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.     

Meiosis,spindle pole body cycle,and taxonomy of the heterobasidiomycetePachnocybe ferruginea

Meiosis and the meiotic spindle pole body cycle were studied electron microscopically in basidia of the heterobasidiomycetePachnocybe ferruginea. Spindle pole body splitting in prometaphase I and II, and intermeiotic and postmeiotic duplication were investigated in particular detail. During prophase, the spindle pole body consists of two three-layered discs connected by a middle piece. At late prophase I and again in prometaphase II, the discs contact the nuclear envelope. Then, the nuclear membrane at the contact area is separated from the non-contacted part of the nuclear envelope and finally disappears. Each disc nests into the nuclear opening of the otherwise intact nuclear envelope. The disc remains in the gap and generates a half spindle. At late metaphase I, a co-disc develops eccentrically within the parent disc. The co-disc detaches from the parent disc during interphase I and becomes one of the metaphase II spindle pole bodies. Co-discs are absent during the second division. A cap of endoplasmic reticulum encloses each disc during prophase I through anaphase I. In the second meiotic division, the caps covering the spindle pole bodies of one nucleus of the pair, are developed from the neighbouring nucleus. Spindle pole bodies ofP. ferruginea are similar to those of the rusts, and especially to those ofEocronartium muscicola andHelicobasidium mompa. Part 73 of the series Studies inHeterobasidiomycetes.     

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.     

Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Intranuclear membranes and the formation of the first meiotic spindle in Xenos peckii (Acroschismus wheeleri) oocytes

The ultrastructure of spindle formation during the first meiotic division in oocytes of the Strepsipteran insect Xenos peckii Kirby (Acroschismus wheeleri Pierce) was examined in serial thick (0.25- micron) and thin sections. During late prophase the nuclear envelope became extremely convoluted and fenestrated. At this time vesicular and tubular membrane elements permeated the nucleoplasm and formed a thin fusiform sheath, 5-7 micron in length, around each of the randomly oriented and condensing tetrads. These membrane elements appeared to arise from the nuclear envelope and/or in association with annulate lamellae in the nuclear region. All of the individual tetrads and their associated fusiform sheaths became aligned within the nucleus subsequent to the breakdown of the nuclear envelope. Microtubules (MTs) were found associated with membranes of the meiotic apparatus only after the nuclear envelope had broken down. Kinetochores, with associated MTs, were first recognizable as electron-opaque patches on the chromosomes at this time. The fully formed metaphase arrested Xenos oocyte meiotic apparatus contained an abundance of membranes and had diffuse poles that lacked distinct polar MT organizing centers. From these observations we conclude that the apparent individual chromosomal spindles--seen in the light microscope to form around each Xenos tetrad during "intranuclear prometaphase" (Hughes-Schrader, S., 1924, J. Morphol. 39:157-197)--actually form during late prophase, lack MTs, and are therefore not complete miniature bipolar spindles, as had been commonly assumed. Thus, the unique mode of spindle formation in Xenos oocytes cannot be used to support the hypothesis that chromosomes (kinetochores) induce the polymerization of their associated MTs. Our observation that MTs appeared in association with and parallel to tubular membrane components of the Xenos meiotic apparatus after these membranes became oriented with respect to the tetrads, is consistent with the notion that membranes associated with the spindle determine the orientation of spindle MTs and also play a part in regulating their formation.     

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.     

Cytoplasmic dynein as a facilitator of nuclear envelope breakdown.

During prophase in higher cells, centrosomes localize to deep invaginations in the nuclear envelope in a microtubule-dependent process. Loss of nuclear membranes in prometaphase commences in regions of the nuclear envelope that lie outside of these invaginations. Dynein and dynactin complex components concentrate on the nuclear envelope prior to any changes in nuclear envelope organization. These observations suggest a model in which dynein facilitates nuclear envelope breakdown by pulling nuclear membranes and associated proteins poleward along astral microtubules leading to nuclear membrane detachment. Support for this model is provided by the finding that interference with dynein function drastically alters nuclear membrane dynamics in prophase and prometaphase.     

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.     

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.     

Mitosis in Barbulanympha. II. Dynamics of a two-stage anaphase, nuclear morphogenesis, and cytokinesis

Anaphase in Barbulanympha proceeds in two discrete steps. In anaphase- A, chromosomal spindle fibers shorten and chromosomes move to the stationary centrosomes. In anaphase-B, the central spindle elongates and ("telophasic") bouquets of chromosomes, with kinetochores still connected by the shortened chromosomal fibers to the centrosomes, are moved far apart. The length, width, and birefringence of the central spindle remain unchanged throughout anaphase-A. In anaphase-B, the central spindle elongates up to fivefold. During elongation, the peripheral fibers of the central spindle splay, first anteriorly and then laterally. The remaining central spindle progressively becomes thinner and the retardation decreases; however, the coefficient of birefringence stays approximately constant. The nuclear envelope persists throughout mitosis in Barbulanympha and the nucleus undergoes an intricate morphological change. In prophase, the nucleus engulfs the spindle; in early anaphase-A, the nuclear envelope forms a seam anterior to the spindle, the nucleus thus transforms into a complete sleeve surrounding the central spindle. In late anaphase-A, the middle of the seam opens up in a cleft as the lips part; in anaphase-B, the cleft expands posteriorly, progressively exposing the central spindle. Finally, the cleft partitions the nucleus into two. The nuclear envelope shows an apparent elasticity and two-dimensional fluidity. Localized, transient deformations of the nuclear envelope indicate poleward and counter-poleward forces acting on the kinetochores embedded in the envelope. These forces appear responsible for nuclear morphogenesis as well as anaphase chromosome movement. At the end of anaphase-B, the two rostrate Barbulanympha may swim apart of be poked apart into two daughter cells by another organism cohabiting the host's hindgut.     

Relationship between the arrangement of microtubules and chromosome behaviour of syntelic autosomal univalents during prometaphase in crane fly spermatocytes

During meiotic prometaphase of crane fly spermatocytes, syntelic autosomal univalents are able to move between the spindle poles several times. The relationship between the behaviour of chromosomes and the arrangement of microtubules during this stage was studied using a fixation technique (Nicklas et al. 1979) which makes it possible to examine a certain cell in an electron microscope after live observation. After reorientation, when a syntelic univalent had started moving towards the opposite spindle pole, a short chromosome fibre was found. When a univalent had reached the equator, a chromosome fibre could be traced up to the spindle apex. During the movement towards the opposite spindle pole the degree of disorder in the chromosome fibre was high, whereas it was low in the fibre of a motionless univalent. The degree of disorder was determined by the relative proportion of skew fibre microtubules. At the beginning of reorientation a chromosome fibre was still present, but later, it was no longer possible to recognize such a fibre. Instead of a chromosome fibre, a bundle of microtubules laterally associated with the kinetochore was observed. Some microtubules of this bundle had a direct contact with the kinetochore. These observations strongly hint that the laterally associated microtubules have an important function in the reorientation of syntelic autosomal univalents.     

Segregation of the amphitelically attached univalent X chromosome in the spittlebug <Emphasis Type="Italic">Philaenus spumarius</Emphasis>

In meiosis I, homologous chromosomes combine to form bivalents, which align on the metaphase plate. Homologous chromosomes then separate in anaphase I. Univalent sex chromosomes, on the other hand, are unable to segregate in the same way as homologous chromosomes of bivalents due to their lack of a homologous pairing partner in meiosis I. Here, we studied univalent segregation in a Hemipteran insect: the spittlebug Philaenus spumarius. We determined the chromosome number and sex determination mechanism in our population of P. spumarius and showed that, in male meiosis I, there is a univalent X chromosome. We discovered that the univalent X chromosome in primary spermatocytes forms an amphitelic attachment to the spindle and aligns on the metaphase plate with the autosomes. Interestingly, the X chromosome remains at spindle midzone long after the autosomes have separated. In late anaphase I, the X chromosome initiates movement towards one spindle pole. This movement appears to be correlated with a loss of microtubule connections between the kinetochore of one chromatid and its associated spindle pole.     

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

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

Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos

The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity maintains the spacing between separated centrosomes during prometaphase and metaphase, and repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.     

Unipolar orientation stability of the sex univalent in the grasshopper (Melanoplus sanguinipes)

The behavior of the X chromosome in living Melanoplus sanguinipes primary spermatocytes was examined utilizing phase contrast microscopy and micromanipulation. During early prometaphase I, the X univalent establishes a unipolar orientation which is normally stable. In 28 cells, the X chromosome did not reorient once during a total of 122 h of observation. Though normally stable, the X chromosome can be induced to reorient by micromanipulation. An anomalous increase in X reorientation was observed in one testicular preparation. Stable unipolar orientation is an intrinsic characteristic of the X chromosome and was not displayed by two small autosomal univalents discovered in one cell. The two autosomal univalents, resulting from asynapsis or desynapsis, oscillated back and forth within the spindle, averaging 26.0 and 17.6 min respectively between reorientation events. The behavior of the M. sanguinipes X chromosome reveals an orientation stability mechanism that does not involve tension created by forces from opposing kinetochoric spindle fibers.     

Formation of division spindles in higher plant meiosis

Depolymerisation of the MT cytoskeleton during late prophase makes it impossible to follow the cytoskeleton cycle in centrosomeless plant meiocytes. This paper describes rearrangements of the MT cytoskeleton during plant meiotic spindle formation in normally dividing pollen mother cells in various higher plant species and forms in which the cytoskeleton does not depolymerise at prophase. In such variants of the wild-type, cytoskeleton rearrangements can be observed at late prophase/early prometaphase. Radial MT bundles coalesce in the meridian plane, reorientate tangentially, curve and give rise to a developed ring-shaped perinuclear cytoskeleton system at the meridian. During nuclear envelope breakdown this ring disintegrates and splits into a set of free MT bundles. Three sub-stages of prometaphase are indicated: early prometaphase (disintegration of perinuclear ring and invasion of MTs into the former nuclear area), middle prometaphase or chaotic stage (formation of bipolar spindle fibres), and late prometaphase (formation of bipolar spindle). Analysis of a range of abnormal phenotypes (disintegrated, multiple, polyarchal, chaotic spindles) reveals two previously unknown processes during late prometaphase: axial orientation and consolidation of the spindle fibres.     

Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.     

Prophase chromosome movements in living house cricket spermatocytes and their relationship to prometaphase,anaphase and granule movements

Chromosome and granule movements in meiotic prophase and prometaphase have been studied by time-lapse cinemicrography in live spermatocytes of the house cricket, Acheta domesticus. Chromosome movements in prophase cells, up to one hour or more before breakdown of the nuclear envelope, are described. These movements are frequent but saltatory; are based mostly at chromosome ends but also at kinetochores; occur in very intimate association with the inside of the nuclear envelope; are directed towards and away from the extranuclear centres (centrioles); tend weakly to accumulate bivalents round the two centres and reach a velocity of 0.65 m/sec. Saltatory movements in granules associated with extranuclear asters are remarkably similar in basic characteristics to the intranuclear chromosome movements. Surprisingly, the chromosome movements (and those of granules) are reversably blocked by colcemid (but not lumi-colcemid), and yet occur in the apparent absence of an intranuclear microtubule array. The movements cease at or shortly after breakdown of the nuclear envelope. However, kinetochore movements in very early prometaphase are similar in velocity and other respects to prophase movements; later prometaphase movements are clearly slower, and those of anaphase very much slower still. — The prophase movements suggest a two component model for motion: a non-microtubule, linear force producer together with microtubules with a skeletal, orientational role. Arguably, both these components are also necessary for chromosome movements in prometaphase and anaphase.This paper is dedicated to Dr. Sally Hughes-Schrader, whose beautiful work in mantids clearly presaged the existence of chromosome movements in late prophase of meiosis; and whose enthusiasm over chromosome movements in general it was my pleasure to share during my stay at Duke.     

The kinesin ATK5 functions in early spindle assembly in Arabidopsis

During cell division, the mitotic spindle partitions chromosomes into daughter nuclei. In higher plants, the molecular mechanisms governing spindle assembly and function remain largely unexplored. Here, live cell imaging of mitosis in Arabidopsis thaliana plants lacking a kinesin-14 (ATK5) reveals defects during early spindle formation. Beginning during prophase and lasting until late prometaphase, spindles of atk5-1 plants become abnormally elongated, are frequently bent, and have splayed poles by prometaphase. The period of spindle elongation during prophase and prometaphase is prolonged in atk5-1 cells. Time-lapse imaging of yellow fluorescent protein:ATK5 reveals colocalization with perinuclear microtubules before nuclear envelope breakdown, after which it congresses inward from the poles to the midzone, where it becomes progressively enriched at regions of overlap between antiparallel microtubules. In vitro microtubule motility assays demonstrate that in the presence of ATK5, two microtubules encountering one another at an angle can interact and coalign, forming a linear bundle. These data indicate that ATK5 participates in the search and capture of antiparallel interpolar microtubules, where it aids in generating force to coalign microtubules, thereby affecting spindle length, width, and integrity.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.