Polo-like kinase 4 controls centriole duplication but does not directly regulate cytokinesis.

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

PLK4 trans-Autoactivation Controls Centriole Biogenesis in Space

The deposited article is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.The deposited article version contains attached the supplementary materials within the pdf.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.Fundação Portuguesa para a Ciência e Tecnologia grants: (SFRH/BPD/87479/2012, PTDC/BBB-BEP/1724/2012, HMSP-CT/SAU-ICT/0075/2009, PTDC/SAU-OBD/105616/2008, EXPL/BIM-ONC/0830/2013, PTDC/BBB-BEP/1724/2012); EMBO installation grant; ERC starting grant: (PFE-GI-UE-ERC-2010-StG-261344).info:eu-repo/semantics/publishedVersio

The maintenance of centriole appendages and motile cilia basal body anchoring relies on TBCCD1

Centrosomes are organelles consisting of two structurally and functionally distinct centrioles, with the mother centriole having complex distal (DA) and subdistal appendages (SDA). Despite their importance, how appendages are assembled and maintained remains unclear. This study investigated human TBCCD1, a centrosomal protein essential for centrosome positioning, to uncover its localization and role at centrioles. We found that TBCCD1 localizes at both proximal and distal regions of the two centrioles, forming a complex structure spanning from SDA to DA and extending inside and outside the centriole lumen. TBCCD1 depletion caused centrosome mispositioning, which was partially rescued by taxol, and the loss of microtubules (MTs) anchored to centrosomes. TBCCD1 depletion also reduced levels of SDA proteins involved in MT anchoring such as Centriolin/CEP110, Ninein, and CEP170. Additionally, TBCCD1 was essential for the correct positioning of motile cilia basal bodies and associated structures in Paramecium. This study reveals that TBCCD1 is an evolutionarily conserved protein essential for centriole and basal body localization and appendage assembly and maintenance. A BioID screening also linked TBCCD1 to ciliopathy-associated protein networks.info:eu-repo/semantics/publishedVersio

Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number

The deposited article is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.This deposit is composed by the main article plus the supplementary materials of the publication.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/βTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/βTrCP 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/βTrCP 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/βTrCP regulation as it operates in both soma and germline. As βTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.Fundação para a Ciência e Tecnologia grants: (SFRH/BD/33213/2007, PTDC/SAU-OBD/73194/2006, PTDC/SAU-OBD/105616/2008); ERC grant:(ERC-2010-StG grant number 261344); EMBO installation grant.info:eu-repo/semantics/publishedVersio

Regulation of Autophosphorylation Controls PLK4 Self-Destruction and Centriole Number

The deposited article is a post-print version and has been submitted to peer review.This publication hasn't any creative commons license associated.This deposit is composed by the main article plus the supplementary materials of the publication.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/βTrCP-E3 ubiquitin ligase controls PLK4 levels through recognition of a conserved phosphodegron. SCF-Slimb/βTrCP 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/βTrCP 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/βTrCP regulation as it operates in both soma and germline. As βTrCP, PLK4, and centriole number are deregulated in several cancers, our work provides novel links between centriole number control and tumorigenesis.Fundação para a Ciência e Tecnologia grants: (SFRH/BD/33213/2007, PTDC/SAU-OBD/73194/2006, PTDC/SAU-OBD/105616/2008); ERC grant:(ERC-2010-StG grant number 261344); EMBO installation grant.info:eu-repo/semantics/publishedVersio

CDK1 Prevents Unscheduled PLK4-STIL Complex Assembly in Centriole Biogenesis

The deposited article is a post-print version (author's manuscript from PMC and available in PMC 2017 May 9).This publication hasn't any creative commons license associated.This deposit is composed by the main article and the supplementary materials are present in the publisher's page in the following link: https://www.sciencedirect.com/science/article/pii/S0960982216303001?via%3Dihub#sec4Centrioles 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.ERC grant: (ERC-2010-StG-261344); FCT grants: (FCT Investigator, EXPL/BIM-ONC/0830/2013, PTDC/SAU-BD/105616/2008); EMBO installation grant.info:eu-repo/semantics/publishedVersio

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

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

Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores

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

Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase and the PCM

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