Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: anaphase B but not anaphase A requires ATP

Permeabilized PtK1 cells continue to undergo anaphase chromosome movements provided MgATP is included in the lysis medium. However, chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) differ with respect to nucleotide requirements. The rate of anaphase B depends on the concentration of ATP in the lysis medium; two-thirds the maximal rate is observed in 0.2 mM ATP. However, other nucleotides, such as ITP, CTP and GTP, cannot substitute for ATP. Spindle elongation is blocked by the addition of nonhydrolyzable ATP analogs. ADP, AMP and inhibitors such as vanadate, the magnesium chelator EDTA and sulfhydryl reagents. Anaphase does no require exogenous ATP and is unaffected by these inhibitors. These results are consistent with "dynein-like" ATPase involvement during spindle elongation, and rule out the possibility of tubulin-dynein and actomyosin mechanochemistry during anaphase A. I suggest that chromosome-to-pole movement involves the collapse of an elastic component in the spindle. Force generation could be provided by microtubule depolymerization or by the contraction of a nonmicrotubule microtrabecular lattice.     

Vanadate inhibits saltatory organelle movement in a permeabilized cell model

A method has been developed for sustaining saltatory organelle movement in permeabilized cells. Fibroblasts are permeabilized using the nonionic detergent, Brij 58, with a 2-step procedure modified from the methods of Cande [1–4]. Saltatory movement continues for 20–40 min in the permeabilized cells, provided that ATP is present in the lysis medium, although the saltations are less frequent and slower than those seen before detergent treatment. Vanadate rapidly and reversibly inhibits saltatory movement in the permeabilized cells. Inhibition by vanadate suggests that a dynein-like molecule may be involved in the mechanism of saltatory movement.     

Anaphase motions in dilute colchicine. Evidence of two phases in chromosome segregation

We have examined the rates of chromosome and pole motion during anaphase in HeLa cells using differential interference contrast and polarization optics. In early anaphase both chromosomes and poles move apart. When the chromosomes are separated by a distance about equal to the metaphase spindle length, both chromosomes and poles slow but continue to move at a reduced rate. Throughout anaphase, the chromosomes move faster than the poles, so the chromosome-to-pole distance decreases. Treatment of the cells with about 5 × 10^?8 M colchicine up to 45 min before observation tends to block normal formation of metaphase spindles, but more than half of the cells in metaphase go on through anaphase. In these cells, both chromosome and pole motions are essentially normal until the chromosomes are separated by a distance equal to the length of the metaphase spindle. After that time, chromosome motion is supressed and the poles move slowly toward one another. These data suggest that the mechanism of anaphase motion changes character when the chromosomes become spaced by the metaphase spindle length. We call anaphase before and after that time phase 1 and phase 2, respectively. The results are discussed in the light of a sliding tubule model for chromosome motion.     

The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement

At anaphase, the linkage betweeh sister chromatids is dissolved and the separated sisters move toward opposite poles of the spindle. We developed a method to purify metaphase and anaphase chromosomes from frog egg extracts and identified proteins that leave chromosomes at anaphase using a new form of expression screening. This approach identified Xkid, a Xenopus homolog of human Kid (kinesin-like DNA binding protein) as a protein that is degraded in anaphase by ubiquitin-mediated proteolysis. Immunodepleting Xkid from egg extracts prevented normal chromosome alignment on the metaphase spindle. Adding a mild excess of wild-type or nondegradable Xkid to egg extracts prevented the separated chromosomes from moving toward the poles. We propose that Xkid provides the metaphase force that pushes chromosome arms toward the equator of the spindle and that its destruction is needed for anaphase chromosome movement.     

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.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

The mechanism of anaphase spindle elongation

At anaphase chromosomes move to the spindle poles (anaphase A) and the spindle poles move apart (anaphase B). In vitro studies using isolated diatom spindles demonstrate that the primary mechanochemical event responsible for spindle elongation is the sliding apart of half-spindle microtubules. Further, these forces are generated within the zone of microtubule overlap in the spindle mid-zone.     

In vivo effects of ortho-vanadate on spindle structure and dynamics of locust spermatocytes I

After external application of vanadate, a potent inhibitor of several ATPases including dynein, the following effects on living spermatocytes I are detectable: spherical metaphase cells change to a lemon shape due to a concentration dependent elongation of the spindle, apparently achieved by pulling the plasma membrane-inserted poles apart, presumably through the assistance of cytoskeletal filaments. The observed dismembering of the spindle seems to be due to the separation of the half-spindle fibres, composed of usually interdigitating kinetochore microtubules (kMTs), free MTs (fMTs) and polar MTs (pMTs). As revealed by microcinematographic recordings, the lengthening of the half-spindles is accompanied by counter-clockwise twisting movements of the polar regions which, after prolonged vanadate treatment, lead to the formation of filiform appendices. Bundles of 5 nm microfilaments, which could be identified by indirect immunofluorescence microscopy (IIF) as actin, are concentrated within these appendices. In spite of a certain derangement of spindle architecture, half of the metaphases in 1 mM vanadate are capable of entering anaphase, but the rates of chromosome-to-pole movement have changed depending on the incubation time and the cell shape developed, respectively. Thus, chromosomes move with the highest speed in lemon-shaped cells but lag in cells with filiform appendices. However, it remains an open question whether the acceleration of chromosome migration is the result of spindle dismemberment or whether the slowing of anaphase motion is the consequence of a far-reaching displacement of the filamentous component from the spindle framework.     

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.     

Chromosome elimination in Heteropeza pygmaea

Chromosome elimination in the 3rd cleavage division of the gall midge Heteropeza pygmaea was observed with the Differential Interference Contrast method and recorded with photomicrography and time-lapse cinémicrography. The chromosomes which move all the way to the poles (S-chromosomes) are included in the presumptive somatic nuclei while the lagging chromosomes are eliminated (E-chromosomes). In early prometaphase of an elimination division the nuclear envelope is replaced by the spindle envelope which persists until late telophase and separates nucleoplasm and cytoplasm. In prometaphase the volume of the spindle decreases considerably. Until mid-anaphase the E and the S-chromosomes cannot be distinguished from each other either morphologically or topologically and they both behave like chromosomes in a normal cleavage division. In early anaphase the velocity of the E-chromosomes is usually less than that of the S-chromosomes. After variable amounts of anaphase movement the E-chromosomes return towards the equator with a velocity which is less than their velocity in early anaphase. Their kinetochores are still oriented towards the poles. The two chromatids of an E-chromosome usually move symmetrically towards the poles and back to the equator. At the time when the E-chromosomes stop moving towards the poles the S-chromosomes sometimes accelerate.     

A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence

Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.     

Verteilung der Mikrotubuli in Metaphase- und Anaphase-Spindeln der Spermatocyten von Pales ferruginea

One metaphase I spindle, seven anaphase I spindles of different stages, and one metaphase II spindle were sectioned in series. The ultrastructure of chromosomes was examined and microtubules (MTs) were counted. The main results of the study are summarized as follows: 1. The autosomes move at the periphery of the continuous MTs during anaphase while the sex chromosomes move more or less within this group of MTs. 2. In metaphase the antosomes have few coarse surface projections, in anaphase many, but more delicate projections of irregular shape which seem to transform into regular radial lamellae at the end of movement. 3. In metaphase continuous MTs have no contact with the chromosomal surface, while during anaphase movement continuous MTs lie closer to the chromosomes, and finally arrange themselves between the radial surface lamellae. There they show lateral filamentous connections with the chromosomal surface. 4. The MT distribution profiles of metaphase and anaphase are different. While the highest density of MTs is observed in the middle region of the spindle in metaphase, there are two density zones during autosomal movement, each in one half spindle in front of the autosomes. After the autosomes have reached the poles the distribution profile is again similar to the metaphase condition. The MT distribution in metaphase II is the same as in metaphase I. Possible explanations for these observations are discussed in detail. 5. There is an overall decrease in MT content during anaphase. 6. With the onset of anaphase MTs are seen within the spindle mantle, closely associated with mitochondria. — Several theoretical aspects of anaphase mechanism are briefly discussed.     

Mitosis in Tilia americana endosperm

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

Cell division and the microtubular cytoskeleton]

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.     

Single particle tracking of surface receptor movement during cell division

We have used fluorescent latex beads to label membrane receptors on cultured NRK cells. Movement of individual beads during cell division was recorded with digital imaging techniques. Surface-bound beads showed no organized movement during metaphase but started to migrate toward the equator approximately 1 min after anaphase onset, when chromosomes moved out of the equatorial region to create the interzone. The movement was most active in the central region of the cell near separating chromosomes, while beads located near the poles of the cell underwent primarily random motion. Most beads showed a surge in speed upon the passage of chromosomes, suggesting a possible link between chromosome separation and cortical reorganization. Furthermore, treatment of anaphase cells with cytochalasin D induced a rapid, simultaneous collapse of beads and cortical actin filaments into aggregates, indicating that the movement of beads was closely related to the reorganization of the actin cortex. In contrast to normal directional movement, cytochalasin-induced movement occurred in random directions and caused some beads in the equatorial region to move toward poles. Our results indicate that cytokinesis involves contractile activities, not only along the equator, but over a wide area of the actin-containing cortex. In addition, organized cortical activities appear to be temporally activated at anaphase onset, and spatially modulated by the spindle interzone or separating chromosomes.     

Anaphase B precedes anaphase A in the mouse egg

Segregation of chromosomes at the time of cell division is achieved by the microtubules and associated molecules of the spindle. Chromosomes attach to kinetochore microtubules (kMTs), which extend from the spindle pole region to kinetochores assembled upon centromeric DNA. In most animal cells studied, chromosome segregation occurs as a result of kMT shortening, which causes chromosomes to move toward the spindle poles (anaphase A). Anaphase A is typically followed by a spindle elongation that further separates the chromosomes (anaphase B). The experiments presented here provide the first detailed analysis of anaphase in a live vertebrate oocyte and show that chromosome segregation is initially driven by a significant spindle elongation (anaphase B), which is followed by a shortening of kMTs to fully segregate the chromosomes (anaphase A). Loss of tension across kMTs at anaphase onset produces a force imbalance, allowing the bipolar motor kinesin-5 to drive early anaphase B spindle elongation and chromosome segregation. Early anaphase B spindle elongation determines the extent of chromosome segregation and the size of the resulting cells. The vertebrate egg therefore employs a novel mode of anaphase wherein spindle elongation caused by loss of k-fiber tension is harnessed to kick-start chromosome segregation prior to anaphase A.     

A note on the behaviour of spindle fibres at mitosis

Summary Measurements done on mitosis in plant endosperm with the use of Dr. Inoué's polarizing microscope indicate that the whole chromosomal spindle fibre is shifted during anaphase, i.e. the distance between the spindle pole and the polar end of the spindle fibre decreases during anaphase. Consequently in endosperm mitosis the chromosomes move faster to the poles than the chromosomal spindle fibres decrease in length. As the chromosomal spindle fibres in animal materials presumably extend to the spindle poles already from the beginning of anaphase, the chromosomes will here approach the poles with the same speed, as the spindle fibres contract.This paper is dedicated to Professor Franz Schrader on the occasion of his seventieth birthday.     

Metabolic inhibitors and mitosis: I. Effects of dinitrophenol/deoxyglucose and nocodazole on the live spindle

Summary Dinitrophenol and deoxyglucose (DNP/DOG) were used to investigate the effects of ATP depletion on mitotic PtK₁ cells. Direct determination of cellular ATP levels showed that the drop of ATP induced by DNP/DOG was rapid; recovery to normal ATP levels was equally rapid once DNP/DOG was removed. On addition of DNP/DOG to live cells, cytoplasmic activity ceased; interphase and prophase cells showed little other response to DNP/DOG. During prometaphase, DNP/DOG induced a pronounced movement of oscillating, monopolar chromosomes towards the spindle poles. As chromosomes became bipolarly attached, DNP/DOG caused the spindle poles themselves to move together. By metaphase, DNP/DOG-treatment led to significant shortening of the spindle which remained intact. DNP/DOG rapidly stopped anaphase chromosome movement and cytokinesis.Nocodazole (NOC) caused the rapid breakdown of the mitotic spindle; prometaphase chromosomes clustered at the poles and in metaphase cells, the poles were drawn towards the chromosomes as the spindle became disorganized. When cells were pretreated with DNP/DOG and then NOC/DNP/DOG, nocodazole did not break down the spindle. When nocodazole was applied first to break down spindle MTs then DNP/DOG was added to the nocodazole, a second contraction was often induced by the DNP/DOG in the absence of spindle microtubules (MTs). Chromosomes expanded appreciably outwards from the poles when the DNP/DOG was removed, even when the cells remained in nocodazole.     

A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered. After lysis, the mitotic apparatus is functional; chromosomes move poleward and the spindle elongates. Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage. On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions. These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements.     

Micromanipulation of mitotic chromosomes in PTK2 cells using laser-induced optical forces ("optical tweezers")

To study the potential use of optical forces to manipulate chromosome movement, we have used a Nd:YAG laser at a wavelength of 1.06 microns focused into a phase contrast microscope. Metaphase and anaphase chromosomes were exposed while being monitored by video microscopy. The results indicated that when optical forces were applied to late-moving metaphase chromosomes on the side closest to the nearest spindle pole, the trapped chromosomes initiated movement to the metaphase plate. The chromosome velocities were two to eight times the normal rate depending on the chromosome size, geometry, and trapping site. At the initiation of anaphase, a pair of chromatids could be held by the optical trap and kept motionless throughout anaphase while the other pairs of chromatids separated and moved to opposite spindle poles. As a result, the trapped chromosome either was incorporated into one of the daughter cells or was lost in the cleavage furrow, or the two chromatids eventually separated and moved to their respective daughter cells. If the trap was removed at the beginning of anaphase B, the chromosome moved back to the poles. Our experiments demonstrate that the laser-induced optical force trap is a potential new technique to study noninvasively the mitotic spindle of living cells.