The Mechanics of Anaphase B in a Basidiomycete as Revealed by Laser Microbeam Microsurgery

Bayles, C. J., Aist, J. R., and Berns, M. W. 1993. The mechanics of anaphase B in a basidiomycete as revealed by laser microbeam microsurgery. Experimental Mycology 17, 191-199. Cytoplasmic forces were found to be actively pulling on the spindle pole bodies during anaphase B in the dikaryotic, basidiomycete fungus, Helicobasidium mompa. When the spindle of one nucleus was severed with a laser microbeam at mid anaphase B, its two spindle pole bodies separated at a much faster rate than did those of the intact spindle in the other nucleus of the same cell. Since astral microtubule populations apparently reach their maximum during anaphase B in this fungus, we suggest that these microtubules may be involved in the cytoplasmic pulling forces. The spindle appears to act primarily as a governor, regulating the rate at which the spindle pole bodies are separated.     

The force-producing mechanism for centrosome separation during spindle formation in vertebrates is intrinsic to each aster

A popular hypothesis for centrosome separation during spindle formation and anaphase is that pushing forces are generated between interacting microtubules (MTs) of opposite polarity, derived from opposing centrosomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prometaphase when their MT arrays no longer overlap (i.e., during anaphase-like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is mediated by a common mechanism we compared their behavior in vivo at a high spatial and temporal resolution. We found that the two centrosomes possess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome does not impart a predictable behavior to the other, and both exhibit frequent and rapid (4-6 microns/min) displacements toward random points within the cell including the other centrosome. The kinetic behavior of individual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of motion toward and away from the other centrosome, the frequency of toward and away motion as well as motion not contributing to separation, and the rate contributed by each centrosome to the separation process. By contrast, when compared with prometaphase, anaphase centrosomes separated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in prophase/prometaphase. The difference in separation rates arises because anaphase centrosomes spend less time moving toward one another than in prophase/prometaphase, and at a significantly slower rate. From our data we conclude that the force for centrosome separation during vertebrate spindle formation is not produced by MT-MT interactions between opposing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.     

Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells

When viewed by light microscopy the mitotic spindle in newt pneumocytes assembles in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which can be much larger than the forming spindle. This unique optical property has allowed us to examine the behavior of individual microtubules, at the periphery of asters in highly flattened living prometaphase cells, by video-enhanced differential interference-contrast light microscopy and digital image processing. As in interphase newt pneumocytes (Cassimeris, L., N. K. Pryer, and E. D. Salmon. 1988. J. Cell Biol. 107:2223-2231), centrosomal (i.e., astral) microtubules in prometaphase cells appear to exhibit dynamic instability, elongating at a mean rate of 14.3 +/- 5.1 microns/min (N = 19) and shortening at approximately 16 microns/min. Under favorable conditions the initial interaction between a kinetochore and the forming spindle can be directly observed. During this process the unattached chromosome is repeatedly probed by microtubules projecting from one of the polar regions. When one of these microtubules contacts the primary constriction the chromosome rapidly undergoes poleward translocation. Our observations on living mitotic cells directly demonstrate, for the first time, that chromosome attachment results from an interaction between astral microtubules and the kinetochore.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.     

Spindle positioning in fibroblasts supports an astral microtubule length dependent force generation at the basal membrane.

V79 Chinese hamster fibroblasts that maintain an elongated shape in metaphase occur at a low frequency and often show the spindle asymmetrically positioned. We show here that this aberrant position is corrected in anaphase by an external force, pulling the spindle into place. The force was applied on astral microtubules because spindle motility was hampered when astral microtubules were poorly developed spontaneously, or destroyed by colcemid. Colcemid also abolished the observed downward positioning of centrosomes in anaphase. One pole of the spindle was usually dominant during correction, but occasionally both poles could become subject to pulling making the spindle move perpendicular to the long axis of the cell, which induced reshaping of the cell. The pulling force acted unevenly with short intervals of resting between the pulling. Spindle elongation also varied in rate but showed a different periodicity than translocation of the spindle, and therefore appeared independently regulated. The length of the spindle increased with the length of the cell, and the rate of spindle elongation and pole movement increased with distance moved, indicating that the forces mediated by astral microtubules increase with their length. Arp1/dynactin, not colocalising with tubulin, was more often continuous with microtubules in anaphase B than in metaphase, and was primarily located at the bottom of the cell. Further, shifts in the geometric gravity centre of the cell occurred in the same direction as migration of the spindle. To explain these results, we suggest that astral microtubles transiently anchored at the bottom of the cell are of particular importance for spindle translocation in fibroblasts.     

Reorientation of mispositioned spindles in short astral microtubule mutant spc72Delta is dependent on spindle pole body outer plaque and Kar3 motor protein

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic gamma-tubulin complex, can only generate very short (<1 microm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.     

Microtubule- and actin filament-dependent motors are distributed on pollen tube mitochondria and contribute differently to their movement

The pollen tube exhibits cytoplasmic streaming of organelles, which is dependent on the actin-myosin system. Although microtubule-based motors have also been identified in the pollen tube, many uncertainties exist regarding their role in organelle transport. As part of our attempt to understand the role of microtubule-based movement in the pollen tube of tobacco, we investigated the cooperation between microtubules and actin filaments in the transport of mitochondria and Golgi vesicles, which are distributed differently in the growing pollen tube. The analysis was performed using in vitro motility assays in which organelles move along both microtubules and actin filaments. The results indicated that the movement of mitochondria and Golgi vesicles is slow and continuous along microtubules but fast and irregular along actin filaments. In addition, the presence of microtubules in the motility assays forces organelles to use lower velocities. Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different organelles bind to identical myosins but associate with specific kinesins. We found that a 90 kDa kinesin (previously known as 90 kDa ATP-MAP) is associated with mitochondria but not with Golgi vesicles, whereas a 170 kDa myosin is distributed on mitochondria and other organelle classes. In vitro and in vivo motility assays indicate that microtubules and kinesins decrease the speed of mitochondria, thus contributing to their positioning in the pollen tube.     

Microtubule-dependent pushing forces contribute to long-distance aster movement and centration in Xenopus laevis egg extracts

During interphase of the eukaryotic cell cycle, the microtubule (MT) cytoskeleton serves as both a supportive scaffold for organelles and an arborized system of tracks for intracellular transport. At the onset of mitosis, the position of the astral MT network, specifically its center, determines the eventual location of the spindle apparatus and ultimately the cytokinetic furrow. Positioning of the MT aster often results in its movement to the center of a cell, even in large blastomeres hundreds of microns in diameter. This translocation requires positioning forces, yet how these forces are generated and then integrated within cells of various sizes and geometries remains an open question. Here we describe a method that combines microfluidics, hydrogels, and Xenopus laevis egg extract to investigate the mechanics of aster movement and centration. We determined that asters were able to find the center of artificial channels and annular cylinders, even when cytoplasmic dynein-dependent pulling mechanisms were inhibited. Characterization of aster movement away from V-shaped hydrogel barriers provided additional evidence for a MT-based pushing mechanism. Importantly, the distance over which this mechanism seemed to operate was longer than that predicted by radial aster growth models, agreeing with recent models of a more complex MT network architecture within the aster.     

In vitro reconstitution of fish melanophore pigment aggregation

Movement and positioning of melanophore pigment organelles depend on microtubule- and actin-dependent motors, but the regulation of these forces are poorly understood. Here, we describe a cell free and fixed time motility assay for the study of the regulation of microtubule-dependent pigment organelle positioning in vitro. The assay involves introduction of microtubule-asters assembled in clam oocyte lysates into lysates prepared from Fundulus heteroclitus melanophores with either aggregated or dispersed pigment. When asters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distributed throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organelles accumulated around the centrosome, mimicking a pigment aggregate. The addition of anti-dynein intermediate chain antibody (m74-1), previously shown to interfere with binding of dynactin to dynein and thereby causing detachment of dynein from organelles, blocked the ATP-dependent aggregation of pigment in vitro and induced a depletion of pigment from the centrosomal area. The results show that dynein is essential for pigment aggregation and involved in maintenance of evenly dispersed pigment in vitro, analogous to cellular evidence, and suggest a possible role for dynactin in these processes as well.     

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster. Received: 29 September 1999 / Accepted: 24 November 1999     

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.     

Microinjected carboxylated beads move predominantly poleward in sea urchin eggs

Observations on living mitotic cells have suggested that material in the spindle moves poleward during mitosis. In order to investigate this movement, sea urchin eggs have been microinjected with 0.25-micron diameter carboxylated fluorescent beads. When fluorescent beads were injected into unfertilized Lytechinus variegatus eggs, no motility was detected. When injected into mitotic cells, beads moved to the spindle poles. Individual beads moved rapidly, in a saltatory fashion, and followed generally linear paths. Beads appeared to move along astral fibers, were generally excluded from the spindle proper, and accumulated at the spindle poles. Some dispersion of the beads away from the pole was observed as cells completed mitosis, but the majority of beads retained a polar location. After depolymerization of spindle microtubules with nocodazole, some dispersion of beads into the cytoplasm was also observed. Beads moved along taxol-induced astral microtubules and accumulated at astral centers. These observations reveal that negatively charged beads accumulate rapidly at mitotic centers, moving toward the minus end of the microtubules. Neither the bidirectional motility of similar beads in interphase cells nor the plus-end-directed bead motility seen in axons was observed in these mitotic cells.     

A Drosophila orthologue of larp protein family is required for multiple processes in male meiosis

It is important for the proper execution of cell division in both mitosis and meiosis that the chromosome segregation, cytokinesis, and partition of cell organelles progress in smooth coordination. We show here that the mitochondria inheritance is closely linked with microtubules during meiotic divisions in Drosophila males. They are first clustered in a cell equator at metaphase associated with astral microtubules and then distributed along central spindle microtubules after anaphase. The molecular mechanism for the microtubule-dependent inheritance of mitochondria in male meiosis has not been demonstrated yet. We first isolated mutations for a larp gene that is highly conserved among eukaryotes and showed that these mutant males exhibited multiple meiotic phenotypes such as a failure of chromosome segregation, cytokinesis, and mitochondrial partition. Our cytological examination revealed that the mutants showed defects in spindle pole organization and spindle formation. The larp encodes a Drosophila orthologue of a La-related protein containing a domain exhibiting an outstanding homology with a La type RNA-binding protein. Surprisingly, the dLarp protein is localized in the cytoplasm of the male germ line cells, as observed by its distinct co-localization with mitochondria in early spermatocytes and during meiotic divisions. We discuss here the essential role that dLarp plays in multiple processes in Drosophila male meiosis.     

Regulation of mitotic spindle asymmetry by SUMO and the spindle-assembly checkpoint in yeast

During mitosis, the kinetochore microtubules capture and segregate chromosomes, and the astral microtubules position the spindle within the cell. Although the spindle is symmetric, proper positioning of the spindle in asymmetrically dividing cells generally correlates with the formation of morphologically and structurally distinct asters [1]. In budding yeast, the spindle-orientation proteins Kar9 and dynein decorate only one aster of the metaphase spindle and direct it toward the bud [2, 3]. The mechanisms controlling the distribution of Kar9 and dynein remain unclear. Here, we show that SUMO regulates astral-microtubule function in at least two ways. First, Kar9 was sumoylated in vivo. Sumoylation and Cdk1-dependent phosphorylation of Kar9 independently promoted Kar9 asymmetry on the spindle. Second, proper regulation of kinetochore function by SUMO was also required for Kar9 asymmetry. Indeed, activation of the spindle-assembly checkpoint (SAC) due to SUMO and kinetochore defects promoted symmetric redistribution of Kar9 in a Mad2-dependent manner. The control of Kar9 distribution by the SAC was independent of Kar9 sumoylation and phosphorylation. Together, our data reveal that three independent mechanisms contribute to Kar9 asymmetry: Cdk1-dependent phosphorylation, sumoylation, and SAC signaling. Hence, the two seemingly independent spindle domains, kinetochores and astral microtubules, function in a tightly coordinated fashion.     

NuMA is required for the organization of microtubules into aster-like mitotic arrays

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.     

Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase. The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase. Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole. The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis. After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers. There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form. In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosome displays many features of a standard anaphase and may be followed by a false cleavage. The process of polar separation during prometaphase occurs without any visible interzonal structures. Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement. The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.     

Asymmetric division in mouse oocytes: with or without Mos

In both vertebrates and invertebrates, meiotic divisions in oocytes are typically asymmetric, resulting in the formation of a large oocyte and small polar bodies. The size difference between the daughter cells is usually a consequence of asymmetric positioning of the spindle before cytokinesis. Spindle movements are often related to interactions between the cell cortex and the spindle asters [1,2]. The spindles of mammalian oocytes are, however, typically devoid of astral microtubules, which normally connect the spindle to the cortex, suggesting that another mechanism is responsible for the unequal divisions in these oocytes. We observed the formation of the first polar body in wild-type oocytes and oocytes derived from c-Mos knockout mice [3]. In wild-type oocytes, the meiotic spindle formed in the centre of the cell and migrated to the cortex just before polar-body extrusion. The spindle did not elongate during anaphase. In mos-/- oocytes, the spindle formed centrally but did not migrate, although an asymmetric division still took place. In these oocytes, the spindle elongated during anaphase and the pole closest to the cortex moved while the other remained in place. Thus, a compensation mechanism exists in mouse oocytes and formation of the first polar body can be achieved in two ways: either after migration of the spindle to the cortex in wild-type oocytes, or after elongation, without migration, of the first meiotic spindle in mos-/- oocytes.     

Light and electron microscopy of Rat kangaroo cells in mitosis

Rat kangaroo (PtK₂) cells were fixed and embedded in situ. Cells in mitosis were studied with the light microscope and thin sections examined with the electron microscope. Pericentriolar, osmiophilic material, rather than the centrioles, is probably involved in the formation of astral microtubules during prophase. Centriole migration occurs during prophase and early prometaphase. The nuclear envelope ruptures first in the vicinity of the asters. Nuclear pore complexes disintegrate as envelope fragments are dispersed to the periphery of the mitotic spindle. Microtubules invade the nucleus through gaps of the fragmented envelope. The number of microtubules and the degree of spindle organization increase during prometaphase and are maximal at metaphase. At this stage, chromosomes are aligned on the spindle equator, sister kinetochores facing opposite poles. Cytoplasmic organelles are excluded from the spindle. Prominent bundles of kinetochore microtubules converge towards the poles. Spindles in cold-treated cells consist almost exclusively of kinetochore tubules. Separating daughter chromosomes in early anaphase are connected by chromatin strands, possibly reflecting the rupturing of fibrous connections occasionally observed between sister chromatids in prometaphase. Breakdown of the spindle progresses from late anaphase to telophase, except for the stem bodies. Chromosomes decondense to form two masses. Nuclear envelope reconstruction, probably involving endoplasmic reticulum, begins on the lateral faces. Nuclear pores reappear on membrane segments in contact with chromatin. Microtubules are absent from reconstructed daughter nuclei.This report is to a large part based on a dissertation submitted by the author to the Graduate Council of the University of Florida in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Formation and function of the polar body contractile ring in Spisula

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility-previously assumed-was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis.