PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

PLK1-mediated phosphorylation of pericentrin induces proper organization of the spindle pole–specific pericentriolar matrix and subsequent centrosome maturation

Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody

BackgroundMutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis.MethodsWe immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles.ResultsNinein signals were significantly impaired in CPAP-depleted cells.ConclusionThe results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells

BOULE, a Deleted in Azoospermia Homolog, Is Recruited to Stress Granules in the Mouse Male Germ Cells.

High temperature adversely affects normal development of male germ cells in mammals. Acute heat stress induces the formation of stress granules (SGs) in a set of male germ cells, and the SGs have been proposed to protect those cells from heat-induced apoptosis. DAZL, one of DAZ (Deleted in Azoospermia) family proteins, was shown to be an essential component of SGs, which is required for SG formation in the mouse testis. In the present study, we asked whether BOULE, the founding member of DAZ family proteins, is a component of the SGs. We show that BOULE is recruited to the SGs upon heat stress, and that these SGs are developmental stage-specific. These results suggest that DAZ family proteins may have conserved roles in the SGs of male germ cells

Triple deletion of TP53, PCNT, and CEP215 promotes centriole amplification in the M phase

Supernumerary centrioles are frequently observed in diverse types of cancer cells. In this study, we investigated the mechanism underlying the generation of supernumerary centrioles during the M phase. We generated the TP53;PCNT;CEP215 triple knockout (KO) cells and determined the configurations of the centriole during the cell cycle. The triple KO cells exhibited a precocious separation of centrioles and unscheduled centriole assembly in the M phase. Supernumerary centrioles in the triple KO cells were present throughout the cell cycle; however, among all the centrioles, only two maintained an intact composition, including CEP135, CEP192, CEP295 and CEP152. Intact centrioles were formed during the S phase and the rest of the centrioles may be generated during the M phase. M-phase-assembled centrioles lacked the ability to organize microtubules in the interphase; however, a fraction of them may acquire pericentriolar material to organize microtubules during the M phase. Taken together, our work reveals the heterogeneity of the supernumerary centrioles in the triple KO cells.N

CEP90 is required for the assembly and centrosomal accumulation of centriolar satellites, which is essential for primary cilia formation.

Centriolar satellites are PCM-1-positive granules surrounding centrosomes. Proposed functions of the centriolar satellites include protein targeting to the centrosome, as well as communication between the centrosome and surrounding cytoplasm. CEP90 is a centriolar satellite protein that is critical for spindle pole integrity in mitotic cells. In this study, we examined the biological functions of CEP90 in interphase cells. CEP90 physically interacts with PCM-1 at centriolar satellites, and this interaction is essential for centrosomal accumulation of the centriolar satellites and eventually for primary cilia formation. CEP90 is also required for BBS4 loading on centriolar satellites and its localization in primary cilia. Our results imply that the assembly and transport of centriolar satellites are critical steps for primary cilia formation and ciliary protein recruitment

Importance of the CEP215-Pericentrin Interaction for Centrosome Maturation during Mitosis

<div><p>At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar material (PCM) and a robust increase in microtubule-organizing activity. CEP215 is one of the major PCM components which accumulates at the centrosome during mitosis. The depletion phenotypes indicate that CEP215 is essential for centrosome maturation and bipolar spindle formation. Here, we performed a series of knockdown-rescue experiments to link the protein-protein interaction properties of CEP215 to its biological functions. The results showed that CEP215 and pericentrin, another major PCM component, is interdependent for their accumulation at the spindle poles during mitosis. As a result, The CEP215-pericentrin interaction is required for centrosome maturation and subsequent bipolar spindle formation during mitosis. On the other hand, CEP215 interaction with γ-tubulin is dispensable for centrosome maturation. Our results provide an insight how PCM components are assembled to form a spindle pole during mitosis.</p></div

Spindle pole formation with cells rescued with a CEP215 mutant devoid of γ-tubulin interaction.

<p>(A) HEK293T cells transfected with FLAG-tagged GFP, CEP215 (WT) and CEP215^F75A (F75A) were treated with STLC for 16 h to synchronize the cells at mitosis. The mitotic cell lysates were subjected to immunoprecipitation with FLAG resin followed by immunoblot analysis with γ-tubulin and FLAG antibodies. (B) The CEP215-depleted cells were rescued with FLAG-tagged CEP215 (WT) and CEP215^F75A (F75A). The cells were treated with RO3306 for 16 h then removed for 40 min to allow accumulation of mitotic cells. The cells were coimmunostained with γ-tubulin (red) and FLAG (green) antibodies. Scale bar, 10 µm. (C, D) The intensities of ectopic CEP215 (C) and endogenous γ-tubulin (D) at the spindle poles were quantified in more than 40 cells per group in three independent experiments. Error bars, SEM. The paired t-test was performed with p value indicated.</p

Integrity of the Pericentriolar Material Is Essential for Maintaining Centriole Association during M Phase.

A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit

A pericentrin mutant devoid of CEP215 interaction.

<p>(A) HEK293T cells transfected with FLAG-tagged GFP, PCNT (WT), PCNT^1235,1241AA (AA) and PCNT^{Δ2390–2406} (Δ17) were treated with STLC for 16 h to synchronize the cells at mitosis. The mitotic cell lysates were subjected to immunoprecipitation with FLAG resin followed by immunoblot analysis with CEP215 and FLAG antibodies. (B–D) The pericentrin-depleted HeLa cells were rescued with FLAG-GFP-tagged PCNT (WT), PCNT^1235,1241AA (AA) and PCNT^{Δ2390–2406} (Δ17). The cells were treated with RO3306 for 16 h and subsequently removed for 40 min to allow accumulation of mitotic cells. The cells were coimmunostained with CEP215 (red) and FLAG (green) antibodies. Scale bar, 10 µm. The intensities of ectopic pericentrin (C) and endogenous CEP215 (D) at the spindle poles were quantified in more than 40 cells per group in three independent experiments. Error bars, SEM. The paired t-test was performed with p value indicated.</p