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.     

Three-dimensional structure of the central mitotic spindle of Diatoma vulgare

Central mitotic spindles in Diatoma vulgare have been investigated using serial sections and electron microscopy. Spindles at both early stages (before metaphase) and later stages of mitosis (metaphase to telophase) have been analyzed. We have used computer graphics technology to facilitate the analysis and to produce stereo images of the central spindle reconstructed in three dimensions. We find that at prometaphase, when the nuclear envelope is dissassembling, the spindle is constructed from two sets of polar microtubules (MTs) that interdigitate to form a zone of overlap. As the chromosomes become organized into the metaphase configuration, the polar MTs, the spindle, and the zone of overlap all elongate, while the number of MTs in the central spindle decreases from greater than 700 to approximately 250. Most of the tubules lost are short ones that reside near the spindle poles. The previously described decrease in the length of the zone of overlap during anaphase central spindle elongation is clearly demonstrated in stereo images. In addition, we have used our three- dimensional data to determine the lengths of the spindle MTs at various times during mitotis. The distribution of lengths is bimodal during prometaphase, but the short tubules disappear and the long tubules elongate as mitosis proceeds. The distributions of MT lengths are compared to the length distributions of MTs polymerized in vitro, and a model is presented to account for our findings about both MT length changes and microtubule movements.     

Spindle microtubule dynamics following ultraviolet-microbeam irradiations of mitotic diatoms

Lesions ("ARBs") generated in metaphase and anaphase central spindles of Hantzschia by an ultraviolet microbeam are devoid of microtubules previously present. In vivo, the poleward transverse edge of the lesion invariably loses birefringence poleward, until this segment has vanished; the loss is slow during metaphase and faster at anaphase. The other transverse edge, proximal to the overlap, remains stable until disassembly of the whole spindle. We conclude that the central spindle microtubules are not in flux during metaphase to telophase, and that depolymerization of these microtubules takes place only from the end distal to the pole, as during normal spindle disassembly. Microtubule polarity and the creation of free ends may determine which microtubules are disassembled during later mitosis and how disassembly proceeds.     

Analysis of the distribution of spindle microtubules in the diatom Fragilaria.

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.     

ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO : Direct Microtubule Counts

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK₁) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.     

Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. Evidence for specific interactions between antiparallel microtubules

During the transition from prometaphase to metaphase, the cross- sectional area of the central spindle of Diatoma decreases by a factor of nearly two, both at the poles and at the region of overlapping microtubules (MTs) near the spindle equator. The density of spindle MT packing stays approximately constant throughout mitosis. Optical diffraction analysis of electron micrographs shows that the packing of the MTs at the poles at all stages of mitosis is similar to that expected for a two-dimensional liquid. Analysis of the region of overlap reveals more packing regularity: during prometaphase, a square packing emerges that displays sufficient organization by late metaphase to generate five orders of diffraction; during anaphase the packing in the overlap region shifts to hexagonal; at telophase, it returns to square. From the data provided by serial section reconstructions of the central spindle, it is possible to identify the polarity of almost every spindle MT, that is, to identify one pole with which the MT is associated. Near neighbor analyses of MTs in cross sections of the overlap region show that MTs prefer antiparallel near neighbors. These near neighbors are most often found at a spacing of approximately 40 nm center-to-center, while parallel near neighbors in the zone of overlap are spaced essentially at random. These results are evidence for a specific interaction between antiparallel MTs. In some sections definite bridges between MTs can be seen. Our findings show that certain necessary conditions for a sliding filament model of anaphase spindle elongation are met.     

Directly probing the mechanical properties of the spindle and its matrix

Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.     

The mitotic apparatus during unequal microspore division observed by a confocal laser scanning microscope

Summary The structure of the mitotic apparatus during the microspore division ofTradescantia paludosa, which has a distinctively unequal division of large vegetative and small generative cells, was studied using -tubulin immunofluorescence methods and confocal laser scanning microscopy. Mitotic apparatuses began to develop asynchronously during early prophase at the vegetative pole (VP) and during prometaphase at the generative pole (GP). Both, however, reached completion together at the same time during metaphase. At the VP from prophase to prometaphase, microtubules (MTs) did not converge on the pole, and there was a circular area containing only a few MTs. The prophase spindles on the VP side were in the form of domes or cones that lacked the top. In the metaphase, however, the MTs concentrated at the pole to form a representative cone-shaped half-spindle. At the GP from prometaphase to metaphase, the MTs did not concentrate, and a circular area existed that lacked MTs. The half-spindles formed truncated cones. When the phragmoplast developed and curved around the generative nucleus during the telophase. it first grew toward the long axis of the ellipsoidal-shaped microspore; and after it arrived at the inner membrane of the microspore, it again curved past the generative nucleus toward the short axis. In conclusion, it was found that the mitotic apparatus ofT. paludosa microspores with its asynchronous growth and asymmetrical spindle structure and with its three dimensional growth of phragmoplasts had a peculiar developmental manner related to unequal division.     

Confocal laser scanning microscopy of microtubule organizational changes in isolated generative cells of Allemanda neriifolia during mitosis

Summary Organizational changes in the microtubules of isolated generative cells of Allemanda neriifolia during mitosis were examined using anti--tubulin and confocal laser scanning microscopy. Due to an improved resolution and a lack of out-of-focus interference, the images of the mitotic cytoskeleton obtained using the confocal microscope are much clearer than those obtained using the non-confocal fluorescence systems. In the confocal microscope one can see clearly that the spindle-shaped interphase cells contain a cage-like cytoskeleton consisting of numerous longitudinally oriented microtubule bundles and some associated smaller bundles. At prophase, the shape of the cells invariably becomes spherical. The microtubule cytoskeleton inside the cells concomitantly changes into a less organized form — consisting of thick bundles, patches, and dots. This structural form is not very stable, and soon afterwards the cytoskeleton changes into a reticulate network. Then the nuclear envelope breaks down, and the microtubules become randomly dispersed throughout the cell. Afterwards, the microtubules reorganize themselves into a number of half-spindle-like structures, each possessing a microtubule-nucleating center. The locations of these centres mark out the positions of the presumptive spindle poles. Numerous microtubules radiate from these centres toward the opposite pole. At metaphase, the microtubules form a number of bipolar spindles. Each spindle has two half-spindles, and each half-spindle has a sharply focused microtubule centre at the pole region. From the centres, kinetochore and non-kinetochore microtubules radiate toward the opposite half-spindle. At anaphase A, sister chromatids separate, the cells elongate, and the kinetochore microtubules disappear; the non-kinetochore microtubules, however, remain, and a new array of microtubules, in the form of a cage, appears. The peripheral cage bundles and the non-kinetochore bundles coverge into a sharp point at the pole region. Later, at anaphase B the microtubule cytoskeleton undergoes reorganization giving rise to a new array of longitudinally oriented microtubule bundles in the cell centre and a cage-like cytoskeleton in the periphery. At telophase, some of the cells elongate further, but some become spherical. The microtubules in the central region of the elongated cell become partially disrupted due to the formation of a phragmoplast-junction-like structure in the mid-interzone region. The microtubule bundles at the periphery are spirally organized, and they appear not to be disrupted by the phragmoplast-like junction. The microtubules in the spherical telophase cells (unlike those seen in the elongated telophase cells) are arranged differently, and no phragmoplast-junction-like structure forms in the spherical cells. The structural and functional significances of some of these new features of the organization of the microtubule cytoskeleton as revealed by the confocal microscope are discussed.     

The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation

To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the "new" overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole.     

Spindle and kinetochore morphology of Dictyostelium discoideum

The metaphase spindle of haploid Dictyostelium discoideum (n = 7) is 2 mum long. It consists of some 20 microtubules which seem continuous between the spindle pole bodies and there are about 20 chromosomal microtubules at each end of the spindle. During anaphase the central spindle elongates and the chromosomal microtubules shorten. The spindle length and structure at this stage suggests that lengthening is caused by elongation as well as parallel sliding of the nonchromosomal microtubules. The nuclear envelope remains mostly intact during mitosis, and nuclear separation through medial constriction takes place when the spindle is 6 mum long. Cytokinesis occurs when the spindle is 10 mum long. At that time the kinetochores double in size. During interphase, the spindle pole body separates from the nucleus to a distance of 0.7 mum, and it returns at the onset of the next prophase when it becomes functionally double, thereby starting the formation of a central spindle. When comparing mitosis in the cellular slime molds Polysphondylium violaceum and D. discoideum, several similarities and some differences are apparent.     

Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments

The anaphase-telophase spindle usually elongates, and it has been assumed that the spindle pushes the incipient daughter nuclei apart. To test this assumption, we used a laser microbeam to sever the central spindle of the fungus, Fusarium solani, and measured the rate of separation of incipient daughter nuclei. When the microbeam was aimed beside the spindle separation occurred at a rate (8.6 micrometer/min) that did not differ significantly from the rate (7.6 micrometer/m) in unirradiated cells. But when the spindle was irradiated, it broke, and the separation was much faster (22.4 micrometer/min). Irradiation of cytoplasm lateral to one spindle pole resulted in a 1.5 micrometer/min reduction in the rate (6.1 micrometer/min) of separation. From these and other data, we infer that extranuclear forces, presumably involving astral microtubules, pull on the incipient daughter nuclei and that the central spindle limits the separation rate. Astral microtubules are associated with the plasma membrane or, sometimes, with the rough endoplasmic reticulum. Most of the spindle microtubules that are present at metaphase are depolymerized during anaphase and early telophase.     

Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.     

Ultrastructure of mitosis in the cowpea rust fungus Uromyces phaseoli var. Vignae

Aspects of the ultrastructure of mitotic nuclei of the fungus Uromyces phaseoli var. vignae are described from both intercellular hyphae in the cowpea host and infection structures induced to differentiate in vitro. The interphase nucleus-associated organelle (NAO) consists of two trilamellar acircular disks connceted by an osmiophilic bar. The intranuclear spindle develops between these disks when they separate. The spindle contains pole to pole, interdigitating, chromosomal, and fragmentary microtubules arranged to form a central bundle along the surface of which lie the metaphase chromosomes. No metaphase plate is found. There are up to three microtubules per kinetochore and approximately 14 chromosomes on the haploid spindle. Telophase elongation appears to involve extension of pole to pole microtubules with no evidence for the remaining presence of interdigitating microtubules. Concomitantly, numerous cytoplasmic microtubules develop from each NAO disk where few or none are present in other phases. Reformation of the interphase NAO involves the formation of a sausage- shaped intermediate at late telophase. The nuclear envelope remains intact and the nucleolus persists throughtout division. Various aspects of the spindle and NAOs appear to be evolutionary intermediates between Ascomycetes and higher Basidiomycetes, thus supporting the theory of Basidiomycete evolution from the former group and demonstrating an encouraging correlation between mitotic characteristics and other phylogenetic markers.     

ULTRASTRUCTURAL FEATURES OF MITOSIS IN PLOEOTIA COSTATA (HETERONEMATALES,EUGLENOPHYTA)1

The ultrastructural features of mitosis in the colorless phagotrophic euglenoid, Ploeotia costata (Farmer and Triemer 1988bn; syn: Serpenomonas costata, Triemer 1986) are described. During interphase the nucleus is rounded and lies adjacent to the reservoir and the four basal bodies, two of which bear flagella. At the onset of mitosis, two additional flagella are generated from the accessory basal bodies such that four basal bodies with flagella now lie at one pole of the prophase nucleus. Microtubules develop in the nucleus prior to migration of one of the basal body pairs to the opposite pole of the nucleus. By metaphase, chromosomes with layered kinetochores are aligned on the equator of the spindle, and a dumbbellshaped nucleolus stretches from pole to pole. Continued elongation of the nucleus results in the separation of the chromosomal masses at anaphase. The distance between the nuclear poles from metaphase to anaphase changes little although the overall length of the nucleus nearly doubles. By telophase a large interzonal spindle develops between the forming daughter nuclei. The extended interzonal spindle breaks near the center prior to cell cleavage.     

Physiological and ultrastructural analysis of elongating mitotic spindles reactivated in vitro

We have developed a simple procedure for isolating mitotic spindles from the diatom Stephanopyxis turris and have shown that they undergo anaphase spindle elongation in vitro upon addition of ATP. The isolated central spindle is a barrel-shaped structure with a prominent zone of microtubule overlap. After ATP addition greater than 75% of the spindle population undergoes distinct structural rearrangements: the spindles on average are longer and the two half-spindles are separated by a distinct gap traversed by only a small number of microtubules, the phase-dense material in the overlap zone is gone, and the peripheral microtubule arrays have depolymerized. At the ultrastructural level, we examined serial cross-sections of spindles after 1-, 5-, and 10-min incubations in reactivation medium. Microtubule depolymerization distal to the poles is confirmed by the increased number of incomplete, i.e., c-microtubule profiles specifically located in the region of overlap. After 10 min we see areas of reduced microtubule number which correspond to the gaps seen in the light microscope and an overall reduction in the number of half-spindle microtubules to about one-third the original number. The changes in spindle structure are highly specific for ATP, are dose-dependent, and do not occur with nonhydrolyzable nucleotide analogues. Spindle elongation and gap formation are blocked by 10 microM vanadate, equimolar mixtures of ATP and AMPPNP, and by sulfhydryl reagents. This process is not affected by nocodazole, erythro-9-[3-(2-hydroxynonyl)]adenine, cytochalasin D, and phalloidin. In the presence of taxol, the extent of spindle elongation is increased; however, distinct gaps still form between the two half-spindles. These results show that the response of isolated spindles to ATP is a complex process consisting of several discrete steps including initiation events, spindle elongation mechanochemistry, controlled central spindle microtubule plus-end depolymerization, and loss of peripheral microtubules. They also show that the microtubule overlap zone is an important site of ATP action and suggest that spindle elongation in vitro is best explained by a mechanism of microtubule-microtubule sliding. Spindle elongation in vitro cannot be accounted for by cytoplasmic forces pulling on the poles or by microtubule polymerization.     

Substructural morphogenesis of the spindle apparatus in meiotic basidia ofCoprinus micaceus (Agaricales)

The spindle apparatus ofCoprinus micaceus begins to develop from the diglobular polar body outside the nucleus. During both meiotic divisions it operates inside the nuclear envelope and consists of two amorphous poles, a central bundle of interpolar microtubules, and chromosomal microtubules. A metaphase plate cannot exist because the interpolar strand of fibers is persistent throughout the division process. Within the spindle axis more than 100 microtubules can be estimated. They are encircled by a ring of chromatic structures. During the telophase the former spindle pole is evaginated from the nuclear envelope and contacts the plasmalemma near the cell wall.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA (CHLOROPHYTA)1

Cell division is described in the octaflagellate prasinophyte Pyramimonas amylifera Conrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.     

Dynamics of a fluorescent calmodulin analog in the mammalian mitotic spindle at metaphase

We have compared the exchange kinetics of fluorescein-labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady-state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells pre-equilibrated to either 26 +/- 2 degrees C or 36 +/- 2 degrees C. A pulse of laser light focused to a 5-microns diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half-spindles and from the two polar regions were calculated for each image in a post-bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26 degrees C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 +/- 2 degrees C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.