The acentriolar state of the Drosophila cell lines 1182

A Drosophila melanogaster cell line devoid of centrioles has been recently described. In order to achieve an easier characterization of these acentriolar cells, we used the monoclonal antibody Bx 63 of M. Frasch which recognizes the Drosophila centrosome. Although centrosomes are detected at every mitotic pole in Drosophila cells with centrioles, no such structure has been observed in 1182-4 acentriolar cells. The antigenic material is, however, present in these cells. Moreover, we noticed a certain proportion of acentriolar cells in 4 other 1182 lines. The lack of centrioles previously found only in the 1182-4 cells seems therefore not accidental and should be linked to their particular origin.     

Plk4 triggers autonomous de novo centriole biogenesis and maturation

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.     

Klp10A, a microtubule-depolymerizing kinesin-13, cooperates with CP110 to control Drosophila centriole length

Klp10A is a kinesin-13 of Drosophila melanogaster that depolymerizes cytoplasmic microtubules. In interphase, it promotes microtubule catastrophe; in mitosis, it contributes to anaphase chromosome movement by enabling tubulin flux. Here we show that Klp10A also acts as a microtubule depolymerase on centriolar microtubules to regulate centriole length. Thus, in both cultured cell lines and the testes, absence of Klp10A leads to longer centrioles that show incomplete 9-fold symmetry at their ends. These structures and associated pericentriolar material undergo fragmentation. We also show that in contrast to mammalian cells where depletion of CP110 leads to centriole elongation, in Drosophila cells it results in centriole length diminution that is overcome by codepletion of Klp10A to give longer centrioles than usual. We discuss how loss of centriole capping by CP110 might have different consequences for centriole length in mammalian and insect cells and also relate these findings to the functional interactions between mammalian CP110 and another kinesin-13, Kif24, that in mammalian cells regulates cilium formation.     

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.     

SAK/PLK4 is required for centriole duplication and flagella development

BACKGROUND: SAK/PLK4 is a distinct member of the polo-like kinase family. SAK-/- mice die during embryogenesis, whereas SAK+/- mice develop liver and lung tumors and SAK+/- MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. RESULTS: Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. CONCLUSIONS: SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.     

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.     

The centrosome cycle in syncytial Drosophila embryos analyzed by energy filtering transmission electron microscopy.

We have investigated the centrosome cycle in Drosophila syncytial embryos at the ultrastructural level by using a transmission electron microscope equipped with an electron energy filtering device (Omega filter). This new technique allows the study of uncontrasted thick sections with a high resolution. We have been able to characterize two classes of filamentous structures in the centrosomal apparatus that were not detectable on ultrathin sections. These new filamentous structures are: 1) a very orderly lattice that connects the two centrioles during mitosis; and 2) a fibrogranular connection between the centrosome and the nuclear envelope. The intercentriolar linkage could be involved in the precise timing of separation of the centrioles during late anaphase. The centrosome-nuclear envelope connection probably prevents the loss of centrosomes in this syncytial environment, and ensures the proper migration of the centrosomes along the surface of the nucleus. This connection may also couple the nuclei to the cytoskeleton, thus allowing their migration and their anchorage to the cortex at the blastoderm stage. This thick section analysis has also allowed us to precisely reconstitute the centrosome cycle. From their separation at telophase and throughout most of interphase, centrosomes are composed of a single centriole. We conclude that in the early Drosophila embryo there is an unusual delay between the separation of the parent centrioles and their duplication. This leaves a surprisingly short time to assemble a daughter centriole.     

The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547-1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous "precentrioles" become morphologically recognizable centrioles before mitosis. De novo-assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo-formed centrioles do not mature if they are assembled in S phase-arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.     

Centrioles in the cell cycle of L cells

The structure of centrosome in non-synchronous L-cells culture during the cell cycle has been studied. In mitosis, mother and daughter centrioles, which differ in their ultrastructure, are located perpendicularly in the pole of the spindle. Microtubules, meeting in the pole area terminate mainly in electron-dense clottings of fibrillar matter surrounding the diplosoma. In telophase, disjunction of mother and daughter centrioles begins. At the beginning of G1-period, centrioles move off from each other for several micron, and then draw together again without forming diplosome. Pericentriolar satellites form on mother centriole of some cells at this time, they disappear at the beginning of S-period, replication of centrioles begins; daughter centrioles reach the size of mother centrioles in anaphase. During growth and maturation, centrioles in L-cells undergo structural changes similar to those described for SPEV cells (Vorob'ev, Chentsov, 1982). Several types of meeting points for microtubules exist in L-cells during the whole interphase: surface of centrioles per se, pericentriolar satellites, free foci.     

Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication

In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 [1-8]. These proteins are widely believed to comprise a conserved core duplication module [3, 9-14]. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles [1, 4, 15, 16]. Here, we show that Drosophila Spd-2 (DSpd-2) is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. DSpd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in DSpd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, DSpd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. DSpd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. We speculate that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm.     

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification.     

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.     

Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation

BACKGROUND: Centrosomes have important roles in many aspects of cell organization, and aberrations in their number and function are associated with various diseases, including cancer. Centrosomes consist of a pair of centrioles surrounded by a pericentriolar matrix (PCM), and their replication is tightly regulated. Here, we investigate the effects of overexpressing the three proteins known to be required for centriole replication in Drosophila-DSas-6, DSas-4, and Sak. RESULTS: By directly observing centriole replication in living Drosophila embryos, we show that the overexpression of GFP-DSas-6 can drive extra rounds of centriole replication within a single cell cycle. Extra centriole-like structures also accumulate in brain cells that overexpress either GFP-DSas-6 or GFP-Sak, but not DSas-4-GFP. No extra centrioles accumulate in spermatocytes that overexpress any of these three proteins. Most remarkably, the overexpression of any one of these three proteins results in the rapid de novo formation of many hundreds of centriole-like structures in unfertilized eggs, which normally do not contain centrioles. CONCLUSIONS: Our data suggest that the levels of centriolar DSas-6 determine the number of daughter centrioles formed during centriole replication. Overexpression of either DSas-6 or Sak can induce the formation of extra centrioles in some tissues but not others, suggesting that centriole replication is regulated differently in different tissues. The finding that the overexpression of DSas-4, DSas-6, or Sak can rapidly induce the de novo formation of centriole-like structures in Drosophila eggs suggests that this process results from the stabilization of centriole-precursors that are normally present in the egg.     

Identification of a polo-like kinase 4-dependent pathway for de novo centriole formation

Supernumerary centrosomes are a key cause of genomic instability in cancer cells. New centrioles can be generated by duplication with a mother centriole as a platform or, in the absence of preexisting centrioles, by formation de novo. Polo-like kinase 4 (Plk4) regulates both modes of centriole biogenesis, and Plk4 deregulation has been linked to tumor development. We show that Plx4, the Xenopus homolog of mammalian Plk4 and Drosophila Sak, induces de novo centriole formation in vivo in activated oocytes and in egg extracts, but not in immature or in vitro matured oocytes. Both kinase activity and the polo-box domain of Plx4 are required for de novo centriole biogenesis. Polarization microscopy in "cycling" egg extracts demonstrates that de novo centriole formation is independent of Cdk2 activity, a major difference compared to template-driven centrosome duplication that is linked to the nuclear cycle and requires cyclinA/E/Cdk2. Moreover, we show that the Mos-MAPK pathway blocks Plx4-dependent de novo centriole formation before fertilization, thereby ensuring paternal inheritance of the centrosome. The results define a new system for studying the biochemical and molecular basis of de novo centriole formation and centriole biogenesis in general.     

Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.     

Spatial relationships between the anterior centriole and the mitotic center during interphase in the amoebae of the myxomycete Physarum polycephalum

Amoebae of the Myxomycete Physarum polycephalum in the interphase state typically contain only one proflagellar apparatus in which the anterior kinetosome (anterior centriole) is attached to the microtubule organizing center 1 (mtoc 1). We built strains possessing more than one mtoc 1 and a variable number of anterior centrioles to allow the appearance of new structures. In 8% of the amoebae of these strains, the 1:1 attachment between the anterior centriole and the mtoc 1 is not always respected. In nine cases studied using tridimensional reconstructions from ultrastructural thin sections, the pattern of attachment was more complex. A mtoc 1 could be linked to several anterior centrioles, and/or reciprocally an anterior centriole could be linked to several mtoc 1. In one case, an anterior centriole was not linked to a mtoc 1 and in three cases, a single centriole exhibited anterior and posterior characteristics. These observations suggest that (1) each pair of centrioles constitutes a morphological and physiological entity that is distinct from the mitotic center (mtoc 1); (2) the attachment of the anterior centriole to the mtoc 1 occurs at the end of each mitosis; (3) there is an inductory process during the morphogenesis of the link between the anterior centriole and the mtoc 1; (4) the anterior characteristics of a centriole can be present in the absence of the link with the mtoc 1; (5) the anterior and posterior characteristics of a centriole are not exclusive of each other, ruling out the existence of a lineage corresponding to the anterior centriole and a lineage corresponding to the posterior centriole; and (6) the differences between anterior and posterior centrioles result from a maturation process.     

Analysis of centriole elimination during C. elegans oogenesis

Centrosomes are the principal microtubule organizing centers (MTOCs) of animal cells and comprise a pair of centrioles surrounded by pericentriolar material (PCM). Centriole number must be carefully regulated, notably to ensure bipolar spindle formation and thus faithful chromosome segregation. In the germ line of most metazoan species, centrioles are maintained during spermatogenesis, but eliminated during oogenesis. Such differential behavior ensures that the appropriate number of centrioles is present in the newly fertilized zygote. Despite being a fundamental feature of sexual reproduction in metazoans, the mechanisms governing centriole elimination during oogenesis are poorly understood. Here, we investigate this question in C. elegans. Using antibodies directed against centriolar components and serial-section electron microscopy, we establish that centrioles are eliminated during the diplotene stage of the meiotic cell cycle. Moreover, we show that centriole elimination is delayed upon depletion of the helicase CGH-1. We also find that somatic cells make a minor contribution to this process, and demonstrate that the germ cell karyotype is important for timely centriole elimination. These findings set the stage for a mechanistic dissection of centriole elimination in a metazoan organism.     

Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption

The effects of colcemid (0.16-1.0 microM) and taxol (10 microM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high-voltage electron microscopy of serial 0.25-micron sections. Although these drugs induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubules, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5-7 h at 37 degrees C and then form 4N "micronucleated" restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration-dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16-0.25 microM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (greater than or equal to 1.0 microM) concentrations. However, even in the presence of 1.0 microM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell-cycle-dependent assembly and disassembly of axonemal microtubules in vivo.     

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.