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Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% +/- 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% +/- 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% +/- 6.27% for PL+F and 47.2% +/- 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% +/- 8.89% for PL+F and 39.6% +/- 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a 'pre-defined' apical location; (ii) translocated to a 'newly defined' basal location upon stimulation with immobilized antigen-antibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.
Positioning of the microtubule-organizing center (MTOC) in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as defined by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.
The nuclear division of E. histolytica trophozoites was analyzed by using specific stains for DNA, with the aim to define the sequential changes of chromatin during its life cycle. Furthermore, we characterized the internal structural arrangements of microtubules in the microtubular organizing center (MTOC) and determined the number of chromosomes and its association with the spindle. The MTOC is formed by multiple microtubule-nucleating centers, that are involved in the displacement of DNA during nuclear division. We found the existence of a single MTOC in one pole of the nucleus at early anaphase. Our results lead us to propose a new hypothesis in which it is suggested that metaphase corresponds to the arrangement of condensed DNA bodies, or "chromosomes" around the MTOC and, through the assembly of microtubules, one set of uncondensed chromatin is displaced to the opposite pole of the nucleus, while the other remains condensed and associated to the original MTOC. We observed six chromosomes in our preparations, corroborating previous observations (2,3). Whether or not a new MTOC is formed during nuclear division remains to be clarified.
Cytoplasmic tubulin and the microtubule organizing centers (MTOCs) for the cytoskeletal microtubule system of the flagellate Polytomella have been isolated. The isolated MTOCs serve as sites for the in vitro assembly of the purified tubulin protein. The major proteins (four polypeptides of molecular weights 190,000-210,000) functioning in this assembly have been extracted from the MTOCs and purified. Kinetic studies and experiments with in vivo 35S-labeled MTOC proteins (or 35S-labeled tubulin) demonstrate that these proteins function specifically in microtubule initiation and do not contribute to microtubule elongation. The results indicate that microtubule assembly in vivo is controlled by microtubule initiating proteins associated with the organelles termed MTOCs.
Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.
As mammalian cells enter mitosis, the Golgi complex is disorganized and the remnants are dispersed throughout the cytoplasm in the form of a few short cisternae and small clusters of vesicles. Once the separation of the chromosomes is completed and nuclei reform, stacks of flattened cisternae reappear and a united Golgi complex of interphase type starts to be reorganized. This process is believed to ensure an approximately equal partitioning of the Golgi complex on the daughter cells. Here, the configuration of the Golgi complex and its relation to the cytoplasmic microtubule system was studied at the end of cytokinesis using synchronized cultures of L929 mouse fibroblasts and rat dermal fibroblasts. One hour after the release of the mitotic block, the Golgi complex (visualized immunocytochemically with antibodies against mannosidase II) was most frequently located on the proximal side of the nucleus as related to the intercellular bridge (visualized immunocytochemically with antibodies against tyrosinated alpha-tubulin). One hour later, it was preferentially found on the distal side of the nucleus as related to the intercellular bridge. Immunocytochemical demonstration of the radiating pattern of microtubules, and direct demonstration of the centrioles using antibodies against detyrosinated or acetylated alpha-tubulin, showed that the microtubule organizing center (MTOC) shifted position in a similar manner as the Golgi complex. Moreover, double staining with antibodies against mannosidase II and tyrosinated alpha-tubulin revealed that the Golgi complex and the MTOC codistributed at both times after the release of the mitotic block. Electron microscopic analysis confirmed that the reforming Golgi stacks first gathered close to the centrosome (a pair of centrioles with associated structures, constituting the main MTOC in the cell) on the proximal side of the nucleus and that the Golgi stacks and the centrosome were subsequently both relocated to the distal side of the nucleus as related to the intercellular bridge. Taken together, the findings indicate that the Golgi complex goes through a characteristic translocation in the terminal phase of cytokinesis and confirm the idea that the cytoplasmic microtubule system plays an important role in the organization of this organelle system. A possible function of the shift in location of the Golgi complex at the end of cytokinesis could be to direct membrane traffic first to the elongating intercellular bridge and thereafter to the leading edge as the cells are about to separate and move away from each other.
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 in Plasmodium spp., the causative agent of malaria, is fundamentally different from model eukaryotes, proceeding via a bipartite microtubule organising centre (MTOC) and lacking canonical regulators such as Polo and Bub1 kinases. During schizogony, asynchronous nuclear replication produces a multinucleate schizont, while rapid male gametogony generates an octaploid nucleus before gamete formation. Here, we identify Aurora-related kinase 1 (ARK1) as a key component of inner MTOC and spindle formation, controlling kinetochore dynamics and driving mitotic progression. Conditional ARK1 depletion disrupts spindle biogenesis, kinetochore segregation, karyokinesis and cytokinesis in both stages, and affects parasite transmission. Interactome analysis reveals ARK1 as the catalytic core of a non-canonical chromosomal passenger complex (CPC) containing two divergent inner centromere proteins (INCENPs) but lacking Survivin and Borealin. Comparative genomics indicates this CPC architecture arose early in Apicomplexa, replacing canonical centromere-targeting modules. These findings uncover a distinct mitotic machinery in Plasmodium and identify the ARK1-INCENP interface as a potential multistage target for malaria therapeutic intervention.
The olfactory neuron is specialized along its length into highly determined morphological regions. These regions include the dendritic cilia, dendritic vesicle, dendritic shaft proper, perikaryon, axon, zone of transition where the axon widens as it approaches its termination, and the axon terminal. Except for the zone of transition and the terminal, characteristic populations of microtubules occur in these compartments. In the olfactory vesicle, three discrete microtubule organizing centers (MTOCs) nucleate microtubules: the basal body, the lateral foot associated with the body, and dense masses of nearby material. Little is known about MTOCs elsewhere in the neuron, although the polarity of the axonal microtubules indicate that they originate at or near the perikaryon. An attempt is made to summarize what is known of the origin, structure, distribution, and function of microtubules in vertebrate olfactory neurons, which are useful model systems in which to study microtubules. Information about olfactory neuron microtubules may be applicable to neurons in general (e.g., the discovery that axons contain microtubules of uniform polarity was first made in the olfactory neuron) or to microtubules in other eukaryotic cells.
We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.
Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.
The distribution of ribonucleoprotein (RNP) within the mitotic spindle of newt lung epithelial cells was studied with the high voltage electron microscope (HVEM) using Bernhard's uranyl-EDTA-lead staining of thick sections in conjunction with the ribonuclease digestion of fixed cells. The results indicate that aside from ribosomes, the major RNP-containing components of the spindle are the kinetochores and centrioles, both of which stain electron-opaque after EDTA treatment. In both cases, the electron-opaque material associated with these microtubule organizing centers (MTOC's) can be removed by RNAse digestion and cold perchloric acid (PCA) extraction under conditions which leave the spindle microtubules (Mts) centrioles, and kinetochores intact. The staining reaction is not abolished by cold PCA extraction alone or by substituting other positively charged proteins (i.e., cytochrome c or lysozyme) for RNAse. The RNP component of the kinetochore is closely associated with the bases of the kinetochore microtubules. The RNP component of the centriole can be seen to surround the microtubules of the triplet blades. No evidence was found to indicate the presence of RNP in the pericentriolar material. The possible function of both kinetochore and centriolar RNP is discussed.
A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.
This investigation provides evidence that pericentriolar material is divorced from the immediate vicinities of centrioles and becomes functionally associated with the plasmalemma during the differentiation of a mammalian cell type. Such events occur prior to the assembly of large transcellular microtubule bundles in columnar epithelial cells called inner pillar cells in the mouse organ of Corti. The microtubules do not radiate from a typical centrosome and its centrioles. They elongate from a microtubule-organizing centre (MTOC), which is deployed as a subapical cell surface-associated layer in each cell. Most of the dense material of this layer, and the tops of most of the microtubules, are initially concentrated around the sides of a cell about 1 microns below its apical surface. In addition, a pair of centrioles is located above the layer, which acts as if it is a pericellular concentration of the pericentriolar material of a modified centrosome. Although microtubule nucleation takes place in a centrosome-like region, 13 protofilament fidelity is not exercised. Most of the microtubules have 15 protofilaments. Microtubule assembly progresses in these cells after the organ of Corti has been isolated for in vitro culture. However, large numbers of microtubules elongate from pericentriolar material juxtaposed against the centrioles. Hence, there is some reversion by the centrosomes of cultured cells to the operational configuration regarded as typical for animal tissue cells in general.
Spermatogonia, nutritive cells, and developmental stages of spermatids were observed with the electron microscope. Spermatogonia are near the surface of the testis and contain large nuclei and comparatively little cytoplasm. Nutritive or supporting cells are associated with the spermatogonia. Early spermatids are characterized by a circle of mitochondria around the nucleus. Late spermatids have 2 parallel free flagella separated by a cytoplasmic process, and a nucleus containing electron-dense strands of chromatin arranged in coils or concentric layers. Mature sperm have 2 flagella enclosed by cytoplasm. Their nuclei contain dense, fibrillar chromatin. A microtubule-organizing center (MTOC) found between basal bodies of spermatids is described. Descriptions are presented of the seminal reservoir, seminal vesicle, and the sperm found in those organs.
Mouse neuroblastoma N-18 cells can be induced by serum deprivation to sprout multiple neurite-like processes which contain many microtubules. Mitotic drugs such as colcemid and colchicine depolymerize these microtubules and the cells lose their processes. Reappearance of microtubules after removal of the drugs was followed by immunofluorescence microscopy using tubulin specific antibodies. At early recovery times multiple star-like structures which contained tubulin were detected in the perinuclear are and in the cytoplasm of individual cells. The mean number seen per cell as approximately 5. Their formation preceeded the organization of the complex microtubular networks typical of N-18 cells. The probable action of these structures as microtubular organization centers (MTOCs) is discussed. Multiple structures were detected during recovery from the influence of mitotic drugs both in previously induced and non-induced N-18 cells, suggesting that N-18 cells harbour the potential of formation of multiple organization centers even without previous induction. We discuss the possibility that differentiation of neuroblastoma N-18 cells may require microtubular organization centers.