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

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

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated α-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 M nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 M nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.On leave of absence from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland     

Temporal and spatial pattern of differences in microtubule behaviour during Drosophila embryogenesis revealed by distribution of a tubulin isoform

Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated alpha-tubulin has revealed that acetylated and nonacetylated alpha-tubulin isoforms have different patterns of distribution during early development. Acetylated alpha-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated alpha-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated alpha-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated alpha-tubulin appeared in early embryos when micro-tubules were stabilized by taxol treatment or anoxia. Because acetylated alpha-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated alpha-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of alpha-tubulin is not merely a consequence of cellularization. After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated alpha-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated alpha-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated alpha-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated alpha-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated alpha-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4 degrees C.     

Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties

A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha-tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B-1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules.     

Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes.

Previous studies suggested that the transition from an incompetent to a competent meiotic state during the course of oogenesis in the mouse involved a G2/M-like cell cycle transition (Wickramasinghe et al, 1991. Dev. Biol. 143, 162). The present studies tested the hypothesis that centrosome phosphorylation, an event normally induced by MPF, is required for this developmental transition and the expression of meiotic competence in cultured growing mouse oocytes. Multiple fluorescence labeling techniques were used to evaluate centrosome number, phosphorylation status, and microtubule nucleating capacity in competent and incompetent oocytes. Experimental conditions were established for reversibly altering the phosphorylation status of the centrosomes and the effects of these treatments on meiotic resumption were examined. Phosphorylated centrosomes nucleating short microtubules were observed in competent oocytes, whereas nonphosphorylated centrosomes and interphase microtubule arrays were found in incompetent oocytes. Upon recovery from nocodazole-induced microtubule depolymerization, short microtubules formed from centrosomes in competent oocytes, whereas long microtubules reappear in the cytoplasm of incompetent oocytes. Perturbation of the phosphorylation state of oocytes with activators of protein kinase A or protein kinase C resulted in the formation of long interphase microtubules in competent oocytes while centrosome phosphorylation was maintained. Treatment of competent oocytes with the phosphorylation inhibitor 6-dimethylaminopurine also led to formation of long microtubules, although under these conditions centrosomes were dephosphorylated. When competent oocytes were treated simultaneously with puromycin and the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) for 6 hr, centrosomes became dephosphorylated; centrosomes were rephosphorylated when competent oocytes were further cultured in IBMX without puromycin. Conditions that induced centrosome dephosphorylation in competent oocytes resulted in the loss of the ability to express meiotic competence in culture, whereas maintenance of centrosome phosphorylation in these oocytes was correlated with the ability to resume meiosis. These results suggest that the G2/M transition that occurs when mouse oocytes progress from an incompetent to a competent state in vivo involves the phosphorylation of centrosomes and that the maintenance of centrosome phosphorylation is required for the in vitro expression of meiotic competence.     

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.     

Cell cycle-dependent behavior of microtubules in hybrids of mouse oocytes and blastomeres.

The behavior of microtubules was studied in hybrids formed between mouse oocytes arrested in metaphase II or activated parthenogenetically and mouse embryo interphase blastomeres. In all cases the interphase blastomere's network of microtubules disassembles rapidly after fusion with oocytes. Introduction of interphase cytoplasm and nuclei to metaphase oocytes during fusion induces the polymerization of new microtubules in the cytoplasm and in the meiotic spindle. The degree and the duration of this facilitated polymerization of microtubules was positively correlated with the volume of blastomeres used for fusion. The blastomere nuclei induce the formation of microtubular frames, which become more evident when the chromatin undergoes premature condensation. Finally, spindle-like structures are formed around the prematurely condensed chromosomes. In hybrids activated around the time of fusion, the blastomere nuclei undergo pronuclear-like transformation. These hybrids develop an interphase network of microtubules typical for activated oocytes. These results are discussed with regards to the cell cycle control of microtubule behavior.     

Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.     

The nucleoporin Nup358 associates with and regulates interphase microtubules

The nucleoporin Nup358 resides on the cytoplasmic face of the interphase nuclear pore complex (NPC). During metaphase, its recruitment to kinetochores is important for correct microtubule-kinetochore attachment. Here, we report that a fraction of endogenous Nup358 interacts with interphase microtubules through its N-terminal region (BPN). Cells overexpressing the microtubule targeting domain of Nup358 displayed dramatic alteration in the microtubule organization including increased microtubule bundling and stability. Ectopic expression of BPN and full-length Nup358 exhibited significantly higher levels of acetylated microtubules that were resistant to nocodazole, a microtubule depolymerizing agent. Furthermore, RNAi mediated depletion of Nup358 affected polarized stabilization of microtubules during directed cell migration, confirming the in vivo role of Nup358 in regulating interphase microtubules.     

Acetylation of α-tubilin in different bovine cell types: implications for microtubule dynamics in interphase and mitosis.

Previous work on five cell types isolated from the bovine corpus luteum showed that the mass of acetylated microtubules (acet-MTs) in interphase differed. Endothelial cells, termed type 3, showed few acet-MTs, whereas the interphase cytoskeleton of granulosal-like cells, termed type 5, was rich in acet-MTs. In the present study, these cultured cells were used to determine whether the degree of α-tubulin acetylation in interphase had consequences on mitosis. To this end, the distribution of acet-MTs was determined throughout the cell cycle using a monoclonal antibody, 6-11B-1, directed against acetylated α-tubulin. For comparison, tyrosinated MTs were visualized with another monoclonal antibody, YL1/2, detecting tyrosinated α-tubulin. Although the amount of acet-MTs in interphase differed significantly between both cell types, major differences in the appearance of acet-MTs during mitosis were only apparent in prophase and during transition from late telophase to interphase. Thus, irrespective of different α-tubulin acetylation in interphase, spindle structure is uniform. Since acetylation of α-tubulin is believed to indicate the presence of relatively stable MTs, we conclude that MT dynamics is differently controlled in interphase and mitosis. Thereby interphase cells are able to carry out functions which involve stable MTs and the cells progress through mitosis in the presence of more dynamic MTs.     

Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis

Katanin is a heterodimer that exhibits ATP-dependent microtubule-severing activity in vitro. In Xenopus egg extracts, katanin activity correlates with the addition of cyclin B/cdc2, suggesting a role for microtubule severing in the disassembly of long interphase microtubules as the cell prepares for mitosis. However, studies from plant cells, cultured neurons, and nematode embryos suggest that katanin could be required for the organization or postnucleation processing of microtubules, rather than the dissolution of microtubule structures. Here we reexamine katanin's role by studying acentrosomal female meiotic spindles in C. elegans embryos. In mutant embryos lacking katanin, microtubules form around meiotic chromatin but do not organize into bipolar spindles. By using electron tomography, we found that katanin converts long microtubule polymers into shorter microtubule fragments near meiotic chromatin. We further show that turning on katanin during mitosis also creates a large pool of short microtubules near the centrosome. Furthermore, the identification of katanin-dependent microtubule lattice defects supports a mechanism involving an initial perforation of the protofilament wall. Taken together, our data suggest that katanin is used during meiotic spindle assembly to increase polymer number from a relatively inefficient chromatin-based microtubule nucleation pathway.     

Plastid polarity and meiotic spindle development in microsporogenesis ofSelaginella

Summary Microsporogenesis inSelaginella was studied by fluorescence light microscopy and transmission electron microscopy. As in other examples of monoplastidic meiosis the plastids are involved in determination of division polarity and organization of microtubules. However, there are important differences: (1) the meiotic spindle develops from a unique prophase microtubule system associated with two plastids rather than from a typical quadripolar microtubule system associated with four plastids; (2) the division axes for first and second meiotic division are established sequentially, whereas as in all other cases the poles of second division are established before those of first division; and (3) the plastids remain in close contact with the nucleus throughout meiotic prophase and provide clues to the early determination of spindle orientation. In early prophase the single plastid divides in the plane of the future division and the two daughter plastids rotate apart until they lie on opposite sides of the nucleus. The procytokinetic plate (PCP) forms in association with the two slender plastids; it consists of two spindle-shaped microtubule arrays focused on the plastid tips with a plate of vesicles at the equatorial region and a picket row of microtubules around one side of the nucleus. Second plastid division occurs just before metaphase and the daughter plastids remain together at the spindle poles during first meiotic division. The meiotic spindle develops from merger of the component arrays of the PCP and additional microtubules emanating from the pair of plastid tips located at the poles. After inframeiotic interphase the plastids migrate to tetrahedral arrangement where they serve as poles of second division.Abbreviations AMS axial microtubule system - FITC fluorescein isothiocyanate - MTOC microtubule organizing center - PCP procytokinetic plate - QMS quadripolar microtubule system - TEM transmission electron microscope (microscopy)     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Cytoskeletal organization of rat oocytes during metaphase II arrest and following abortive activation: A study by confocal laser scanning microscopy

In metaphase II arrested rat oocytes (M il), microtubles were found in the taper-shaped meiotic spindle and in the cytoplasm as asters and free microtubules. Whereas spindle microtubules were acetylated, those located in the cytoplasm were not. Cytoplasmic microtubules were also labile as assessed by mild cooling. In contast to mouse oocytes, rat microtubule organizing centers (MTOCs) did not react with MPM-2 antibody by immunofluorescence despite the fact that this antibody reacts with several proteins as shown by immunoblot. However, cytoplasmic MTOCs in M II-arrested rat oocytes could be detected by their nucleating capacity in the presence of taxol, a drug that induced the formation of numerous cytoplasmic asters. In addition, taxol caused a change in the spindle shape and the formation of astral microtubules at the spindle poles. Meiotic spindles (as well as chromosomes devoid of microtubules after nocodazoletreatment) were overlaid by an actin-rich domain. Spontaneous abortive activation led to the extrusion of the second polar body followed by another metaphase arrest— metaphase III; however, normal spindles did not form and dispersed chromosomes surrounded by microtubles were observed. Electron microscopic studies confirmed these observations and revealed that the kinetochores are located deep within the chromosomes in contrast to mouse kinetochores, and this might be responsible for the absence of a metaphase III spindle in the rat oocyte. Induced activation caused transition to interphase with the formation of a characteristic microtubule network. This study shows that there are several significant differences in the cytoskeletal organization of rat and mouse oocytes. © 1993 Wiley-Liss, Inc.     

The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.     

Stability of cyclin B protein during meiotic maturation and the first mitotic cell division in mouse oocytes

This report examines in detail the metabolism of the cyclin protein B1 during meiotic maturation and following the activation of mature mouse oocytes using immunoprecipitation of the radiolabelled protein. The net synthesis of cyclin B increases progressively during meiotic maturation, reaching its maximum levels at least 1 h before oocytes exit into metaphase of meiosis II (MII). This increase correlates with the rise in cdc2 kinase activity reported previously and suggests an association between the length of the first meiotic M phase (MI) and the net synthesis of cyclin B, that seems to regulate the time required for the cdc2 kinase to reach its maximum activity. Moreover, no marked degradation of cyclin B was observed before the MI to MII transition and that which occurs does so independently of the presence of microtubules, which are essential for cyclin degradation during metaphase II arrest and exit of oocytes into interphase of the first mitotic cell cycle. Cyclin B is degraded rapidly during the transitions MI to MII, MII to the first mitotic interphase and MII to an abortive third metaphase state (MIII). However, whilst its degradation was incomplete during the MI to MII transition, virtually no cyclin B protein was detected following both the MII to interphase and MII to MIII transitions. Thus, the decision of oocytes to exit into MIII, or interphase is not controlled at the level of cyclin B degradation. Lastly, in aging, non-activated oocytes, the net synthesis of cyclin B declines. Whereas, in activated eggs cultured in parallel although the rate of net synthesis declines initially, it is effectively ‘rescued’ being two-fold greater than in non-activated oocytes of an equivalent age. This gradual fall in the net synthesis of cyclin B observed in aging oocytes may contribute to the increasing ease with which they become activated, compared to recently ovulated oocytes.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum

Summary Changes in the spindle pole body (SPB) and meiotic nuclei from interphase I through interphase II in the hollyhock rustPuccinia malvacearum are analyzed ultrastructurally by three-dimensional reconstructions from serial sections. Interphase I nuclei undergo a coordinated migration and rotation during which the SPBs approach the convex face of the lateral promycelial wall. During the transition from interphase I to prometaphase II, the collateral disc (co-disc) apparently enlarges and fuses with the main disc of the SPB. The resulting single SPB nucleates two confluent half spindles and about 225 astral microtubules (MTs). Co-discs and middle pieces (MPs) are absent during division II. SPBs separate and form metaphase II intranuclear spindles oriented in a predictable manner. Tubular cisternae are present within the spindle at early metaphase II. The architecture of the spindle at division II is essentially identical to that reported for division I except that the spindle is about half as long. Anaphase-telophase II nuclear envelope constriction, separation of the sibling nuclei, and externalization of the SPBs is identical to that reported for division I. Genesis of the duplicated interphase II SPB apparently occurs rapidly and involves formation of the MP followed by the three-layered SPB discs. General aspects of the division II spindle are discussed. A model for the meiotic SPB cycle in a rust is presented and its phylogenetic and functional significance in relation to other basidiomycetes and ascomycetes is discussed.     

The effect of the UV microirradiation of the centrosome on cell behavior. III. The ultrastructure of the centrosome after irradiation]

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.     

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.