The role of the centriolar region in animal cell mitosis. A laser microbeam study

An argon ion laser microbeam (488 and 514 nm) was used to selectively irradiate one of the two centriolar regions of rat kangaroo Potorous tridactylis (PtK2) prophase cells in vitro. The cells were sensitized to the laser radiation by treatment with acridine orange (0.1-0.2 mug/ml). Ultrastructural examination of the irradiated centriolar regions demonstrated that the primary site of damage was the pericentriolar material. This result suggests that nucleic acid is present in the pericentriolar material. Behavioral and ultrastructural analysis demonstrated that cells with one damaged pericentriolar zone could undergo (a) nuclear membrane breakdown, (b) chromosome condensation, (c) metaphase plate formation, and (d) cytokinesis. However, the chromosomes neither separated nor exhibited any anaphase movements. Detailed ultrastructural analysis revealed the presence of kinetochore microtubules on both sides of chromosome mass and a lack of microtubules in the cytokinesis constriction. These results indicate that the pericentriolar material is important in spindle orgainization and essential for the formation of the interpolar microtubules.     

Inhibition of proteasome activity impairs centrosome-dependent microtubule nucleation and organization

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.     

Centrosome size is controlled by centriolar SAS-4

The centrosome consists of a pair of centrioles and a surrounding matrix of pericentriolar material that anchors microtubule nucleation sites and consequently determines the number and organization of microtubules in interphase and mitotic cells. Recent studies utilizing a functional genomics approach in the nematode worm Caenorhabditis elegans and sophisticated light and electron microscopy techniques provide new insight into how centrioles act as centrosomal organizers and use a centriolar structural element to dictate centrosome size by defining their capacity to recruit pericentriolar material.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Identification of Vinculin as a Pericentriolar Component in Mammalian Cells

Previous studies have shown that centrosome position and structure can be influenced by actin filaments, that centrosomes can influence actin organization, and that an actin homologue is associated with centrosomes. Such observations suggest the existence of connections between centrosomes and actin networks. In keeping with such observations, we show that the pericentriolar material, a main component of centrosomes, contains vinculin, a well-known component of cell adhesion plaques and of adherens cell junctions. We find that in various cell types, centrosomes are specifically stained by five different anti-vinculin antibodies. In adherent cell lines, these antibodies also stained adhesion plaques, but in thymocytes, a cell type devoid of adhesive structures, such antibodies stained only centrosomes. Isolated centrosomes also reacted with the anti-vinculin antibodies and immuno-electron microscopy showed apparent localization of vinculin in the pericentriolar material. Immunoblot analysis confirmed the presence of vinculin in purified centrosomal protein preparations. In such protein fractions, anti-vinculin antibodies reacted with a single polypeptide with an apparent molecular weight similar to that of vinculin. Stepwise solubilization of centrosomal structures using urea showed that high urea concentrations were required to solubilize centrosomal vinculin, suggesting tight association of vinculin with the pericentriolar material. The identification of vinculin as a component of centrosomes provides a possible molecular basis for interaction between F-actin and centrosomes.     

Interaction proteomics identify NEURL4 and the HECT E3 ligase HERC2 as novel modulators of centrosome architecture

Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.     

Centriolar satellites: molecular characterization, ATP-dependent movement toward centrioles and possible involvement in ciliogenesis

We identified Xenopus pericentriolar material-1 (PCM-1), which had been reported to constitute pericentriolar material, cloned its cDNA, and generated a specific pAb against this molecule. Immunolabeling revealed that PCM-1 was not a pericentriolar material protein, but a specific component of centriolar satellites, morphologically characterized as electron-dense granules, approximately 70-100 nm in diameter, scattered around centrosomes. Using a GFP fusion protein with PCM-1, we found that PCM-1-containing centriolar satellites moved along microtubules toward their minus ends, i.e., toward centrosomes, in live cells, as well as in vitro reconstituted asters. These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization. Next, to understand the relationship between centriolar satellites and centriolar replication, we examined the expression and subcellular localization of PCM-1 in ciliated epithelial cells during ciliogenesis. When ciliogenesis was induced in mouse nasal respiratory epithelial cells, PCM-1 immunofluorescence was markedly elevated at the apical cytoplasm. At the electron microscopic level, anti-PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis. These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.     

Centrosome reduction during gametogenesis and its significance

Animal spermatids and primary oocytes initially have typical centrosomes comprising pairs of centrioles and pericentriolar fibrous centrosomal proteins. These somatic cell-like centrosomes are partially or completely degenerated during gametogenesis. Centrosome reduction during spermiogenesis comprises attenuation of microtubule nucleation function, loss of pericentriolar material, and centriole degeneration. Centrosome reduction during oogenesis is due to complete degeneration of centrioles, which leads to dispersal of the pericentriolar centrosomal proteins, loss of replicating capacity of the spindle poles, and switching to acentrosomal mode of spindle organization. Oocyte centrosome reduction plays an important role in preventing parthenogenetic embryogenesis and balancing centrosome number in the embryonic cells.     

Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells

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.     

Heat as a probe of centrosomal function: a phase-contrast and immunofluorescent study of human blood monocytes

In normal human blood monocytes, the nucleus is indented by the centrosome, which excludes the phase-dense granules that are spread throughout the cytoplasm. Within this paranuclear region, the paired centrioles are marked by immunofluorescent staining with an anti-centrosome antibody directed against the pericentriolar osmiophilic material that appears to serve as microtubule-organizing centers (MTOCs). Congruent paired structures are seen in phase-contrast. Following heat treatment (45 degrees C, 9 min), granules are retracted about a less indented nucleus, and anti-centrosome immunofluorescence is absent or very weak, even though paired centrosomal structures remain at least as phase-dense as in controls. Immunofluorescent staining with antimicrotubule antibody is also essentially lost following heat treatment. These findings are consistent with a heat-induced lesion in the pericentriolar osmiophilic material, which may prove generally useful as a probe of centrosomal function.     

Identification and function of the centrosome centromatrix

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.     

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole.     

Continuation of mitosis after selective laser microbeam destruction of the centriolar region

The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

Microtubule organization: a pericentriolar material-like structure in yeast meiosis

During meiotic prophase in fission yeast, the nucleus undergoes dramatic oscillatory movements. A newly identified structure, the radial microtubule organizing center (rMTOC), mediates these movements and shares some of the features of the pericentriolar material in higher eukaryotes.     

Does the autoimmune serum 5051 specifically recognise microtubule organising centres in plant cells?

The autoimmune serum 5051 recognises pericentriolar microtubule nucleating sites in animal cells. Because this whole serum immunostains higher plant cells it has previously been suggested that it is detecting microtubule organising centres in plants. From a study comparing immunostaining and immunoblotting activity of 5051 and normal human serum controls, in various plant and animal cells, we argue that there is no clear evidence to support this suggestion. A normal human serum, that did not recognise animal cell pericentriolar material, had a staining pattern in Allium cells indistinguishable from 5051. The spindle poles in Funaria or Chlamydomonas were not recognised by 5051 and several normal human sera mimicked the 5051 staining patterns observed. There was no similarity in the sizes of proteins detected by 5051 in animals and plants, and no proteins were detected only by 5051 and other sera giving the same staining.     

Ultrastructural Study of Asters Induced by Microinjection with Sperm Centriolar Fraction in Sea Urchin Eggs

Multiple asters can be artificially induced in sea urchin fertilized eggs by the microinjection of the centriolar fraction of sperm homogenate. Investigation was continued by the electron microscopy to determine whether the multi-aster formation was due to the centrioles or the contaminants in the injected sperm fraction. Thirty three asters in 3 operated eggs were thoroughly examined, and we confirmed that the presence of centrioles in the central region of 26 asters. We considered that the rest of them might contained the centrioles in the sections lost during the preparation procedures. Fragmented axoneme, the plug of electron dense material, and the centriolar fossa, which were usually accompanied with the isolated centrioles, disappeared from the centrioles in these multiple asters. However, electron dense, amorphous materials were formed associating with the triplet blades and distributed around the centrioles. Many astral microtubules were terminated in these pericentriolar materials. Results obtained suggest that, although the pericentriolar material is acting as the microtubule organizing center, all multiple asters, except those derived from fertilization (2 asters per egg), are most likely induced by the injected centrioles and not by the contaminants.     

Mechanisms of procentriole formation

The centrosome comprises a pair of centrioles and associated pericentriolar material, and it is the principal microtubule-organizing centre of most animal cells. Like the genetic material, the centrosome is duplicated once and only once during the cell cycle. Despite the fact that both doubling events are crucial for genome integrity, the understanding of the mechanisms governing centrosome duplication has lagged behind the fuller knowledge of DNA replication. Here, we review recent findings that provide important mechanistic insights into how a single procentriole forms next to each centriole once per cell cycle, thus ensuring that one centrosome becomes two.     

Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.     

Association of actin with sperm centrioles: isolation of centriolar complexes and immunofluorescent localization of actin

The centrioles of cnidarian sperm associate with striated specializations (pericentriolar processes) during spermiogenesis. Three functions have been proposed for the role of these structures: (a) an anchoring mechanism for the sperm flagellum, (b) a signal-transmitting mechanism for communication between sperm head and tal, and (c) a contractile mechanism involved in motor function of the sperm flagellum. To investigate these proposed functions, we developed a technique for the isolation and purification of Hydractinia sperm distal centriles with attached pericentriolar processes. SDS polyacrylamide electrophoretic profiles of whole sperm and pericentriolar process proteins revealed a prominent protein that comigrates with rabbit and penaeid shrimp muscle actin. To label and localize actin in hydroid spem, we produced in rabbits a highly specific antiserum to invertebrate actin that cross-reacts with both invertebrate and vertebrate muscle and nonmuscle actin. Immunofluorescent double antibody labeling of hydroid sperm with antiactin has demonstrated the presence of actin in the pericentriolar process region of the sperm. In earlier reports, it has been proposed that pericentriolar processes, if contractile, could alter the mid-piece asymmetry of hydroid sperm, facilitating the directional motility that these cells demonstrate in respone to egg-released chemoattractants. The present results support this hypothesis.