OBSERVATIONS ON SPERMIOGENESIS IN THE FUNGUS GNAT SCIARA COPROPHILA

Although 9-membered centrioles are found in somatic tissues of Sciara, the centriole which lies at the spindle pole of the second meiotic division in male Sciara is composed of a row of approximately 70 short tubules in an oval array. Shortly after telophase of this unequal division, in the daughter cell destined to undergo spermiogenesis, microtubules become confluent with the tubules of the centriole. These tubules have the same density as other cytoplasmic microtubules after glutaraldehyde-OsO₄ fixation and, like them, are not preserved with OsO₄ fixation. As the centriole, now with approximately 70 attached, posteriorly directed, doublet tubules, migrates from the polar to the apolar end of the nucleus to take a final position in an oval groove which forms in the nuclear envelope, the tubules lengthen and become demonstrable after OsO₄ fixation and more electron opaque than other cytoplasmic microtubules following glutaraldehyde-OsO₄ fixation. Later, a singlet tubule appears peripherally to each doublet of the oval and 4 "arms" develop at specific sites on the tubules. Posteriorly, where the oval of tubules becomes discontinuous and forms a spiral, the arrangement of arms is different and the singlet tubules are lacking. Dense solid bodies develop inside this odd flagellum and become enclosed by a smooth double membrane. A single mitochondrial derivative has three components: a central area of homogeneous, moderately electron-opaque, proteinaceous material; a peripheral ring of cristae; and a crystalloid which is specifically oriented with respect to the flagellar tubules.     

Centrin-2 is required for centriole duplication in mammalian cells

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.     

Mother centrioles are kicked out so that starfish zygote can grow

Most oocytes eliminate their centrioles during meiotic divisions through unclear mechanisms. In this issue, Borrego-Pinto et al. (2016. J Cell. Biol. http://dx.doi.org/10.1083/jcb.201510083) show that mother centrioles need to be eliminated from starfish oocytes by extrusion into the polar bodies for successful embryo development.Canonical centrosomes contain a pair of centrioles, often made of nine triplets of microtubules and surrounded by the pericentriolar material (PCM). They are the major microtubule organizing centers in most cells, which organize the microtubule spindle required to segregate chromosomes during cell division. Yet, most oocytes get rid of their centrioles. The biological significance of oocyte centriole riddance remains a mystery. Removing centrioles in oocytes could prevent some species, like Xenopus, from undergoing parthenogenetic development (Tournier et al., 1991). Also, eliminating the maternal centrioles is required to prevent the zygote from having an abnormal number of centrioles after fertilization, as sperm contribute two centrioles (motile sperm cells require centriole-based flagellar assembly and must retain their centrioles until fertilization [Manandhar et al., 2005]). In Drosophila, Xenopus, nematode, mouse, and human oocytes, egg centrioles are eliminated during meiotic prophase before oocyte asymmetric divisions (Szollosi et al., 1972; Manandhar et al., 2005; Januschke et al., 2006). Apart from the involvement of a helicase of undefined substrates, the pathway leading to centriole elimination has not been identified (Mikeladze-Dvali et al., 2012).In contrast, starfish oocytes, like sea urchin or mollusk, eliminate their centrioles later in meiotic divisions (Nakashima and Kato, 2001; Shirato et al., 2006). Centrioles are replicated in a semiconservative manner during the S phase of the cell cycle. The old centriole, named the mother, is characterized by the presence of distal and subdistal appendages and serves as a template for the assembly of a new daughter centriole, lacking appendages (Bornens and Gönczy, 2014). However, to become haploid, oocytes undergo two consecutive divisions with no intervening DNA replication. Hence, centrioles are not duplicated between the two meiotic divisions and oocytes keep their number of centrioles limited to four. This also means that starfish oocytes assemble their first meiotic spindle in the presence of a pair of centrioles at each pole (Fig. 1 A). Out of the four centrioles contained in the oocyte, two (one mother and one daughter centriole) are extruded into the first polar body during the first asymmetric division. Subsequently, the second meiotic spindle is formed with only one centriole per pole (Fig. 1 A), and one centriole is extruded in the second polar body. Previous work suggested that the poles of the second meiotic spindle in starfish are not functionally equivalent (Uetake et al., 2002). In this issue, Borrego-Pinto et al. find that the mother centriole retains the ability to nucleate asters but is specifically guided into the second polar body for extrusion, whereas the daughter centriole is inactivated and then eliminated within the oocyte.Open in a separate windowFigure 1.Centriole elimination during meiotic maturation of starfish oocytes. (A) Scheme of starfish oocyte meiotic divisions and early egg development. Oocyte divisions are asymmetric in size; meiotic spindles are off-centered in these large cells; and daughter cells are tiny, tailored to the chromatin mass, and named polar bodies. Microtubules are green, DNA is pink, maternal centrosomes are yellow, and sperm centrosomes are orange. (B) Fate of mother and daughter centrioles during meiotic divisions. Centrosomes are artificially enlarged to emphasize the centrioles. PB1 and PB2, first and second polar body, respectively. During anaphase I, the DNA and centrioles are segregated; one set of chromosomes and one pair of centrioles are extruded into PB1 during anaphase I. The remaining mother centriole separates from its paired daughter and rapidly moves toward the plasma membrane, where it is extruded in the second polar body (PB2) during anaphase II, leaving one set of oocyte chromatids to combine with the sperm chromatids. The remaining oocyte daughter centriole is inactivated and degraded after anaphase II. Therefore, only the sperm centrioles form the first mitotic spindle in the fertilized oocyte. Oocytes forced to retain a mother centriole form a tripolar aster upon fertilization, which stops development.To investigate the mechanism of centriole elimination in the starfish Patiria miniata, Borrego-Pinto et al. (2016) first isolated homologues of centrosomal proteins and constructed fluorescent protein fusions to several centriolar proteins to track centriole fate in 3D time-lapse imaging during oocyte asymmetric divisions. Using specific markers of mother versus daughter centrioles, they established that, in meiosis I, the two spindle poles are equivalent, being constituted of a pair of mother and daughter centrioles. At anaphase I, one pair of mother/daughter centrioles is extruded into the first polar body. Importantly, the authors described an asymmetry in metaphase II, with the second meiotic spindle always having the mother centriole facing the cortex and the daughter centriole deep inside the cytoplasm (Fig. 1 B).Borrego-Pinto et al. (2016) went on to identify the origin of this asymmetry. They show that the mother centriole, but not the daughter one, starts being rapidly transported toward the plasma membrane before completion of meiosis I spindle disassembly in a microtubule- and dynein-dependent manner, as its trafficking could be impaired by the dynein inhibitor ciliobrevin D (Firestone et al., 2012). In a second step, the mother centriole is anchored to the plasma membrane through the second meiotic division. Interestingly, electron microscopy of starfish oocytes revealed electron-dense material as well as vesicles between the mother centriole and the plasma membrane, suggesting that the mother centriole’s plasma membrane anchorage occurs via its appendages (Reiter et al., 2012; Stinchcombe et al., 2015). Whether the mother centriole migrates to the cortex with its appendages facing or opposite the plasma membrane has not been addressed. However, it is reasonable to assume that, in a viscous environment such as the oocyte cytoplasm, a motion with the appendages up would be favored (Fig. 1 B). Moreover, whereas the migration of the mother centriole to the plasma membrane requires microtubules, its anchoring does not depend on microtubules or microfilaments, as shown by the continued tight association between the centriole and the membrane in the presence of microtubule- and/or actin-depolymerizing agents. This close anchoring via the centriole’s appendages is reminiscent of the anchoring of centrioles forming cilia or at the immunological synapse in T cells (Stinchcombe et al., 2015). The precise mechanisms involved in mother centriole anchoring to the plasma membrane in starfish might be conserved in other systems that also require proximity between these two structures. It would be interesting to assess whether astral microtubules emanating from the mother centriole progressively depolymerize as the mother centriole approaches the plasma membrane to allow the intimate anchoring of the appendages with the plasma membrane. If so, Katanin, a microtubule-severing enzyme whose activity is regulated during meiotic divisions in the nematode oocyte, would be a good candidate to promote such a progressive destabilization (Srayko et al., 2000).Future work will tell us why the daughter centriole does not experience such a migration event. This strongly argues for a functional asymmetry between the two types of centrioles. From the work of Borrego-Pinto et al. (2016), it appears that the daughter centriole is passively pushed inside the oocyte cytoplasm as a result of meiosis II spindle assembly and elongation. Dynein, which controls the migration of the mother centriole, could specifically associate with this centriole, like it does in Saccharomyces cerevisiae, by localizing preferentially to the spindle pole body (the yeast equivalent of the centrosome) facing the bud (Grava et al., 2006). Centrosome asymmetry has been described in several stem cell types (Roubinet and Cabernard, 2014) and this asymmetry is often rooted in its activity. However, Borrego-Pinto et al. (2016) show that the microtubule nucleation capacity of the daughter and mother centrioles is equivalent up to the metaphase II stage. It is only after fertilization and anaphase II that a difference in activity is detected between the mother and daughter centrioles. Thus, what underlies the asymmetry in behavior between the mother and daughter centrioles at anaphase I remains to be discovered. One possibility is that the presence of appendages in the mother centriole allows the recruitment of specific factors, such as dynein, which in turn regulate mother centriole migration and anchoring.Borrego-Pinto et al. (2016) also discovered that specific anchoring of the mother centriole to the plasma membrane, at which the second polar body will form, is the mechanism by which oocytes get rid of the remaining mother centriole. Importantly, actively removing the mother centriole after anaphase II is essential for zygotic development. Indeed, the researchers used the actin polymerization inhibitor cytochalasin D to prevent extrusion of the second polar body and artificially retain the mother centriole in the oocyte after anaphase II. When a mother centriole is retained, it keeps its microtubule nucleation capacity and participates in the first mitotic spindle pole organization of the fertilized egg, whereas the daughter centriole is inactivated and dismantled after anaphase II. As a consequence, because of the two centrioles contributed by the sperm cell, the mitotic spindle ends up being tripolar in the presence of an additional mother centriole, precluding correct chromosome segregation and further development (Fig. 1 B).The origin of the difference in behavior between mother and daughter centrioles after anaphase II will require further investigation. To explain the loss in nucleation capacity of the daughter centriole, it will be important to check for the presence of various PCM components. Indeed, it is reasonable to assume that the daughter centriole loses its PCM association. PCM size scales with centriole size; thus, appendages of the mother centriole might possess an innate ability to maintain association with the PCM (Bobinnec et al., 1998; Delattre et al., 2004). A possible cell cycle–dependent enzymatic activity appearing after anaphase II might explain the rapid loss in microtubule nucleation capacity of the daughter centriole. It is surprising that the starfish zygote cannot cluster the mother centriole material with the centrioles from the sperm, unlike mouse oocytes, which, like cancer cells, are able to cluster PCM to regulate the total number of microtubule organizing centers (Kwon et al., 2008; Breuer et al., 2010). It will be interesting to determine whether starfish zygotes express proteins such as HURP or HSET, which are major players in extra-centrosome clustering (Kwon et al., 2008; Breuer et al., 2010).Altogether, the results from Borrego-Pinto et al. (2016) address a major unresolved question: why do oocytes lose or inactivate their canonical centrioles during female meiosis? They show for the first time that maternal centrioles must be extruded from or inactivated in the starfish egg before fertilization so that they do not perturb mitotic spindle assembly. This is a very important step in our understanding of female gamete formation. Moreover, this work establishes starfish oocyte meiosis as a novel model system to study both functional and structural centrosome asymmetry, an essential component of asymmetric divisions.     

Centrioles in the cell cycle. I. Epithelial cells

A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.     

Zoosporulation in Labyrinthula sp.; an Electron Microscope Study1

SYNOPSIS. Zoosporulation in Labyrinthula sp. in monoxenic culture was initiated by aggregation of spindle cells into reticulate sori. The spindle cells then changed into rounded or oval cells and formed, de novo, 2 pairs of centrioles at opposite sides of each nucleus. A pair of granular aggregates (protocentrioles) ～ 240 mμ in diameter served as precursor bodies during centriole formation. Spindle microtubules around the prophase nucleus connected the pairs of centrioles but were not found in the nucleoplasm until nuclear envelope fragmentation occurred. Prophase nuclei of uninucleated sporangia contained synaptinemal complexes; therefore, meiosis is presumed to occur. The envelope fragments moved toward the centrioles and regrouped to form the nuclear membranes of the daughter cells. Alternating nuclear and cytoplasmic divisions subdivided the preparation into 8 cells which differentiated into laterally biflagellated zoospores. Flagellar development involved growth of the kinetosome microtubules into a bud which formed over the kinetosome tangential to the cell surface. Kinetosomes were derived directly from centrioles with little differentiation other than addition of an electron-dense core to the lumen of the centriole. Zoospore ultrastructure included a stigma comprised of a row of electron-dense granules located slightly under the plasmalemma and posterior to the pair of kinetosomes. A single row of 17–21 microtubules lay parallel to the stigma granules, one or more being connected to the anterior kinetosome. A striated fiber apparatus similar to that found in some phytoflagellates connected the midregions of the kinetosomes. Fibers 1.0–1.2 μ long were attached to the plasmalemma around the base of the anterior flagellum. Zoospores settled on the substrate and differentiated directly into spindle cells. Since synaptinemal complexes were observed the planonts are probably haploid zoospores and probably not gametes since planogametic copulation was not observed.     

Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.     

THE STRUCTURE AND FUNCTION OF CENTRIOLES AND THEIR SATELLITES IN THE JELLYFISH PHIALIDIUM GREGARIUM

Testes of jellyfish Phialidium gregarium were fixed in 2 per cent OsO₄ in Veronal-acetate buffer at pH 7.4. Thin sections showed that in young spermatids the spindle fibers of the last maturation division are attached to satellites of the filament-forming centriole. In more mature spermatids this attachment is not observed. During the developmental phase, nine satellites can be observed emanating from the interspaces between the nine tubular triplets of this centriole. A circular region on each of the enlarged distal ends of the satellites attaches them to the cell membrane. The satellites apparently provide a firm anchor for the axial filament. Each of the epithelial cells covering the testis produces a single long flagellum. On the filament-forming centriole often a satellite can be observed to which tubules are attached. These tubules are 180 A in diameter and probably represent remnants of spindle fibers. It is suggested that the distal centriole has the ability to form several satellites or appendages at appropriate times during the cell cycle. These satellites are distinct from the daughter centrioles in that they are supportive structures: in certain phases of cell life, spindle fibers may attach to them, while in other instances the distal centriole and the flagellum it is forming are anchored by them.     

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     

Cnn dynamics drive centrosome size asymmetry to ensure daughter centriole retention in Drosophila neuroblasts

Centrosomes comprise a pair of centrioles surrounded by an amorphous network of pericentriolar material (PCM). In certain stem cells, the two centrosomes differ in size, and this appears to be important for asymmetric cell division [1, 2]. In some cases, centrosome asymmetry is linked to centriole age because the older, mother centriole always organizes more PCM than the daughter centriole, thus ensuring that the mother centriole is always retained in the stem cell after cell division [3]. This has raised the possibility that an "immortal" mother centriole may help maintain stem cell fate [4, 5]. It is unclear, however, how centrosome size asymmetry is generated in stem cells. Here we provide compelling evidence that centrosome size asymmetry in Drosophila neuroblasts is generated by the differential regulation of Cnn incorporation into the PCM at mother and daughter centrioles. Shortly after centriole separation, mother and daughter centrioles organize similar amounts of PCM, but Cnn incorporation is then rapidly downregulated at the mother centriole, while it is maintained at the daughter centriole. This ensures that the daughter centriole maintains its PCM and so its position at the apical cortex. Thus, the?daughter centriole, rather than an "immortal" mother centriole, is ultimately retained in these stem cells.     

Centriole number and the reproductive capacity of spindle poles

The reproduction of spindle poles is a key event in the cell's preparation for mitosis. To gain further insight into how this process is controlled, we systematically characterized the ultrastructure of spindle poles whose reproductive capacity had been experimentally altered. In particular, we wanted to determine if the ability of a pole to reproduce before the next division is related to the number of centrioles it contains. We used mercaptoethanol to indirectly induce the formation of monopolar spindles in sea urchin eggs. We followed individually treated eggs in vivo with a polarizing microscope during the induction and development of monopolar spindles. We then fixed each egg at one of three predetermined key stages and serially semithick sectioned it for observation in a high-voltage electron microscope. We thus know the history of each egg before fixation and, from earlier studies, what that cell would have done had it not been fixed. We found that spindle poles that would have given rise to monopolar spindles at the next mitosis have only one centriole whereas spindle poles that would have formed bipolar spindles at the next division have two centrioles. By serially sectioning each egg, we were able to count all centrioles present. In the twelve cells examined, we found no cases of acentriolar spindle poles or centriole reduplication. Thus, the reproductive capacity of a spindle pole is linked to the number of centrioles it contains. Our experimental results also show, contrary to existing reports, that the daughter centriole of a centrosome can acquire pericentriolar material without first becoming a parent. Furthermore, our results demonstrate that the splitting apart of mother and daughter centrioles is an event that is distinct from, and not dependent on, centriole duplication.     

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new ‘daughter’ centriole is built orthogonally on one side of each radially symmetric ‘mother’ centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.     

FINE STRUCTURE OF SCIARA COPROPHILA SPERM

Though the fagellum of Sciara sperm arises from a blepharoplast and is characterized by doublet tubules with arms, it differs markedly from the familiar type of flagella in the number and arrangement of its tubules. The axial filament complex in sperm from the testis of Sciara consists of approximately 70 doublet tubules, each with an associated singlet tubule. Near the nucleus these tubules are displaced in an oval array. Posteriorly the oval breaks and coils from one free end so that the axial filament complex at posterior levels has the form of a spiral. The singlet tubules do not extend the full length of the sperm but terminate in order from inside the spiral. Farther posteriorly the axial filament complex reverses the direction of coiling, and the doublets terminate from outside the spiral. Four arms are specifically positioned on the singlet and doublet tubules. A single mitochondrial derivative extends most of the length of the sperm; it consists of a large mass of proteinacious material, a crystalloid located adjacent to the axial filament complex, and peripheral cristae. In the female genital tract, sperm undergo gross morphological changes which include sloughing of practically all the mitochondrial material except the crystalloid, repositioning of the crystalloid, and uncoiling and subsequent recoiling of the axial filament complex into a different configuration. From analysis of serial sections it was determined that the orientation of arms, when the axial filament is viewed from base to tip, is the same as in conventional flagella.     

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.     

Daughter Centriole Elongation Is Controlled by Proteolysis

The centrosome is the major microtubule-organizing center of most mammalian cells and consists of a pair of centrioles embedded in pericentriolar material. Before mitosis, the two centrioles duplicate and two new daughter centrioles form adjacent to each preexisting maternal centriole. After initiation of daughter centriole synthesis, the procentrioles elongate in a process that is poorly understood. Here, we show that inhibition of cellular proteolysis by Z-L₃VS or MG132 induces abnormal elongation of daughter centrioles to approximately 4 times their normal length. This activity of Z-L₃VS or MG132 was found to correlate with inhibition of intracellular protease-mediated substrate cleavage. Using a small interfering RNA screen, we identified a total of nine gene products that either attenuated (seven) or promoted (two) abnormal Z-L₃VS–induced daughter centriole elongation. Our hits included known regulators of centriole length, including CPAP and CP110, but, interestingly, several proteins involved in microtubule stability and anchoring as well as centrosome cohesion. This suggests that nonproteasomal functions, specifically inhibition of cellular proteases, may play an important and underappreciated role in the regulation of centriole elongation. They also highlight the complexity of daughter centriole length control and provide a framework for future studies to dissect the molecular details of this process.     

Ciliogenesis and centriole formation in the mouse embryonic nervous system. An ultrastructural analysis

Serial ultrathin sections were used to study the formation of the primary cilium and the centriolar apparatus, basal body, and centriole in the neuroepithelial primordial cell of the embryonic nervous system in the mouse. At the end of mitosis, the centrioles seem to migrate toward the ventricular process of the neuroepithelial cell, near the ventricular surface. One of these centrioles, the nearest to the ventricular surface, begins to mature to form a basal body, since its tip is capped by a vesicle probably originating in the cytoplasm. This vesicle fuses with the plasmalemma and the cilium growth by the centrifugal extension of the 9 sets of microtubule doublets. These 9 sets invade the thick base of the cilium which is initially capped by a ball-shaped tip with the appearance of a mushroom cilium. The secondary extension of 7, then 5, and finally 2 sets of microtubule doublets contribute to form the tip of the mature cilium, which is associated with a mature centriolar apparatus formed by a basal body and a centriole. Centriologenesis occurs before mitosis and is concomitant with the progressive resorption of the cilium. The daughter centriole, or procentriole, begins to take form near the tips of fibrils that extend perpendicularly and at a short distance from the wall of the parent centriole. Osmiophilic material accumulates around these fibrils, and gives rise to the microtubules of the mature daughter centriole. These centrioles formed by a centriolar process are further engaged in mitosis, after the total resorption of the cilium. This pattern of development suggests that in the primordial cells of the embryonic nervous system, centriologenesis and ciliogenesis are 2 independent phenomena.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

Reproductive maternal centrosomes are cast off into polar bodies during maturation division in starfish oocytes

Animal egg inherits a maternal centrosome from the meiosis-II spindle and sperm can introduce another centrosome at fertilization. It has been believed that in most animals only the sperm centrosome provides the division poles for mitosis in zygotes. This uniparental (paternal) inheritance of the centrosome must depend on the loss of the maternal centrosome. In starfish, suppression of polar body (PB) extrusion is a prerequisite for induction of parthenogenesis (Washitani-Nemoto et al. (1994) Dev. Biol. 163, 293-301), suggesting that the centrosomes cast off into PBs have reproducing capacity. Due to the absence of centriole duplication in meiosis II of starfish oocytes, each centrosome of a meiosis-II spindle has only one single centriole, whereas in meiosis I each has a pair of centrioles (Sluder et al. (1989) Dev. Biol. 131, 567-579; Kato et al. (1990) Dev. Growth Differ. 32, 41-49). Hence, the first PB (PB1) has two centrioles, whereas the second PB (PB2) and the mature egg have only one centriole, respectively. The present study examined the reproductive capacity of PB centrosomes by transplanting them into artificially activated eggs, and then the recipient egg nucleus with the surrounding cytoplasm was removed. A transplanted PB2 centrosome with a single centriole formed a monopolar spindle at the first mitosis, followed by formation of a bipolar spindle in the next mitosis, leading to actual cleavage and subsequent development. This proves the reproducing capacity of the single centriole in the PB2 centrosome. The behavior of the transplanted PB1 centrosome was exactly the same as in the PB2 centrosome, in spite of the difference in the number of centrioles. These results clearly show that four maternal centrioles are heterogeneous in duplicating capacity, during meiosis only one centriole in each of the centrosomes of a meiosis-I spindle pole retains duplicating capacity, the reproductive centrioles are successively cast off into PBs, and finally a mature egg inheriting a nonreproductive centriole alone is formed, and the presence of a single reproductive centriole is sufficient condition for embryonic development in starfish.     

The centriole cycle in the amoebae of the myxomycetePhysarum polycephalum

Summary In the amoebae of the myxomycetePhysarum polycephalum, procentrioles are formed on the anterior and posterior centrioles in early prophase. Although the relative position of the parental and procentrioles is fixed, all relative positions of the daughter and parental centrioles were observed. During the different stages of mitosis daughter centrioles elongate and acquire anterior satellites, one of the characteristic features of the anterior centrioles. All other anterior morphological characteristics appear only in telophase and early reconstruction stages. In contrast to the parental posterior centrioles, which do not change morphologically during the successive mitotic stages, the parental anterior centrioles lose their morphological characteristics in late prophase and early prometaphase and then acquire the morphological features characteristic of the posterior centrioles. Thus, the following maturation scheme is suggested: a procentriole becomes an anterior centriole during the first mitosis and a posterior centriole during the second mitosis. Since posterior features are maintained during mitosis, the posterior centriole corresponds to the final state of centriole maturation.     

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.     

Control of daughter centriole formation by the pericentriolar material

Controlling the number of its centrioles is vital for the cell, as supernumerary centrioles cause multipolar mitosis and genomic instability. Normally, one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in S-phase-arrested cells. Under conditions when multiple daughters form simultaneously on a single mother, all of these daughters must be removed to induce reduplication. The number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) by overexpression of the PCM protein pericentrin in S-phase-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.