Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.
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ABSTRACT:
SUBMITTER: Kong D
PROVIDER: S-EPMC4178969 | biostudies-literature | 2014 Sep
REPOSITORIES: biostudies-literature
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Project description:Supernumerary centrioles lead to abnormal mitosis, which in turn promotes tumorigenesis. Thus, centriole duplication must be coordinated with the cell cycle to ensure that the number of centrioles in the cell doubles precisely during each cell cycle. However, in some transformed cells, centrioles undergo multiple rounds of duplication (reduplication) during prolonged interphase. Mechanisms responsible for centriole reduplication are poorly understood. Here, we report that centrioles reduplicate consistently in cancerous and nontransformed human cells during G2 arrests and that this reduplication requires the activity of Polo-like kinase 1 (Plk1). We also find that a cell's ability to reduplicate centrioles during S arrests depends on the presence of activated (Thr210-phosphorylated) Plk1 at the centrosome. In the absence of activated Plk1, nascent procentrioles remain associated with mother centrioles, which prevents centriole reduplication. In contrast, if Plk1(pT210) appears at the centrosome, procentrioles mature, disengage from mother centrioles, and ultimately duplicate. Plk1 activity is not required for the assembly of procentrioles, however. Thus, the role of Plk1 is to coordinate the centriole duplication cycle with the cell cycle. Activation of Plk1 during late S/G2 induces procentriole maturation, and after this point, the centriole cycle can be completed autonomously, even in the absence of cell-cycle progression.
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Project description:Centrosome overduplication promotes mitotic abnormalities, invasion and tumorigenesis. Cells regulate the number of centrosomes by limiting centriole duplication to once per cell cycle. The orthogonal orientation between a mother and a daughter centriole, established at the time of centriole duplication, is thought to block further duplication of the mother centriole. Loss of orthogonal orientation (disengagement) between two centrioles during anaphase is considered a licensing event for the next round of centriole duplication. Disengagement requires the activity of Polo-like kinase 1 (Plk1), but how Plk1 drives this process is not clear. Here we employ correlative live/electron microscopy and demonstrate that Plk1 induces maturation and distancing of the daughter centriole, allowing reduplication of the mother centriole even if the original daughter centriole is still orthogonal to it. We find that mother centrioles can undergo reduplication when original daughter centrioles are only ∼80 nm apart, which is the distance centrioles normally reach during prophase.
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Project description:BackgroundAlthough the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7(th) day and identification of the bacterial isolates was carried out with a molecular technique.MethodsSelf-collected vaginal swabs were obtained daily from 17 non pregnant, menarchal volunteers, and used for daily Gram staining and weekly culture. Bacterial isolates were identified with tDNA-PCR and 16 S rRNA gene sequencing.ResultsNine women presented with predominantly normal VMF and the 8 others had predominantly disturbed VMF. The overall VMF of each volunteer was characteristic and rather stable. Menses and antimicrobials were the major disturbing factors of the VMF. Disturbances were always accompanied by a rise in Gram positive cocci, which also appeared to be a significant group within the VMF in general.ConclusionsWe observed a huge interindividual variability of predominantly stable VMF types. The importance of Gram positive cocci in VMF is underestimated. L. crispatus was the species that was most negatively affected by the menses, whereas the presence of the other lactobacilli was less variable.
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Project description:Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.
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Project description:Centrioles are duplicated and segregated in close link to the cell cycle. During mitosis, daughter centrioles are disengaged and eventually separated from mother centrioles. New daughter centrioles may be generated only after centriole separation. Therefore, centriole separation is considered a licensing step for centriole duplication. It was previously known that separase specifically cleaves pericentrin (PCNT) during mitotic exit. Here we report that PCNT has to be phosphorylated by PLK1 to be a suitable substrate of separase. Phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of a PLK1 inhibitor. On the basis on these results, we propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.
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Project description:Shugoshin 1 (Sgo1) functions as a protector of centromeric cohesion of sister chromatids in higher eukaryotes. Here, we provide evidence for a previously unrecognized role for Sgo1 in centriole cohesion. Sgo1 depletion via RNA interference induces the formation of multiple centrosome-like structures in mitotic cells that result from the separation of paired centrioles. Sgo1+/- mitotic murine embryonic fibroblasts display split centrosomes. Localization study of two major endogenous splice variants of Sgo1 indicates that the smaller variant, sSgo1, is found at the centrosome in interphase and at spindle poles in mitosis. sSgo1 interacts with Plk1 and its spindle pole localization is Plk1 dependent. Centriole splitting induced by Sgo1 depletion or expression of a dominant negative mutant is suppressed by ectopic expression of sSgo1 or by Plk1 knockdown. Our studies strongly suggest that sSgo1 plays an essential role in protecting centriole cohesion, which is partly regulated by Plk1.
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Project description:Mother-daughter centriole disengagement, the necessary first step in centriole duplication, involves Plk1 activity in early mitosis and separase activity after APC/C activity mediates securin degradation. Plk1 activity is thought to be essential and sufficient for centriole disengagement with separase activity playing a supporting but non-essential role. In separase null cells, however, centriole disengagement is substantially delayed. The ability of APC/C activity alone to mediate centriole disengagement has not been directly tested. We investigate the interrelationship between Plk1 and APC/C activities in disengaging centrioles in S or G2 HeLa and RPE1 cells, cell types that do not reduplicate centrioles when arrested in S phase. Knockdown of the interphase APC/C inhibitor Emi1 leads to centriole disengagement and reduplication of the mother centrioles, though this is slow. Strong inhibition of Plk1 activity, if any, during S does not block centriole disengagement and mother centriole reduplication in Emi1 depleted cells. Centriole disengagement depends on APC/C-Cdh1 activity, not APC/C-Cdc20 activity. Also, Plk1 and APC/C-Cdh1 activities can independently promote centriole disengagement in G2 arrested cells. Thus, Plk1 and APC/C-Cdh1 activities are independent but slow pathways for centriole disengagement. By having two slow mechanisms for disengagement working together, the cell ensures that centrioles will not prematurely separate in late G2 or early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion only late in mitosis.
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