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.     

Centriole modification in human aortic endothelial cells.

The structure of centrioles in endothelial cells of embryonic (22-24 weeks old) and definitive (2, 14-17, and 30-40 years) human aorta in situ and also in aortic endothelial cells dividing in organ and cell cultures (donor age 30-40 years) was studied. It was found that in the endothelial cells from definitive aorta the lengths of mother centrioles vary from 0.5 to 2 microns, whereas the length of daughter centrioles remains constant (0.4-0.5 microns). The distal part of the cylinder of long mother centrioles consists of microtubule doublets. In aorta of donors 30-40 years old in multinucleated cells and in one of 30 single-nucleated cells analyzed, C-shaped long centrioles were seen. These centrioles exhibit a doublet organization along all their length. Mitotic cells in organ and cell culture had a nonequal structure of spindle poles: at one pole, the long mother centriole was seen, while at the other a mother centriole of standard size was found. In such cells of organ culture long centrioles make contact with the remnant of primary cilia until the end of anaphase. In cell culture mitotic cells are also observed containing C-shaped centrioles. In these cells the number of mother centrioles is odd and their number is not equal to the number of daughter centrioles. The possible mechanism for transformation of endothelial centrioles and its role in the control of cell-cycle progression are discussed.     

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.     

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.     

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.     

Asymmetric spindle pole formation in CPAP-depleted mitotic cells

CPAP is an essential component for centriole formation. Here, we report that CPAP is also critical for symmetric spindle pole formation during mitosis. We observed that pericentriolar material between the mitotic spindle poles were asymmetrically distributed in CPAP-depleted cells even with intact numbers of centrioles. The length of procentrioles was slightly reduced by CPAP depletion, but the length of mother centrioles was not affected. Surprisingly, the young mother centrioles of the CPAP-depleted cells are not fully matured, as evidenced by the absence of distal and subdistal appendage proteins. We propose that the selective absence of centriolar appendages at the young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells. Our results suggest that the neural stem cells with CPAP mutations might form asymmetric spindle poles, which results in premature initiation of differentiation.     

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.     

Structure of the centriolar complex in the hepatocytes of intact and regenerating mouse live

The structure of the cellular center in polyploid hepatocytes of intact and regenerating liver of adult mice has been studied. It was shown that the structure of the centriolar complex depends on stages of the cellular cycle. No pericentriolar structures (such as satellites, appendages and others) and cytoplasmic microtubules were found in the centriolar complex within G0-period. The satellites and appendages are formed in the half of the centrioles within G1-period. The microtubules can branch off some satellites; the daughter centrioles begin to form within S-period; there are diplosomes in the cells within G2-period, some mother centrioles are surrounded with the fine fibrillar halo. It is concluded that the structure of the centriolar complex within G0-period is distinguished by that within G1-period. The structure of the centriolar complex in polyploid hepatocytes has the same feature of reorganization in certain interphase periods of the cell cycle as in diploid cells of some cultured cells and the thyroid epithelium.     

Centrioles: some self-assembly required

Centrioles play an important role in organizing microtubules and are precisely duplicated once per cell cycle. New (daughter) centrioles typically arise in association with existing (mother) centrioles (canonical assembly), suggesting that mother centrioles direct the formation of daughter centrioles. However, under certain circumstances, centrioles can also selfassemble free of an existing centriole (de novo assembly). Recent work indicates that the canonical and de novo pathways utilize a common mechanism and that a mother centriole spatially constrains the self-assembly process to occur within its immediate vicinity. Other recently identified mechanisms further regulate canonical assembly so that during each cell cycle, one and only one daughter centriole is assembled per mother centriole.     

Spermatogenesis and spermiogenesis in Ascaris lumbricoides Var. suum

Reorganization of the prophase I nucleus marks the beginning of the first meiotic division. A pair of centrioles is present at each pole at metaphase I and mitochondria are not observed in the spindle area. A chromosomal pellicle, which resembles a kinetochore plate but has no apparent association with microtubules, surrounds each autosome at metaphase I and II. The sex body lags behind the autosomes at anaphase I and segregates differentially to one daughter cell. Mitochondria and a pair of centrioles are present in the spindle during the second meiotic division. Localized condensation of chromatin and fusion of the condensed chromatin of the secondary spermatocyte telophase nucleus results in a compact spermatid nucleus. Loss of spermatid cytoplasm is effected by the ejection of a cytophore vesicle.     

Dynamics of spreading of cells of L-929 line after the mitosis

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".     

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.     

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.     

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.     

GIANT CENTRIOLE FORMATION IN SCIARA

Although somatic tissues of Sciara contain 9-membered centrioles, germ line tissues develop giant centrioles with 60–90 singlet tubules disposed in an oval array. Some 9-membered centrioles still may be seen in second instar spermatogonia. Each of these centrioles is associated with a larger "daughter" or secondary centriole at right angles to it. Most centrioles of second instar spermatogonia consist of 20–50 singlet tubules arranged in an oval, sometimes associated with an even larger secondary centriole. The more recently formed centriole of a pair is distinguishable from its partner by a concentric band of electron-opaque material inside its tubules. If a pair of centrioles at right angles to each other is pictured as a "T" formed by two cylinders, the secondary centriole is always the stem of the T; the primary centriole is the top. The two centrioles are oriented at the pole of the mitotic spindle so that the tubules of the primary centriole are parallel to the spindle axis. Each daughter cell receives a pair of centrioles and, during interphase, each of these centrioles gives rise to a new daughter centriole. A Golgi area of characteristic morphology is found in association with centrioles shortly after two new ones have formed. We conclude that in Sciara a centriole may give rise to a daughter morphologically different from itself. Whether the daughter is a 9-membered or giant centriole depends on the tissue type and stage of development.     

Ultrastructure of the centrosome in L cells

Three types of microtubule-organizing centers are present in the interphase L-cells: centriolar matrix, pericentriolar satellites, and electron-dense bodies that are not attached to the centrioles. Different types of microtubule-organizing centers may be present simultaneously in the same centrosome. In most of the cells some microtubules have their proximal ends free, rather than attached to the microtubule-organizing center. A network of intermediate filaments is condensed around the centrosome. The intermediate filaments run from the centrosome parallel to the microtubules. Although the filaments are often in close proximity to the centrioles and microtubules, direct contacts between them are rare. The intermediate filaments have convergence foci of their own in the centrosome.     

THE FINE STRUCTURE OF MITOSIS IN RAT THYMIC LYMPHOCYTES

The fine structure of rat thymic lymphocytes from early prophase to late telophase of mitosis is described, using material fixed at pH 7.3 either in 1 per cent OsO4 or in glutaraldehyde followed by 2 per cent OsO4. The structure of the centriolar complex of interphase thymocytes is analyzed and compared with that of centrioles during division. The appearance of daughter centrioles is the earliest clearly recognizable sign of prophase. Daughter centrioles probably retain a secondary relation to the primary centriole, while the latter appears to be related, both genetically and spatially, to the spindle apparatus. The nuclear envelope persists in recognizable form to help reconstitute the envelopes of the daughter nuclei. Ribosome bodies (dense aggregates of ribosomes) accumulate, beginning at late prophase, and are retained by the daughter cells. Cytokinesis proceeds by formation of a ribosome-free plate at the equator with a central plate of vesicles which may coalesce to form the new plasma membrane of the daughter cells. Stages in the formation of the midbody are illustrated.     

Plk2 regulated centriole duplication is dependent on its localization to the centrosome and a functional polo-box domain

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell-cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G1/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G1 phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated down-regulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.     

Centrobin-Centrosomal Protein 4.1-associated Protein (CPAP) Interaction Promotes CPAP Localization to the Centrioles during Centriole Duplication

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.