Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: evidence that secretory granules move anterogradely along microtubules

During the cell cycle the distribution of the ACTH-containing secretory granules in AtT20 cells, as revealed by immunofluorescence labeling and electron microscopy of thin sections, undergoes a cycle of changes. In interphase cells the granules are concentrated in the Golgi region, where they form, and also at the tips of projections from the cells, where they accumulate. These projections contain many microtubules extending to their tips. During metaphase and anaphase the granules are randomly distributed in the cytoplasm of the rounded-up mitotic cells. On entry into telophase there is a rapid and striking redistribution of the granules, which accumulate in large numbers in the midbody as it develops during cytokinesis. This accumulation of secretory granules in the midbody is dependent upon the presence of microtubules. The changing pattern of distribution of the secretory granules during the cell cycle fulfills the predictions of a model envisaging first that secretory granules associate with and move along interphase microtubules in a net anterograde direction away from the centrioles, and secondly that they do not associate with microtubules of the mitotic spindle during metaphase and anaphase.     

Polarity of spindle microtubules in Haemanthus endosperm

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

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.     

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.     

Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK₁ cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.     

Kinetochore-driven outgrowth of microtubules is a central contributor to kinetochore fiber maturation in crane-fly spermatocytes

We use liquid crystal polarized light imaging to record the life histories of single kinetochore (K-) fibers in living crane-fly spermatocytes, from their origins as nascent K-fibers in early prometaphase to their fully matured form at metaphase, just before anaphase onset. Increased image brightness due to increased retardance reveals where microtubules are added during K-fiber formation. Analysis of experimentally generated bipolar spindles with only one centrosome, as well as of regular, bicentrosomal spindles, reveals that microtubule addition occurs at the kinetochore-proximal ends of K-fibers, and added polymer expands poleward, giving rise to the robust K-fibers of metaphase cells. These results are not compatible with a model for K-fiber formation in which microtubules are added to nascent fibers solely by repetitive “search and capture” of centrosomal microtubule plus ends. Our interpretation is that capture of centrosomal microtubules—when deployed—is limited to early stages in establishment of nascent K-fibers, which then mature through kinetochore-driven outgrowth. When kinetochore capture of centrosomal microtubules is not used, the polar ends of K-fibers grow outward from their kinetochores and usually converge to make a centrosome-free pole.     

Micronuclear division and microtubular stability in the ciliate Colpoda steinii

The first microtubules which appear in the prophase micronucleus of Colpoda steinii are located beneath the nuclear envelope and not connected to the chromosomes. Most microtubules of the metaphase spindle are connected to the tapered tips of the micronucleus and terminate singly at the chromosomes surrounded by a conical, RNA-containing kinetochore which disappears upon cold treatment. During anaphase, an interzonal stembody is formed which is maximally stretched at telophase before the daughter micronuclei are pinched off from its ends. The macronucleus, which also stretches parallel to the micronuclear stembody, has fewer microtubules which insert at the inner nuclear envelope but are not attached to the chromatin. Based upon the effects of depolymerizing factors different classes of microtubules can be distinguished. Kinetochore microtubules are sensitive to cold and vinblastine (VLB). In 2.5×10^–5 M VLB their number is drastically reduced and the interzonal microtubules of early anaphase, which are also highly sensitive to nocodazole, become completely disassembled. The cross-bridged microtubules of the fully formed stembody of late anaphase display the highest resistance to depolymerization. They show signs of partial disassembly only after prolonged cold exposure and withstand higher concentrations of VLB or nocodazole than other micronuclear microtubules. Microtubules in the elongating macronucleus are fairly insensitive to cold but are depolymerized by 5×10^–5 M VLB while 1.66×10^–5 M nocodazole, which leaves only traces of stembody microtubules, merely reduces their number and length. All microtubules are fairly resistant to colchicine since high concentrations (5×10^–2 M) are required to prevent assembly while fully formed stembodies are unaffected. Macronuclear microtubules are depolymerized at this concentration. Nocodazole, which depolymerizes all premetaphase microtubules at 6.6×10^–6 M, leads to multipolar metaphase spindles with numerous microtubules, even at 1.66×10^–5 M, an effect ascribed to the activity of the nuclear envelope as a microtubule organizing centre. At twice this concentration multipolar spindles are no longer found and the remaining microtubules show no apparent order. A stabilizing influence of the micronuclear envelope is indicated by the fact that whenever remnants of microtubules are found after depolymerizing treatments, they are located in its vicinity.     

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.     

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.     

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

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

Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase

In previous work we injected mitotic cells with fluorescent tubulin and photobleached them to mark domains on the spindle microtubules. We concluded that chromosomes move poleward along kinetochore fiber microtubules that remain stationary with respect to the pole while depolymerizing at the kinetochore. In those experiments, bleached zones in anaphase spindles showed some recovery of fluorescence with time. We wished to determine the nature of this recovery. Was it due to turnover of kinetochore fiber microtubules or of nonkinetochore microtubules or both? We also wished to investigate the question of turnover of kinetochore microtubules in metaphase. We microinjected cells with x- rhodamine tubulin (x-rh tubulin) and photobleached spindles in anaphase and metaphase. At various times after photobleaching, cells were detergent lysed in a cold buffer containing 80 microM calcium, conditions that led to the disassembly of almost all nonkinetochore microtubules. Quantitative analysis with a charge coupled device image sensor revealed that the bleached zones in anaphase cells showed no fluorescence recovery, suggesting that these kinetochore fiber microtubules do not turn over. Thus, the partial fluorescence recovery seen in our earlier anaphase experiments was likely due to turnover of nonkinetochore microtubules. In contrast fluorescence in metaphase cells recovered to approximately 70% the control level within 7 min suggesting that many, but perhaps not all, kinetochore fiber microtubules of metaphase cells do turn over. Analysis of the movements of metaphase bleached zones suggested that a slow poleward translocation of kinetochore microtubules occurred. However, within the variation of the data (0.12 +/- 0.24 micron/min), it could not be determined whether the apparent movement was real or artifactual.     

Spindle membranes and calcium sequestration during meiosis ofDysdercus intermedius (Heteroptera)

The fate of intracellular membranes stained by the osmium ferricyanide (OsFeCN) procedure was followed from premeiotic interphase to interkinesis inDysdercus intermedius. During diakinesis the centrioles forming primary cilia attach temporarily with their proximal ends to the nuclear envelope which is stretched from pole to pole. Breakdown of the nuclear envelope is preceded by deep indentations with microtubules from growing asters. Vesicles of smooth endoplasmic reticulum which accumulate gradually in the course of prophase contribute to the ensheathment of the chromosomes with membranes. When the nuclear envelope breaks down, the polar parts of the formerly perinuclear membranes follow the ingrowth of the spindle microtubules towards the cell equator where the seven bivalents are arranged in a circle with the X₁X₂ sex chromosomes in the centre. The metaphase I spindle thus contains longitudinally oriented membranes between the poles, membranous envelopes around all chromosomes and radial connections from the autosomes to the sex chromosomes in the centre. At anaphase the homologues leave their common sheath and a microtubular stembody surrounded by membranes appears between the receding dyads. In the interkinetic nucleus the gonosomes are separated from the autosomes by a common membranous sheath which may be instrumental in their joint assignment to only one pole in the second meiotic division. Calcium sequestering sites visualized by oxalate precipitation are the Golgi lamellae and vesicles derived from them that surround the whole spindle body.     

Acetylated alpha-tubulin in microtubules during mouse fertilization and early development

alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Behavior of kinetochores during mitosis in the fungus Saprolegnia ferax

In rapidly growing hyphae of Saprolegnia ferax, all nuclei contain arrays of kinetochore microtubules, which suggests that the nuclei are all in various phases of mitosis, with no apparent interphase. In prophase nuclei, kinetochore microtubules form a single, hemispherical array adjacent to the centrioles. This array separates into two similar arrays after centriole replication. The two arrays form by separation of the initial group of microtubules, with no kinetochore replication. During metaphase, between 6.5 and 85% of the kinetochores occur as amphitelic pairs, with a slight tendency for pairing to increase as the spindle elongates. 100% pairing has never been observed. The interkinetochore distance in these pairs is consistently similar to or approximately 0.17 microns. Throughout metaphase and early anaphase, there is extensive and increasing diversity in kinetochore microtubule length, so that a true metaphase plate has not been found. During metaphase, anaphase, and telophase, kinetochore numbers vary considerably, with a mean of similar to or approximately 30 per half spindle. A number of artefactual causes for this variability were examined and discarded. Thus, these results are accepted as real, suggesting either variable ploidy levels in the coenocytic hyphae or kinetochore replication during mitosis.     

Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle

The polarity of kinetochore microtubules (MTs) has been studied in lysed PtK1 cells by polymerizing hook-shaped sheets of neurotubulin onto walls of preexisting cellular MTs in a fashion that reveals their structural polarity. Three different approaches are presented here: (a) we have screened the polarity of all MTs in a given spindle cross section taken from the region between the kinetochores and the poles, (b) we have determined the polarity of kinetochore MTs are more stable to cold-treated spindles; this approach takes advantage of the fact that kinetochore MTs are more stable to cold treatment than other spindle MTs; and (c) we have tracked bundles of kinetochore MTs from the vicinity of the pole to the outer layer of the kinetochore in cold- treated cells. In an anaphase cell, 90-95% of all MTs in an area between the kinetochores and the poles are of uniform polarity with their plus ends (i.e., fast growing ends) distal to the pole. In cold- treated cells, all bundles of kinetochore MTs show the same polarity; the plus ends of the MTs are located at the kinetochores. We therefore conclude that kinetochore MTs in both metaphase and anaphase cells have the same polarity as the aster MTs in each half-spindle. These results can be interpreted in two ways: (a) virtually all MTs are initiated at the spindle poles and some of the are "captured" by matured kinetochores using an as yet unknown mechanism to bind the plus ends of existing MTs; (b) the growth of kinetochore MTs is initiated at the kinetochore in such a way that the fast growing MT end is proximal to the kinetochore. Our data are inconsistent with previous kinetochore MT polarity determinations based on growth rate measurements in vitro. These studies used drug-treated cells from which chromosomes were isolated to serve as seeds for initiation of neurotubule polymerization. It is possible that under these conditions kinetochores will initiate MTs with a polarity opposite to the one described here.     

Functional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy

The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is "park," in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.     

Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle

We argue that hypotheses for how chromosomes achieve a metaphase alignment, that are based solely on a tug-of-war between poleward pulling forces produced along the length of opposing kinetochore fibers, are no longer tenable for vertebrates. Instead, kinetochores move themselves and their attached chromosomes, poleward and away from the pole, on the ends of relatively stationary but shortening/elongating kinetochore fiber microtubules. Kinetochores are also "smart" in that they switch between persistent constant-velocity phases of poleward and away from the pole motion, both autonomously and in response to information within the spindle. Several molecular mechanisms may contribute to this directional instability including kinetochore-associated microtubule motors and kinetochore microtubule dynamic instability. The control of kinetochore directional instability, to allow for congression and anaphase, is likely mediated by a vectorial mechanism whose magnitude and orientation depend on the density and orientation or growth of polar microtubules. Polar microtubule arrays have been shown to resist chromosome poleward motion and to push chromosomes away from the pole. These "polar ejection forces" appear to play a key role in regulating kinetochore directional instability, and hence, positions achieved by chromosomes on the spindle.     

The effect of trifluoroperazine on microtubules,nuclear division and the nuclear membranes of the ciliate Nyctotherus ovalis

Exposure of the dividing ciliate Nyctotherus ovalis to the tranquilizer trifluoroperazine (TFP; 10 M) leads to the complete disassembly of kinetochore microtubules in the metaphase micronucleus. Interpolar microtubules located underneath the micronuclear envelope at anaphase and telophase stembody microtubules are more resistant to TFP. However, stembodies of drug-exposed ciliates are much shorter than in the controls. In their centre they contain only a reduced number of widely separated microtubules, indicating that assembly of new tubules or elongation of existing microtubules at this site, which appears essential for further separation of the future daughter nuclei, is blocked by TFP. Although microtubules polymerized in the macronucleus during its elongation include a set of tubules made up of more than 13 protofilaments, comparable to the micronuclear stembody microtubules, they are much more sensitive towards drug treatment. Macronuclear tubules become completely depolymerized resulting in failure of nuclear stretching. Already elongated macronuclei can still become constricted in their centre which suggests that microtubules are not involved in this process. Disassembly and higher sensitivity of macronuclear compared with micronuclear microtubules may be explained by a different composition and behaviour of nuclear membranes towards TFP in the two types of nuclei. While the micronuclear envelope may be only partially destroyed where it is facing the macronucleus, the inner membrane of the macronuclear envelope is severely affected by drug treatment. It shows a multitude of infoldings accompanied by attachment of chromatin to it. Cytoplasmic microtubules which proved resistant to other depolymerizing drugs become partly disassembled during TFP treatment.