Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm

The skeletal framework of cells at the various stages of mitosis are prepared by extraction with nonionic detergent and examined by stereoscopic whole mount electron microscopy. The insoluble filament network remaining after the detergent-extraction and the depolymerization of microtubules is shown. The nonchromatin filament network of the nucleus, or nuclear matrix, becomes visible as the chromatin condenses at prophase. Filaments are associated with the chromosomes throughout mitosis. Parts of the chromosomes are associated with or are near the nuclear lamina at early stages. The nuclear lamina disappears at metaphase while chromosomes remain associated with filaments now continuous with the cytoplasmic network. Microtubules appear to be unnecessary for maintaining the chromosome position in these preparations since comparison of cells with and without microtubules shows no gross change in chromosome arrangement. The cellular filament network at metaphase and anaphase appears continuous from the plasma lamina to the chromosomes. The filament networks visualized here may be responsible for the prometaphase chromosome movement and participate in the formation of the midbody.     

Origin of the mitotic spindle in onion root cells

Summary Immunofluorescence studies on microtubule arrangement during the transition from prophase to metaphase in onion root cells are presented. The prophase spindle observed at late preprophase and prophase is composed of microtubules converged at two poles near the nuclear envelope; thin bundles of microtubules are tracable along the nuclear envelope. Prior to nuclear envelope breakdown diffuse tubulin staining occurs within the prophase nuclei. During nuclear envelope breakdown the prophase spindle is no longer identifiable and prominent tubulin staining occurs among the prometaphase chromosomes. Patches of condensed tubulin staining are observed in the vicinity of kinetochores. At advanced prometaphase kinetochore bundles of microtubules are present in some kinetochore regions. At metaphase the mitotic spindle is mainly composed of kinetochore bundles of microtubules; pole-to-pole bundles are scarce. Our observations suggest that the prophase spindle is decomposed at the time of nuclear envelope breakdown and that the metaphase spindle is assembled at prometaphase, with the help of kinetochore nucleating action.     

Dynamic changes in autosomal spindle fibers during prometaphase in crane fly spermatocytes

Different prometaphase stages of Pales ferruginea spermatocytes were serially sectioned and the regions between kinetochores and poles analysed by counting and measuring spindle microtubules. These regions are characterized by an intermingling of kinetochoric (kMTs) and non-kinetochoric microtubules (nkMTs). A considerable proportion of nkMTs is skewed with respect to kMTs, thus being responsible for microtubule disorder in these spindle areas. The degree of disorder expressed by the percentage of skew microtubules was found to decrease from early prometaphase to metaphase, parallel with an increase in kMT number. A possible causal relation between pulling forces and morphological changes in the spindle is discussed.     

Mitotic Microtubule Development and Histone H3 Phosphorylation in the Holocentric Chromosomes of Rhynchospora Tenuis (Cyperaceae)

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.     

The relationship between nuclear envelope-derived annulate lamellae and the asymmetric division of primary spermatocytes in aphids

The structure of dividing primary spermatocytes of Amphorophora tuberculata (Aphididae, Hemiptera) as determined by electron microscopy and serial sectioning is described. The developmental stages examined extend from late prophase I to late telophase I. We looked for any asymmetric organization that could be causally linked to the differences in chromatin behaviour between the two daughter nuclei towards the end of meiosis I of this species. In late prophase I, evaginations of the nuclear envelope in the vicinity of two neigh-bouring centrosomes develop into closed cytoplasmic compartments with a dense content. The compartments open in prometaphase I and come to lie together with fragments of the nuclear envelope within the spindle area. Since nuclear pores are preserved in the membranes, intraspindle annulate lamellae have formed. These and material of presumed nuclear origin associated with them are asymmetrically distributed within the cell. Although dispersed at stages beyond prometaphase I, the material may be largely incorporated into one of the two daughter cells and thus be decisive for further development. Some annulate lamellae form a cap at the chromosome surface opposite to the neighbouring centrosomes in prometaphase I. These membranes may prevent interaction between spindle microtubules and chromosomes until a bipolar spindle forms in metaphase I. At this stage, both the banana-shaped autosomal bivalent and the X univalent occupy the equatorial plane. This is strange, because the X univalent has microtubular connections with one spindle pole and would be expected to migrate towards that pole. Possibly, the kinetochore of the X chromosome is inactive, and remains so in anaphase I, when the X univalent remains located between the two autosomal half-bivalents.M.F. Trendelenburg     

The intranuclear mitosis of the ciliates Paracineta limbata and Ichthyophtirius multifiliis

Electron microscope studies on the premetaphase stages of micronuclear divisions of Paracineta limbata and Ichthyophtirius multifiliis showed that spindle material also exists during interphase. In the case of I. multifiliis scattered microtubule fragments persist in the nuclear space; in P. limbata the micronuclei contain a small paracrystalloid which is suggested to be microtubular protein. Wide microtubules, varying in diameter from 300 to 400 Å develop during intranuclear prophase near the nuclear envelope in both cases. There are good reasons to assume that they function as a kind of stem body during the enlargement of the surface area of the nuclear envelope. Later micronuclear prophase stages of both species show a some-what different development. In I. multifiliis, there are scattered groups of short microtubular segments, partly in parallel array, whereas in P. limbata the wide tubules are transformed into normal microtubules of 180–200 Å diameter. The nuclei of both species are similar at late prophase and prometaphase stages. Bundles of interpolar microtubules run between the chromosomes, and single microtubules, presumably induced by the chromosomes, cross them at different angles. The chromosome-induced microtubules appear a short time after the interpolars. At prometaphase stage all microtubules show a highly parallel arrangement and therefore it is suggested that chromosomal tubules reach their final polar orientation by the action of cross-bridges.     

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.     

Electron microscopy of dividing cells

During intranuclear mitosis in plasmodia of Physarum polycephalum the primordium of spindle microtubules which is a somewhat electron opaque, amorphous structure with fibrous or granular elements, occurs in the center of early prophase, 20 to 30 minutes before metaphase. Then, the primordium seems to divide into two parts. Spindle microtubules develop radially from the primordia of spindle microtubules. These spindle microtubules increase in number and length during prophase. Spindle microtubules are completed in about five minutes before metaphase. The nuclear envelope remains intact during prophase, but after metaphase it breaks at the polar regions. The nuclear envelope of the daughter nucleus is re-formed from the original nuclear envelope.The authors wish to express their thanks Professor K. Ueda for valuable advice. They would also like to thank Professor N. Kamiya of Osaka University who kindly supplied the material (Physarum polycephalum) used in the present study.     

Dynamics of the nuclear envelope and of nuclear pore complexes during mitosis in the Drosophila embryo

Early embryonic development in Drosophila melanogaster is marked by a series of thirteen very rapid (10-15 min) and highly synchronous nuclear divisions, the last four of which occur just beneath the embryo surface. A total of some 6000 blastoderm nuclei result, which are subsequently enclosed by furrow membranes to form the cellular blastoderm. We have examined the fine structure of nuclear division in late syncytial embryos. The mitotic spindle forms adjacent to the nuclear envelope on the side facing the embryo surface. During prophase, astral microtubules deform the nuclear envelope which then ruptures at the poles at the onset of prometaphase. The nuclear envelope remains essentially intact elsewhere throughout mitosis. A second envelope begins to form around the nuclear envelope in prometaphase and is completed by metaphase; the entire double layered structure, referred to as the spindle envelope, persists through early in the ensuing interphase. Pole cell spindles are enclosed by identical spindle envelopes. Interphase and prophase nuclei contain nuclear pore complexes (PCs) of standard dimensions and morphology. In prometaphase PCs become much less electron-dense, although they retain their former size and shape. By metaphase, no semblance of PC structure remains, and instead, both layers of the spindle envelope are interrupted by numerous irregular fenestrae. PCs are presumably disassembled into their component parts during mitosis, and reassembled subsequently. Yolk nuclei remain among the central yolk mass when most nuclei migrate to the surface, cease to divide, yet become polyploid. These nuclei nonetheless lose and regain PCs in synchrony with the dividing blastoderm nuclei. In addition, they gain and lose a second fenestrated membrane layer with the same timing. Cytoplasmic membranes containing PCs (annulate lamellae) also lose and regain pores in synchrony with the two classes of nuclear envelopes. The factors that affect the integrity of PCs in dividing blastoderm nuclei appear to affect those in other membrane systems to an equivalent degree and with identical timing.     

CELL DIVISION IN SPIROGYRA. I. MITOSIS

Dividing cells of Spirogyra sp. were examined with both the light and electron microscopes. By preprophase many of the typical transverse wall micro-tubules disappeared while others were seen in the thickened cytoplasmic strands. Microtubules appeared in the polar cytoplasm at prophase and by prometaphase they penetrated the nucleus. They were attached to chromosomes at metaphase and early anaphase, and formed a sheath surrounding the spindle during anaphase; they were seen in the interzonal strands and cytoplasmic strands at telophase. The interphase nucleolus, containing 2 distinct zones and chromatinlike material, fragmented at prophase; at metaphase and anaphase nucleolar material coated the chromosomes, obscuring them by late anaphase. The chromosomes condensed in the nucleoplasm at prophase, moving into the nucleolus at prometaphase. The nuclear envelope was finally disrupted at anaphase during spindle elongation; at telophase membrane profiles coated the reforming nuclei. During anaphase and early telophase the interzonal region contained vacuoles, a few micro-tubules, and sometimes eliminated n ucleolar material; most small organelles, including swollen endoplasmic reticulum and tubular membranes, were concentrated in the polar cytoplasm. Quantitative and qualitative cytological observations strongly suggest movement of intact wall rnicrotubules to the spindle at preprophase and then back again at telophase.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum I. Prophase I — Prometaphase I

Summary A thoroughly documented account of the ultrastructure of the meiotic spindle pole body (SPB) cycle in a rust (Basidiomycota, Uredinales) is presented for the first time. The three-dimensional structure of the SPB and spindle during meiosis in the hollyhock rust fungusPuccinia malvacearum is analyzed from serial sections of preselected stages. This paper covers prophase I to prometaphase I. At late prophase I, the nucleolus disperses and does not reappear until the end of meiosis. The SPB at late prophase I consists of two, 4-layered discs, 0.8–1.0 m in diameter, connected by a middle piece (MP). The SPB is associated with a differentiated region of the nuclear envelope and nucleoplasm. At late diplotene to diakinesis, each disc generates a half spindle as it inserts into an otherwise intact nuclear envelope. The MP connecting the interdigitating half spindles elongates and eventually splits transversely during subsequent spindle elongation. Each half MP, which is attached to a SPB disc, becomes inserted in a sheath-like extension of the nuclear envelope. The intranuclear late prometaphase I spindle always becomes oriented perpendicularly to the longitudinal axis and sagittal plane of the metabasidium. There are 200–290 spindle microtubules (MTs) at each SPB at late prometaphase. The nonkinetochore MTs form a coherent central spindle around which the kinetochore MTs and bivalents are spread. A metaphase plate is absent. The results are compared with SPB behavior and spindle structure in early meiosis of other basidiomycetes and ascomycetes.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

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.     

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.     

Open spindle nuclear division in the amoebal phase of the acellular slime moldEchinostelium minutum with chromosomal movement related to the pronounced rearrangement of spindle microtubules

Summary Myxamoebae ofEchinostelium minutum exhibit extranuclear (open spindle) mitosis with centrioles present at the poles. Spindle microtubules are formed in association with a juxtanuclear MTOC which surrounds the cell's complement of centrioles. During late prophase or prometaphase the nuclear envelope breaks down and subsequently a metaphase plate is formed. Two anaphasic movements occur sequentially: firstly, the distance of the chromosomes to the poles shortens; secondly the distance between the spindle poles increases. The arrangement of spindle microtubules during anaphase is consistent with the hypothesis that chromosomal separation is due to lateral interaction (zippering) of microtubules. During telophase, reconstitution of the nuclear envelope usually takes place in the interzonal region prior to reformation in the polar region. Cytokinesis, which begins in anaphase or early telophase involves the participation of vesicles, microfilaments and microtubules.Based on the doctoral dissertation of the first author presented to the Department of Botany, University of Washington, Seattle, WA 98195, U.S.A.     

Transformation of the flagella and associated flagellar components during cell division in the coccolithophoridPleurochrysis carterae

Summary Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.Abbreviations CR1, CR2 crystalline roots 1 and 2 - CT cytoplasmic tongue microtubules - DIC differential interference contrast light microscopy - H haptonema - HVEM high voltage transmission electron microscopy - IMF immunofluorescence microscopy - L left flagellum/basal body - M metaphase plate - MT microtubule - N nucleus - R right flagellum/basal body - R1, R2, R3 roots 1, 2, and 3 - TEM transmission electron microscopy     

Subcellular distribution of aster-nucleated microtubule length: A more or less mitotic status of cytoplasmic areas during meiosis I of starfish oocytes

Summary— Shortening of aster-nucleated microtubules is a mitotic feature, which is proportional to the M-phase promoting factor kinase activity. We have taken advantage of this characteristic to appraise the mitotic status of cytoplasmic territories in polyspermic starfish oocytes. In early prometaphase, asters are small in the nuclear area, where cyclin B accumulated before GVBD, while in the remaining of the oocytes asters are large. In metaphase all asters are small. This coincides with a change in behaviour of cyclin B, whose association with microtubules increases in late prometaphase. We discuss the possibility that uneven distribution of cyclin B-dependent kinase is a general feature of mitosis and that it allows the cell to build the convenient spindle at the right place.     

The ultrastructure of mitosis inChroomonas salina (Cryptophyceae)

Summary A detailed account of the ultrastructure of mitosis in a member of theCryptophyceae is given for the first time. The initial indication of mitosis is the duplication of the flagellar bases. The nucleus migrates towards the anterior of the cell and its envelope and nucleolus break down. The chromatin which at interphase is in the form of scattered clumps, condenses into a solid mass through which run narrow tunnels. Each tunnel allows the passage of one to four microtubules. At metaphase the dense plate of chromatin is situated on the equator and the spindle has a rectangular shape. Individual chromosomes cannot be recognized and no morphologically differentiated kinetochores have been observed. The flagella remain functional, their bases stay at the anterior side of the nucleus and do not move to the poles. At anaphase two plates of chromatin separate and these move apart until they come to lie against the ER sheath surrounding the chloroplasts. The new nuclear envelope starts to form on the opposite side of the daughter nucleus. Cytokinesis may commence early in mitosis and consists of a constriction of the parent cell, starting from the posterior end, followed by separation of the two daughters. The present work supports earlier views that only one chromosome is evident during the nuclear division of these organisms. The mitosis is completely different from that of theDinophyceae with which theCryptophyceae were formerly linked.     

Multiple mechanisms of chromosome movement in vertebrate cells mediated through the Ndc80 complex and dynein/dynactin

Kinetochores bind microtubules laterally in a transient fashion and stably, by insertion of plus ends. These pathways may exist to carry out distinct tasks during different stages of mitosis and likely depend on distinct molecular mechanisms. On isolated chromosomes, we found microtubule nucleation/binding depended additively on both dynein/dynactin and on the Ndc80/Hec1 complex. Studying chromosome movement in living Xenopus cells within the simplified geometry of monopolar spindles, we quantified the relative contributions of dynein/dynactin and the Ndc80/Hec1 complex. Inhibition of dynein/dynactin alone had minor effects but did suppress transient, rapid, poleward movements. In contrast, inhibition of the Ndc80 complex blocked normal end-on attachments of microtubules to kinetochores resulting in persistent rapid poleward movements that required dynein/dynactin. In normal cells with bipolar spindles, dynein/dynactin activity on its own allowed attachment and rapid movement of chromosomes on prometaphase spindles but failed to support metaphase alignment and chromatid movement in anaphase. Thus, in prometaphase, dynein/dynactin likely mediates early transient, lateral interactions of kinetochores and microtubules. However, mature attachment via the Ndc80 complex is essential for metaphase alignment and anaphase A. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Timing the phases of the microtubule cycles involved in cytoplasmic and nuclear divisions in cells of undisturbed onion root meristems

The duration of the different phases of the microtubule and chromosome cycles were estimated in the native diploid cell populations of Allium cepa L root meristems proliferating undisturbed, under steady state conditions, at the physiological temperature of 15°C. The cycles were coupled by considering their fitting in relation to the short process of nuclear envelope breakdown. In the cycle related to cytoplasmic division, the preprophase band which predicts the future position of the phragmoplast made its appearance, as a wide band, 16 mm before the G₂ to prophase transition, ie it was only present during the final 5% of the total G₂ timing (5 h 30 mm). The band became narrow only 6 mm after prophase had started and it was present in this form for the remaining prophase time (2 h 24 mm). Its disappearance occurred strictly coinciding with nuclear envelope breakdown, at the end of prophase. No microtubules related to cytoplasmic division were apparent until 9 mm after telophase had initiated. The two initial stages of phragmoplast formation which followed occupied, respectively, 27 mm and 54.5 mm of the 2-h long telophase. On the other hand, the third and last stage in phragmoplast formation covered both the final 35 mm of mitosis and the 6 initial mm of the G₁ of the next interphase. A very short (less than 4 mm) stage of microtubular nucleation around the nuclear envelope took place immediately afterwards, before the cortical array of microtubules appeared. The microtubule cycle related to nuclear division started with the apparent activation of the future spindle poles 7.4 mm before prophase was over. The mitotic spindle developed in the 5.6 mm long prometaphase. The spindle functioned in metaphase for the 42 mm it lasted, half spindles being separated for the 37 mm anaphase occupied in these cells.