a phosphatase and the substrate protein(s). We show that phosphorylation in vitro is sensitive to tension, as it is in living cells. This makes the conditions required for phosphorylation in vitro relevant to the process in living cells. The phosphatase is ruled out as the tension-sensitive component in vitro, leaving either the kinase or the substrate as the sensitive component. We show that a kinase extracted from mammalian cells in mitosis phosphorylates the kinetochores of insect meiotic chromosomes very effectively. The mammalian kinase under-phosphorylates the kinetochore of the insect's X-chromosome, just as the native insect kinase does. This provides a clue to the evolution of a chromosome that is not detected by the checkpoint. The mammalian kinase is not tightly bound to the chromosome and thus functions primarily in solution. This suggests that the substrate's phosphorylatable groups are freely available to outside constituents, e.g. regulators, as well as to the kinetochore's own kinase and phosphatase.

Improper chromosome attachment to the spindle can lead to daughter cells with missing or extra chromosomes. Such mishaps are avoided in many cells by a checkpoint that detects even a single improperly attached chromosome. What is detected? A misattached chromosome is not under tension from opposed mitotic forces, and in praying mantid spermatocytes, direct experiments show that the absence of tension is what the checkpoint detects. How is the absence of tension detected? Tension-sensitive kinetochore protein phosphorylation is the most likely possibility. We combined micromanipulation with immunostaining for phosphoproteins in order to study the effect of tension on kinetochore phosphorylation in mantid spermatocytes. We confirm earlier observations on mammalian cells and grasshopper spermatocytes that misattached chromosomes have phosphorylated kinetochore proteins. We also confirm experiments in grasshopper spermatocytes showing that tension alters kinetochore chemistry: tension from a micromanipulation needle causes kinetochore protein dephosphorylation, and relaxation of tension causes kinetochore protein rephosphorylation. Beyond confirmation, our results provide fresh evidence for phosphorylation as the signal to the checkpoint. First, mantid cells are the only ones in which an effect of tension on the checkpoint has been directly demonstrated; by equally direct experiments, we now show that tension affects kinetochore phosphorylation in these same cells. Second, sex chromosome behavior in mantids provides a natural experiment to test the relationship between phosphorylation and the checkpoint. In grasshoppers, an unpaired sex chromosome is normal, its kinetochore is under-phosphorylated, and the checkpoint is not activated. In mantids, exactly the opposite is true: an unpaired sex chromosome is abnormal, its kinetochore is phosphorylated and, as predicted, the checkpoint is activated. We conclude that tension-sensitive kinetochore protein phosphorylation very likely is the essential link between proper chromosome attachment and the check-point, the link that permits potential errors in chromosome distribution to be detected and avoided.

When cells divide, the chromosomes must be delivered flawlessly to the daughter cells. Missing or extra chromosomes can result in birth defects and cancer. Chance events are the starting point for chromosome delivery, which makes the process prone to error. Errors are avoided by diverse uses of mechanical tension from mitotic forces. Tension stabilizes the proper chromosome configuration, controls a cell cycle checkpoint, and changes chromosome chemistry.

Anaphase and cytokinesis are key processes in the segregation of replicated chromosomes to the daughter cells: in anaphase, chromosomes move apart; in cytokinesis, a cleavage furrow forms midway between the separated chromosomes. Some evidence suggests that chromosomes may be involved both in controlling the timing of anaphase onset and in dictating the position of the cleavage furrow. Other evidence indicates that the controlling mechanisms are intrinsic to the spindle and the cell. Here we test these possibilities in grasshopper spermatocytes by observing spindles and cells after removal of chromosomes. We found that both anaphase and cytokinesis occur independently of chromosomes: stage-specific changes occur at an appropriate time and in the correct way, despite the absence of chromosomes. This finding is particularly noteworthy because chromosomes have an important impact on spindle microtubule assembly and the timing of anaphase onset in these cells.

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

Some cells have a quality control checkpoint that can detect a single misattached chromosome and delay the onset of anaphase, thus allowing time for error correction. The mechanical error in attachment must somehow be linked to the chemical regulation of cell cycle progression. The 3F3 antibody detects phosphorylated kinetochore proteins that might serve as the required link (Gorbsky, G. J., and W. A. Ricketts. 1993. J. Cell Biol. 122:1311-1321). We show by direct micromanipulation experiments that tension alters the phosphorylation of kinetochore proteins. Tension, whether from a micromanipulation needle or from normal mitotic forces, causes dephosphorylation of the kinetochore proteins recognized by 3F3. If tension is absent, either naturally or as a result of chromosome detachment by micromanipulation, the proteins are phosphorylated. Equally direct experiments identify tension as the checkpoint signal: tension from a microneedle on a misattached chromosome leads to anaphase (Li, X., and R. B. Nicklas. 1995. Nature (Lond.). 373:630-632), and we show here that the absence of tension caused by detaching chromosomes from the spindle delays anaphase indefinitely. Thus, the absence of tension is linked to both kinetochore phosphorylation and delayed anaphase onset. We propose that the kinetochore protein dephosphorylation caused by tension is the all clear signal to the checkpoint. The evidence is circumstantial but rich. In any event, tension alters kinetochore chemistry. Very likely, tension affects chemistry directly, by altering the conformation of a tension-sensitive protein, which leads directly to dephosphorylation.

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video-enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.

Every time a cell divides, the chromosomes must be distributed accurately to the daughter cells. Errors in distribution arise if chromosomes are improperly attached to the mitotic spindle. Improper attachment is detected by a cell-cycle checkpoint in many cells and the completion of cell division is delayed, allowing time for error correction. How is an improperly attached chromosome detected? An absence of tension from mitotic forces is one possibility. Here we test this possibility directly by applying tension to an improperly attached chromosome with a micromanipulation needle. In the absence of tension, the entry into anaphase and the completion of mitosis was delayed by 5-6 hours. When the misattached chromosome was placed under tension, however, the cell entered anaphase in 56 minutes, on average. Tension from mitotic forces or from a micromanipulator's needle evidently signals to the checkpoint that all is in order and that cell division can proceed.

The correction of certain errors in mitosis requires capture and release: new kinetochore microtubules must be captured and old, misdirected ones must be released. We studied capture and release in living grasshopper spermatocytes. Capture is remarkably efficient over a broad range in the angle at which a microtubule encounters a kinetochore. However, capture is inefficient when kinetochores point directly away from the source of properly directed microtubules. Capture in that situation is required for correction of the most common error; microtubule-kinetochore encounters are improbable and capture occurs only once every 8 min, on average. Release from the improper attachment caused by misdirected microtubules allows kinetochore movement and the completion of error correction. We tugged on kinetochores with a micromanipulation needle and found they are free to move less than one time in two. Thus error correction depends on two improbable events, capture and release, and they must happen by chance to coincide. In spermatocytes this will occur only once every 18 min, on average, but a leisurely cell cycle provides ample time. Capture and release generate only change, not perfection. Tension from mitotic forces brings change to a halt by stabilizing the one correct attachment of chromosomes to the spindle. We show that tension directly affects stability, rather than merely constraining kinetochore position. This implies that chromosomes are attached to the spindle by tension-sensitive linkers whose stability is necessary for proper chromosome distribution but whose loss is necessary for the correction of errors.

Errors in chromosome orientation in mitosis and meiosis are inevitable, but normally they are quickly corrected. We find that such errors usually are not corrected in cells treated with protein kinase inhibitors. Highly inaccurate chromosome distribution is the result. When grasshopper spermatocytes were treated with the kinase inhibitor 6-dimethylaminopurine (DMAP), 84% of maloriented chromosomes failed to reorient; in anaphase, both partner chromosomes were distributed to the same daughter cell. These chromosomes were observed for a total of over 60 h, and not a single reorientation was seen. In contrast, in untreated cells, maloriented chromosomes invariably reoriented, and quickly: in 10 min, on average. A second protein kinase inhibitor, genistein, had exactly the same effect as DMAP. DMAP affected PtK1 cells in mitosis as it did spermatocytes in meiosis: improper chromosome orientations persisted, leading to frequent errors in distribution. We micromanipulated chromosomes in spermatocytes treated with DMAP to learn why maloriented chromosomes often fail to reorient. Reorientation requires the loss of improper microtubule attachments and the acquisition of new, properly directed kinetochore microtubules. Micromanipulation experiments disclose that neither the loss of old nor the acquisition of new microtubules is sufficiently affected by DMAP to account for the indefinite persistence of malorientations. Drug treatment causes a novel form of chromosome movement in which one kinetochore moves toward another kinetochore. Two kinetochores in the same chromosome or in different chromosomes can participate, producing varied, dance-like movements executed by one or two chromosomes. These kinetochore-kinetochore interactions evidently are at the expense of kinetochore-spindle interactions. We propose that malorientations persist in treated cells because the kinetochores have numerous, short microtubules with a free end that can be captured by a second kinetochore. Kinetochores capture each other's kinetochore microtubules, leaving too few sites available for the efficient capture of spindle microtubules. Since the efficient capture of spindle microtubules is essential for the correction of errors, failure of capture allows malorientations to persist. Whether the effects of DMAP actually are due to protein kinase inhibition remains to be seen. In any case, DMAP reveals interactions of one kinetochore with another, which, though ordinarily suppressed, have implications for normal mitosis.

We used an evolutionary test to ask whether the congression of chromosomes to the spindle equator is important in itself or just a mitotic happenstance. If congression matters, then it might evolve if absent initially. Previous workers established that newly made trivalents, meiotic units of three chromosomes, generally do not congress to the spindle equator. Instead, these young trivalents lie close to the pole to which two of the three chromosomes are oriented. We studied ancient sex-chromosome trivalents that arose hundreds of thousands to several million years ago in several species of praying mantids and one grasshopper. All these old trivalents lie near the spindle equator at metaphase; some of them congress as precisely to the equator as the ordinary chromosomes in the same cells. We conclude that congression evolved independently two or three times in the materials studied. Therefore, the metaphase position of chromosomes midway between the poles appears to matter, but why? In the praying mantids, the evident answer is that metaphase is a quality-control checkpoint. Sometimes the three chromosomes are not associated in a trivalent but rather are present as a bivalent plus an unpaired chromosome, which lies near one pole. Earlier workers showed that such cells are blocked in metaphase and eventually degenerate; this prevents the formation of sperm with abnormal combinations of sex chromosomes. We suggest that the quality-control system would have trouble distinguishing an unpaired chromosome from an uncongressed, newly arisen trivalent, both of which would lie near a spindle pole. If so, the confused quality-control system would block anaphase imprudently, causing a loss of cells that would have produced normal sperm. Hence, we conclude that the congression of the trivalent to the equator probably evolved along with the metaphase quality-control checkpoint. The mechanism of congression in old trivalents is uncertain, but probably involves an interesting force-sensitive regulation of the motors associated with particular chromosomes. We also examined the congression of two newly made quadrivalents when they orient with three kinetochores to one pole and one to the other. As others have described, one of these quadrivalents does not congress, while the other quadrivalent comes closer than expected to the spindle equator. Such variation in the extent of congression may provide materials on which natural selection can act, leading to the evolution of congression. The trivalents of praying mantids are attractive materials for further studies of the mechanism of congression and of the idea that metaphase is a checkpoint for progression through the cell cycle.

We studied the orientation and segregation of a particular quadrivalent in living grasshopper spermatocytes. Quadrivalents were detached from the spindle by micromanipulation, then placed and bent as desired. The detached quadrivalents reattach and orient on the spindle. Their orientation is determined by the same principles that apply to ordinary chromosomes in mitosis and meiosis, but the outcome is different. Certain characteristics of the quadrivalent lead to a variety of orientations rather than the single one typical of ordinary chromosomes. Two kinetochores in the quadrivalent are linked to the others by unusually long, flexible chromosome arms. These kinetochores may face either the same pole or opposite poles and tend to orient initially to the pole toward which they face. Consequently, the initial orientation of the flexibly linked kinetochores is variable, and, moreover, they frequently reorient. In contrast, the other two kinetochores are as rigidly connected as those in a small bivalent and so display the typical back-to-back arrangement. Usually, this arrangement leads quickly to a stable orientation of the two kinetochores to opposite poles. Sometimes, however, the back-to-back arrangement changes to a side-by-side arrangement so that the orientation of both kinetochores to the same pole is favored. The combined effect of this diverse behavior is that the quadrivalent has four stable orientations, each leading to a different distribution of chromosomes in anaphase. The result is genetic chaos. Ironically, this chaos is produced by the same mechanisms that, in ordinary bivalents and mitotic chromosomes, produce a single stable orientation and genetically appropriate chromosome distribution.

Most aspects of chromosome distribution to the daughter cells in meiosis and mitosis are now understood, at the cellular level. The most striking evidence that the proposed explanation is valid is that it correctly predicts the outcome of experiments on living cells in which the experimenter (1) can determine the distribution of any chosen chromosome to a chosen daughter cell, (2) can induce a mal-orientation, and (3) can stabilize a mal-orientation, causing non-disjunction of a chosen bivalent. Recent reviews of chromosome distribution mechanisms are also considered, in an attempt to clarify the remaining unsolved problems.

The basis for stable versus unstable kinetochore orientation was investigated by a correlated living-cell/ultrastructural study of grasshopper spermatocytes. Mal-oriented bivalents having both kinetochores oriented to one spindle pole were induced by micromanipulation. Such mal-orientations are stable while the bivalent is subject to tension applied by micromanipulation but unstable after tension is released. Unstable bivalents always reorient with movement of one kinetochore toward the opposite pole. Microtubules associated with stably oriented bivalents, whether they are mal-oriented or in normal bipolar orientation, are arranged in orderly parallel bundles running from each kinetochore toward the pole. Similar orderly kinetochore microtubule arrangements characterized mal-oriented bivalents fixed just after release of tension. A significantly different microtubule arrangement is found only some time after tension release, when kinetochore movement is evident. The microtubules of a reorienting kinetochore always include a small number of microtubules running toward the pole toward which the kinetochore was moving at the time of fixation. All other microtubules associated with such a moving kinetochore appear to have lost their anchorage to the original pole and to be dragged passively as the kinetochore proceeds to the other pole. Thus, the stable anchorage of kinetochore microtubules to the spindle is associated with tension force and unstable anchorage with the absence of tension. The effect of tension is readily explained if force production and anchorage are both produced by mitotic motors, which link microtubules to the spindle as they generate tension forces.

Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.

A new method is offered for combined living cell and electron-microscopic studies of spermatocytes (or other cells) which normally do not adhere to glass. The key step is micro-injection of glutaraldehyde near the target cell whenever desired during observation in life. Fixation begins and simultaneously the cell is stuck very firmly to the underlying coverslip. The method is easy and reliable: cells are almost never lost and are well preserved, except for membranes. The application of the method is illustrated by studies of micromanipulated grasshopper spermatocytes. A chromosome was detached from the spindle and placed in the cytoplasm. Before or after the beginning of chromosome movement back toward the spindle, the cell was fixed, sectioned and the manipulated chromosome observed in the electron microscope. If the detached chromosome had not moved by the time of fixation, no or only one or two microtubules were seen at its kinetochore, but if movement had occurred, a few microtubules were always present. The arrangement of these microtubules corresponded to the direction of movement, but they commonly were at an unusual angle relative to the kinetochore. The origin and role in chromosome movement of the microtubules seen near moving chromosomes far from the spindle is not yet established, but a speculation is offered. A goal for future work is the detailed analysis of the microtubules associated with individual moving chromosomes. Such an analysis is feasible because the moving chromosome is far removed from the confusing mass of spindle microtubules, and its value is enhanced because the direction of movement at the time of fixation is known.

We have discovered an easy way to cut through the mitotic spindle at any desired place. Spindles of demembranated cricket or grasshopper spermatocytes were severed with a microneedle between the chromosomes and one pole, and the cut-off polar piece was swept away. Spindle structure and microtubule dynamics in cut spindles were studied by anti-tubulin immunostaining and electron microscopy. The cut is clean: all microtubules are severed and only a few extend beyond the others. This provides the basis for a clear test of whether traction fibers pull chromosomes to the pole in anaphase, because the putative traction fiber is cleanly severed. Cutting creates new plus ends on microtubules in the cut-off polar piece and new minus ends on microtubules in the main spindle body. The microtubules with new plus ends are unstable, as expected from the dynamic instability of microtubules. However, the microtubules with new minus ends are as stable as uncut microtubules in the same spindle. Our mechanical method of cutting microtubules very likely creates native, reactive ends, and therefore the surprising stability of new minus ends is genuinely interesting, not an artifact of cutting.

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

We extracted chromosomes by micromanipulation from Melanoplus differentialis spermatocytes, producing metaphase spindles with only one or a few chromosomes instead of the usual complement of 23. Cells with various numbers of chromosomes were prepared for electron microscopy, and spindle microtubule length was measured. A constant increment of microtubule length was lost upon the removal of each chromosome; we estimate that only approximately 40% of the original length would remain in the total absence of chromosomes. Unexpectedly, kinetochore microtubules were not the only ones affected when chromosomes were removed: nonkinetochore microtubules accounted for a substantial fraction of the total length lost. No compensatory increase in microtubule length outside the spindle was found. Studies by others show that the kinetochore microtubules of extracted chromosomes are left behind in the cell and dissassemble. The resulting increase in subunit concentration would be expected from in vitro studies to drive microtubule assembly until the original total microtubule length was restored, but that did not happen in these living cells. We conclude that the assembly of a certain, large fraction of microtubule subunits into stable microtubules is dependent on the presence of chromosomes. Possible explanations include (a) limits on microtubule length that prevent any net assembly of the subunits released after chromosomes are removed or (b) a promotion of microtubule assembly by chromosomes, which therefore is reduced in their absence. Chromosome-dependent regulation of microtubule length may account for some features of normal mitosis.

I have tested two contending views of chromosome-to-pole movement in anaphase. Chromosomes might be pulled poleward by a traction fiber consisting of the kinetochore microtubules and associated motors, or they might propel themselves by a motor in the kinetochore. I cut through the spindle of demembranated grasshopper spermatocytes between the chromosomes and one pole and swept the polar region away, removing a portion of the would-be traction fiber. Chromosome movement continued, and in the best examples, chromosomes moved to within 1 micron of the cut edge. There is nothing beyond the edge to support movement, and a push from the rear is unlikely because cuts in the interzone behind the separating chromosomes did not stop movement. Therefore, I conclude that the motor must be in the kinetochore or within 1 micron of it. Less conclusive evidence points to the kinetochore itself as the motor. The alternative is an external motor pulling on the kinetochore microtubules or directly on the kinetochore. A pulling motor would move kinetochore microtubules along with the chromosome, so that in a cut half-spindle, the microtubules should protrude from the cut edge as chromosomes move toward it. No protrusion was seen; however, the possibility that microtubules depolymerize as they are extruded, though unlikely, is not ruled out. What is certain is that the motor for poleward chromosome movement in anaphase must be in the kinetochore or very close to it.

A microheater was used to produce a temperature gradient within the mitotic spindle of living cells. The slope of the temperature gradient was estimated from thermal conductivity calculations and confirmed by measurements of spindle birefringence and by experiments on striated muscle. When the microheater was placed at one spindle pole or at one group of kinetochores, the gradient was steep enough to cause a large difference in birefringence between the two half-spindles, but the velocity of chromosome movement in anaphase was nearly the same in the warmer and cooler half-spindles. When the heater was shifted from the pole toward the interzone, the average velocity of chromosome movement increased approximately two-fold but was, again, nearly uniform in the two half-spindles. The rate of spindle elongation was especially sensitive to the site of heating, increasing ten-fold when the heater was shifted from the pole to the interzone. Regardless of heater position, the rate of chromosome movement was determined largely by the temperature of the coolest spindle region--chromosomes in the warmer half-spindle moved more slowly than expected from estimates of the temperature in that region. Since the microheater produces a substantial temperature gradient within the spindle, the near uniformity of chromosome velocity in both half-spindles must be due to some biological property of the spindle. Two very different explanations for the results are considered the most likely. According to one explanation, the near uniformity of velocity in both half-spindles is determined by the structure of the interpolar spindle, while changes in velocity involve force producers located both in the half-spindles and in the interzone. On the other explanation, the velocity is nearly the same in both half-spindles because the force producers are located exclusively in the interzone (Margolis et al., 1978).

Ostergren (1951) provided a simple explanation for both chromosome distribution in mitosis and chromosome segregation in meiosis, and more recently a molecular extension of his hypothesis has been possible. This report focuses on experimental tests of these ideas. Micromanipulation experiments on cell hybrids containing both meiotic and mitotic spindles demonstrate that differences in meiotic and mitotic chromosome behavior are determined by something intrinsic to the chromosome: meiotic chromosomes transferred to a mitotic spindle (or vice versa) behave just as they normally would. The molecular explanation postulates polarized growth or binding of microtubules at kinetochores. This has just been tested in vitro by McGill & Brinkley (1975) and by Telzer, Moses & Rosenbaum (1975), and their results are reviewed. In addition, a novel method for in vitro studies is described - mechanical demembranation of cells which is compatible with quite normal chromosome movement in anaphase. After addition of microtubule subunits to a demembranated prophase cell, chromosome orientation and movement toward an aster was observed for the first time in vitro. It is concluded that important aspects of chromosome distribution are probably understood at both the cellular and molecular levels, but final tests are still required. The outlook is hopeful indeed because the gaps in our knowledge are well known - the necessity of observations on prophase is a recurrent theme - and the means of filling the gaps are in hand.

Cellular motile systems as diverse as muscle and the mitotic spindle have been compared by their specific power output: the maximum power they develop per unit of engine volume. Striated muscles and flagella have high specific output; their performance is comparable to that of typical automobile engines. The cytokinetic furrow and the mitotic spindle have very much lower specific power output. The furrow's output is 7,000 times lower than muscle and the spindle's is 300,000 times lower. Different macromolecules have been used to generate power in systems with similar output (muscles and flagella) and, conversely, the same macromolecular motor has been used in systems with very different output (muscles and cytokinetic furrows). The common feature amid this diversity is adaptation to a particular biological role, which specific power output reflects very well. High values are found where a powerful, compact engine should be advantageous, while low values are found where precision, not power, matters most.

Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle. We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure.

The fabrication and use of resistance wire microheaters is described and the results obtainable with cells are illustrated. The microheaters permit localized warming of a small area, wherever desired, within a single living cell. This is directly shown by the production of localized structural changes in the mitotic spindle. Thus in this case, subcellular areas respond independently to the local temperature. The method is applicable to a variety of cellular processes, providing clues to cellular control mechanisms.

The X chromosome in Melanoplus differentialis is negatively heteropycnotic in the early spermatogonial cell generations but positively heteropycnotic in the final premeiotic interphase. Autoradiography after administration of tritiated thymidine reveals that this condensation change is paralleled by a change in the time of DNA replication in the X chromosome relative to that in the autosomes.

Kinetochore reorientation is the critical process ensuring normal chromosome distribution. Reorientation has been studied in living grasshopper spermatocytes, in which bivalents with both chromosomes oriented to the same pole (unipolar orientation) occur but are unstable: sooner or later one chromosome reorients, the stable, bipolar orientation results, and normal anaphase segregation to opposite poles follows. One possible source of stability in bipolar orientations is the normal spindle forces toward opposite poles, which slightly stretch the bivalent. This tension is lacking in unipolar orientations because all the chromosomal spindle fibers and spindle forces are directed toward one pole. The possible role of tension has been tested directly by micromanipulation of bivalents in unipolar orientation to artificially create the missing tension. Without exception, such bivalents never reorient before the tension is released; a total time "under tension" of over 5 hr has been accumulated in experiments on eight bivalents in eight cells. In control experiments these same bivalents reoriented from a unipolar orientation within 16 min, on the average, in the absence of tension. Controlled reorientation and chromosome segregation can be explained from the results of these and related experiments.

Chromosome velocity has been studied in living Melanoplus differentialis spermatocytes by phase contrast cinemicrography. Melanoplus chromosomes (and bivalents) differ in length by as much as 1:3.5. As expected, no size-dependent velocity differences were detected in anaphase, and this is also shown to be true for the less predictable movements during prometaphase congression. The size of the X chromosome can change during observation following x-irradiation, but this is equally without influence on velocity. However, an effect of position on velocity is found in both prometaphase and in anaphase: the chromosomes furthest from the central interpolar axis move 25 per cent faster than more central chromosomes. A simple mechanical model relating frictional resistance and mitotic forces to chromosome velocity is discussed in detail. Calculations from the model suggest that a significant difference in the force acting on a large, as compared with a small chromosome is necessary to account for the observed similarity in velocity. Therefore, it is concluded that the mitotic forces are so organized or regulated that velocity is, within limits, independent of load. The implications of velocity-load independence in relation to the molecular origin of mitotic forces are discussed.