RESUMO
Centrioles are part of centrosomes and cilia, which are microtubule organising centres (MTOC) with diverse functions. Despite their stability, centrioles can disappear during differentiation, such as in oocytes, but little is known about the regulation of their structural integrity. Our previous research revealed that the pericentriolar material (PCM) that surrounds centrioles and its recruiter, Polo kinase, are downregulated in oogenesis and sufficient for maintaining both centrosome structural integrity and MTOC activity. We now show that the expression of specific components of the centriole cartwheel and wall, including ANA1/CEP295, is essential for maintaining centrosome integrity. We find that Polo kinase requires ANA1 to promote centriole stability in cultured cells and eggs. In addition, ANA1 expression prevents the loss of centrioles observed upon PCM-downregulation. However, the centrioles maintained by overexpressing and tethering ANA1 are inactive, unlike the MTOCs observed upon tethering Polo kinase. These findings demonstrate that several centriole components are needed to maintain centrosome structure. Our study also highlights that centrioles are more dynamic than previously believed, with their structural stability relying on the continuous expression of multiple components.
Assuntos
Centríolos , Centrossomo , Proteínas de Drosophila , Proteínas Associadas aos Microtúbulos , Centríolos/metabolismo , Centrossomo/metabolismo , Oócitos/metabolismo , Oogênese , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Drosophila melanogaster , Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , HumanosRESUMO
The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral-facial-digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein Cep164. Myosin VI depletion in non-tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be, at least partially, ascribed to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.
Assuntos
Ciliopatias , Proteínas Associadas aos Microtúbulos , Centríolos/metabolismo , Cílios/metabolismo , Ciliopatias/genética , Ciliopatias/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Proteínas/metabolismoRESUMO
Centrosomes function in key cellular processes ranging from cell division to cellular signaling. Their dysfunction is linked to cancer and developmental disorders. Here, we identify CCDC57 as a pleiotropic regulator of centriole duplication, mitosis, and ciliogenesis. Combining proximity mapping with superresolution imaging, we show that CCDC57 localizes to the proximal end of centrioles and interacts with the microcephaly protein CEP63, centriolar satellite proteins, and microtubules. Loss of CCDC57 causes defects in centriole duplication and results in a failure to localize CEP63 and CEP152 to the centrosome. Additionally, CCDC57 depletion perturbs mitotic progression both in wild-type and centriole-less cells. Importantly, its centrosome-targeting region is required for its interaction with CEP63 and functions during centriole duplication and cilium assembly, whereas the microtubule-targeting region is required for its mitotic functions. Together, our results identify CCDC57 as a critical interface between centrosome and microtubule-mediated cellular processes that are deregulated in microcephaly.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Microcefalia/metabolismo , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Animais , Proteínas de Ciclo Celular/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Microcefalia/genética , Proteínas dos Microtúbulos/genética , TransfecçãoRESUMO
Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation.
Assuntos
Proteína Quinase CDC2/metabolismo , Centríolos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteína Quinase CDC2/genética , Ciclo Celular/fisiologia , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , XenopusRESUMO
Centrioles are essential for cilia and centrosome assembly. In centriole-containing cells, centrioles always form juxtaposed to pre-existing ones, motivating a century-old debate on centriole biogenesis control. Here, we show that trans-autoactivation of Polo-like kinase 4 (PLK4), the trigger of centriole biogenesis, is a critical event in the spatial control of that process. We demonstrate that centrioles promote PLK4 activation through its recruitment and local accumulation. Though centriole removal reduces the proportion of active PLK4, this is rescued by concentrating PLK4 to the peroxisome lumen. Moreover, while mild overexpression of PLK4 only triggers centriole amplification at the existing centriole, higher PLK4 levels trigger both centriolar and cytoplasmatic (de novo) biogenesis. Hence, centrioles promote their assembly locally and disfavor de novo synthesis. Similar mechanisms enforcing the local concentration and/or activity of other centriole components are likely to contribute to the spatial control of centriole biogenesis under physiological conditions.
Assuntos
Centríolos/genética , Proteínas de Drosophila/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Espermatogênese/genética , Animais , Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Peroxissomos/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Polo-like kinase 4 (PLK4) is a major player in centriole biogenesis: in its absence centrioles fail to form, while in excess leads to centriole amplification. The SCF-Slimb/ßTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/ßTrCP substrate binding and targeting for degradation is normally regulated by phosphorylation cascades, controlling complex processes, such as circadian clocks and morphogenesis. Here, we show that PLK4 is a suicide kinase, autophosphorylating in residues that are critical for SCF-Slimb/ßTrCP binding. We demonstrate a multisite trans-autophosphorylation mechanism, likely to ensure that both a threshold of PLK4 concentration is attained and a sequence of events is observed before PLK4 can autodestruct. First, we show that PLK4 trans-autophosphorylates other PLK4 molecules on both Ser293 and Thr297 within the degron and that these residues contribute differently for PLK4 degradation, the first being critical and the second maximizing auto-destruction. Second, PLK4 trans-autophosphorylates a phospho-cluster outside the degron, which regulates Thr297 phosphorylation, PLK4 degradation, and centriole number. Finally, we show the importance of PLK4-Slimb/ßTrCP regulation as it operates in both soma and germline. As ßTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.
Assuntos
Centríolos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica , Masculino , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Serina/metabolismo , Treonina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Microtubule plus-end tracking proteins (+TIPs) are structurally and functionally diverse factors that accumulate at the growing microtubule plus-ends, connect them to various cellular structures, and control microtubule dynamics [1, 2]. EB1 and its homologs are +TIPs that can autonomously recognize growing microtubule ends and recruit to them a variety of other proteins. Numerous +TIPs bind to end binding (EB) proteins through natively unstructured basic and serine-rich polypeptide regions containing a core SxIP motif (serine-any amino acid-isoleucine-proline) [3]. The SxIP consensus sequence is short, and the surrounding sequences show high variability, raising the possibility that undiscovered SxIP containing +TIPs are encoded in mammalian genomes. Here, we performed a proteome-wide search for mammalian SxIP-containing +TIPs by combining biochemical and bioinformatics approaches. We have identified a set of previously uncharacterized EB partners that have the capacity to accumulate at the growing microtubule ends, including protein kinases, a small GTPase, centriole-, membrane-, and actin-associated proteins. We show that one of the newly identified +TIPs, CEP104, interacts with CP110 and CEP97 at the centriole and is required for ciliogenesis. Our study reveals the complexity of the mammalian +TIP interactome and provides a basis for investigating the molecular crosstalk between microtubule ends and other cellular structures.
Assuntos
Motivos de Aminoácidos , Proteínas Associadas aos Microtúbulos/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Mamíferos , Espectrometria de Massas , Proteínas Associadas aos Microtúbulos/análise , Microtúbulos/química , Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteoma/análise , Proteômica/métodos , Transdução de SinaisRESUMO
Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis.
Assuntos
Centríolos/metabolismo , Citocinese , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Centrossomo/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Camundongos , Microtúbulos/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Transporte Proteico , Interferência de RNA , Fuso Acromático/metabolismo , Imagem com Lapso de TempoRESUMO
One of the most extraordinary events in the lifetime of a cell is the coordinated separation of sister chromatids during cell division. This is truly the essence of the entire mitotic process and the reason for the most profound morphological changes in cytoskeleton and nuclear organization that a cell may ever experience. It all occurs within a very short time window known as "anaphase", as if the cell had spent the rest of its existence getting ready for this moment in an ultimate act of survival. And there is a good reason for this: no space for mistakes. Problems in the distribution of chromosomes during cell division have been correlated with aneuploidy, a common feature observed in cancers and several birth defects, and the main cause of spontaneous abortion in humans. In this paper, we critically review the mechanisms of anaphase chromosome motion that resisted the scrutiny of more than 100 years of research, as part of a tribute to the pioneering work of Miguel Mota.
Assuntos
Anáfase , Animais , Cromátides/fisiologia , Cromossomos/fisiologia , Drosophila , Dineínas/metabolismo , Cinetocoros/metabolismoRESUMO
The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro(71) and Chro(612) have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Mitose , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/fisiologia , Animais , Drosophila/genética , Imuno-Histoquímica , Larva/metabolismo , Microtúbulos/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore-microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore-microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase.
Assuntos
Segregação de Cromossomos , Drosophila melanogaster/metabolismo , Cinetocoros/metabolismo , Anáfase , Animais , Fenômenos Biomecânicos , Linhagem Celular , Movimento Celular , Polaridade Celular , Cromossomos/metabolismo , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Metáfase , Microcirurgia , Microtúbulos/metabolismo , Modelos Biológicos , Fenótipo , Interferência de RNA , Fuso Acromático/metabolismoRESUMO
Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscopy and state-of-the-art laser microsurgery, combined with RNA interference (RNAi) in Drosophila cell lines stably expressing fluorescent markers. This technological synergism allows the specific targeting and manipulation of several structural components of the mitotic apparatus in different genetic backgrounds, at the highest spatial and temporal resolution. Finally, we demonstrate the successful adaptation of agar overlay flattening techniques to human HeLa cells and discuss the advantages of its use for laser micromanipulation and molecular studies of mitosis in mammals.
Assuntos
Drosophila melanogaster/citologia , Mitose/fisiologia , Interferência de RNA , Fuso Acromático/fisiologia , Ágar , Animais , Técnicas de Cultura de Células , Linhagem Celular , Células HeLa , Humanos , Lasers , Microcirurgia , Fuso Acromático/ultraestruturaRESUMO
A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible "wait anaphase" signal to the vicinity of the spindle.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Mitose/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/ultraestrutura , Cromossomos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Recuperação de Fluorescência Após Fotodegradação , Genes cdc/fisiologia , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Proteínas Mad2 , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Fuso Acromático/ultraestruturaRESUMO
CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown. Here, we show that CLASP2 localization at kinetochores, centrosomes, and spindle throughout mitosis is remarkably similar to CLASP1, both showing fast microtubule-independent turnover rates. Strikingly, primary fibroblasts from Clasp2 knockout mice show numerous spindle and chromosome segregation defects that can be partially rescued by ectopic expression of Clasp1 or Clasp2. Moreover, chromosome segregation rates during anaphase A and B are slower in Clasp2 knockout cells, which is consistent with a role of CLASP2 in the regulation of kinetochore and spindle function. Noteworthy, cell viability/proliferation and spindle checkpoint function were not impaired in Clasp2 knockout cells, but the fidelity of mitosis was strongly compromised, leading to severe chromosomal instability in adult cells. Together, our data support that the partial redundancy of CLASPs during mitosis acts as a possible mechanism to prevent aneuploidy in mammals.