Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope

A group of antigens related by their reactivity with monoclonal antibodies MPM-1 and MPM-2 appear as cells enter mitosis. These antibodies bind to a phosphorylated epitope on certain proteins, and therefore the antigens are presumed to be a group of phosphoproteins. A subset of these proteins has been shown previously to be components of mitotic microtubule organizing centers in PtK1 cells. We present here evidence that the mitosis-specific appearance of these phosphoproteins is a phenomenon common to all eukaryotic cells. The MPM reactive phosphoproteins were localized to mitotic spindle poles regardless of whether the spindle formed in the cytoplasm after nuclear envelope breakdown (open mitosis) or within the nucleus (closed mitosis). This reactivity was not dependent upon the presence of centrioles at the spindle poles. Proteins that contained the phosphorylated epitope were not, however, restricted to mitotic cells. Cells of neuronal derivation and flagellated cells showed specific localization of MPM antibody to the microtubule network and basal bodies respectively. On immunoblots, the MPM antibody reacted with brain MAP-1 among a number of other phosphoproteins. The identification of microtubule-associated protein (MAP)-1 correlates with the localization of the antibody to microtubules of neuroblastoma cells. These results suggest, that different phosphoprotein molecules detected by the MPM antibody may be specific for different mitotic microtubule organizing centers, basal bodies, and other specialized cytoskeletal structures; and the presence of a related phosphorylated domain on these proteins may be important for their proper function and/or interaction with microtubules.     

Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.     

Protein phosphorylation and the regulation of basal body microtubule organizing centres in Tetrahymena.

Previous work suggests that changes in the phosphorylation state of some centrosomal proteins regulate centrosomal activity. The hypothesis that changes in the phosphorylation state of one or more basal body microtubule organizing centre (MTOC) components regulate its ability to nucleate cilia assembly in Tetrahymena thermophila was tested. The MPM-2 antibody, which recognizes phosphorylated epitopes in MTOCs in a variety of organisms, was used to probe immunoblots of cytoskeletal frameworks prepared from starved Tetrahymena, from starved deciliated Tetrahymena, and from a starved deciliated mutant Tetrahymena which failed to initiate ciliogenesis following deciliation. The MPM-2 antibody recognized an identical array of proteins in all blots. These results suggest that, unlike centrosomes, basal body MTOC activity is not regulated by changes in the phosphorylation state of component proteins.     

Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos

By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

APC is a component of an organizing template for cortical microtubule networks

A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. Here, we investigate the proteins that are involved in organizing this network, and we show that end-binding protein 1 (EB1), adenomatous polyposis coli protein (APC) and p150Glued - although considered to be microtubule plus-end-binding proteins - are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation.     

Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.     

Fine oral filaments in Paramecium: a biochemical and immunological analysis

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.     

Probing microtubule organizing centres with MPM-2 in dividing cells of higher plants using immunofluorescence and immunogold techniques

Summary The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnospermPicea glauca and in cells of the angiospermVicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) - FITC fluorescein isothiocyanate - MT microtubule - MTOC microtubule organizing centre - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PBSB phosphate buffered saline with bovine serum albumin - PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - PPB preprophase band - SPB spindle pole body - TRITC tetramethylrhodamine isothiocyanate     

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.     

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.     

Structural and immunocytochemical characterization of microtubule organizing centers in pteridophyte spermatogenous cells

Summary During the development of the spermatogenous cells, the pteridophyteCeratopteris richardii produces three structurally well-defined microtubule organizing centers (MTOCs). The blepharoplast, a spherical body that occurs during the last two spermatogenous divisions, organizes two microtubule (MT) arrays, one associated with a nuclear indentation and the other that organizes the spindle apparatus for the final divisions. After the last spermatogenous division, the blepharoplast reorganizes to produce two new putative MTOCs: the lamellar strip (LS) of the multilayered structure (MLS), which apparently organizes the spline microtubule array, and an amorphous zone (AM), that connects the basal bodies. Thin and semi-thin sections of this tissue were probed with antisera which recognize MTOCs in lower eukaryotes and animals to determine if any of these structures contain MTOC-associated proteins or epitopes recognized by monoclonal antisera. Gamma tubulin antibodies, which recognizeonly the minus ends of MTs in mammalian cells, label along the MT in all arrays found in the pteridophyte spermatogenous cells. Kinetochore MTs are unlabelled near the kinetochore, however. The monoclonal antibodies MPM-2 and C-9, that recognize centrosomal and nuclear epitopes in mammalian cells, label the interphase nucleus, the cytoplasm of mitotic cells, and the blepharoplast during both nuclear indentation and spindle formation. Double labelling of the blepharoplast-containing cells with anti-tubulin and either MPM-2 or C-9 reveals that the blepharoplast-associated fluorescence is the focus of the tubulin arrays. Centrin labels the reorganizing blepharoplast, the MLS, the AM, and a stellate pattern in the transition region of the flagella. These data indicate the usefulness of the structurally well-recognized MTOCs in pteridophyte spermatogenous cells in investigation of land plant MTOCs.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.     

Regulation of microtubule nucleation mediated by γ-tubulin complexes

The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.     

A Novel 95-kD Protein Is Located in a Linker between Cytoplasmic Microtubules and Basal Bodies in a Green Flagellate and Forms Striated Filaments In Vitro

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH₂- and COOH-terminal domains flanking a central domain of ~650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.     

Redistribution of microtubules and pericentriolar material during the development of polarity in mouse blastomeres

The distribution of microtubules and microtubule organizing centers (MTOCs) during the development of cell polarity in eight-cell mouse blastomeres was studied by immunofluorescence and immunoelectron microscopy using monoclonal anti-tubulin antibodies and an anti-pericentriolar material (PCM) serum. In early eight-cell blastomeres microtubules were found mainly around the nucleus and in the cell cortex, whereas PCM foci were observed dispersed in the cytoplasm. During the eight-cell stage, microtubules disappeared from the area adjacent to the zone of intercellular contact and accumulated in the apical part of the cell while their number decreased in the basal domain. The PCM also relocalized to the apical domain of the cell, but this occurred after the redistribution of the microtubules by a mechanism that involved the microtubule network. The possible roles of both MTOCs and microtubules in establishing cell polarity are discussed.     

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.     

Increased cell cycle-dependent staining of plant cells by the antibody MPM-2 correlates with preprophase band formation

Cytoplasmic microtubules of animal cells catastrophically depolymerize upon entry into mitosis but in higher plants there is a longer transition during which cortical microtubules form an increasingly narrow preprophase band, and the chromatin gradually condenses. Progression towards mitosis in onion root tip cells was analysed using a CCD camera and image processing to quantify fluorescence staining by the monoclonal antibody MPM-2, which recognizes mitotic phosphoproteins in a range of eukaryotic cells. MPM-2 fluorescence, which was predominantly nuclear, was categorized relative to the stage of the DNA cycle (using DAPI), and to the microtubule cycle (using anti-tubulin) in individual cells. Cells with the characteristic interphase cortical microtubule arrays had a bimodal distribution of DAPI fluorescence, indicating that some were in G1 (2C DNA) whilst the double value suggested the others to be in G2 (4C). There was no difference in MPM-2 fluorescence between 2C and 4C cells possessing the cortical array in which microtubules were evenly distributed. However, in 4C cells possessing a preprophase band MPM-2 values doubled; this relationship applied not only to tight PPBs but to early, broad PPBs in which the individual microtubules could still be distinguished. Since alkaline phosphatase abolished MPM-2 reactivity it is concluded that mitotic phosphoproteins do not necessarily begin to accumulate in G2 per se , but during that part of G2 when the preprophase band first becomes recognizable as a distinct entity.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.