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Centrosomes consist of two centrioles and the surrounding pericentriolar material (PCM). The PCM expands during mitosis in a process called centrosome maturation, in which PCM scaffold proteins play pivotal roles to recruit other centrosomal proteins. In Caenorhabditis elegans, the scaffold protein SPD-5 forms a PCM scaffold in a polo-like kinase 1 (PLK-1) phosphorylation-dependent manner. However, how phosphorylation of SPD-5 promotes PCM scaffold assembly is unclear. Here, we identified three functional domains of SPD-5 through in vivo domain analyses, and propose that sequential domain interactions of SPD-5 are required for mitotic PCM scaffold assembly. Firstly, SPD-5 is targeted to centrioles through a direct interaction between its centriole localization (CL) domain and the centriolar protein PCMD-1. Then, intramolecular and intermolecular interactions between the SPD-5 phospho-regulated multimerization (PReM) domain and the PReM association (PA) domain are enhanced by phosphorylation by PLK-1, which leads to PCM scaffold expansion. Our findings suggest that the sequential domain interactions of scaffold proteins mediated by PLK-1 phosphorylation is an evolutionarily conserved mechanism of PCM scaffold assembly. This article has an associated First Person interview with the first author of the paper.

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Centrosomes are the main microtubule-organizing centres, playing essential roles in the organization of the cytoskeleton during interphase, and in the mitotic spindle, which controls chromosome segregation, during cell division. Centrosomes also act as the basal body of cilia, regulating cilium length and affecting extracellular signal reception as well as the integration of intracellular signalling pathways. Centrosomes are self-replicative and duplicate once every cell cycle to generate two centrosomes. The core support structure of the centrosome consists of two molecularly distinct centrioles. The mother (mature) centriole exhibits accessory appendages and is surrounded by both pericentriolar material and centriolar satellites, structures that the daughter (immature) centriole lacks. In this Review, we discuss what is currently known about centrosome duplication, its dialogue with the cell cycle and the sequential acquisition of specific components during centriole maturation. We also describe our current understanding of the mature centriolar structures that are required to build a cilium. Altogether, the built-in centrosome asymmetries that stem from the two centrosomes inheriting molecularly different centrioles sets the foundation for cell division being an intrinsically asymmetric process.

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At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT, enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs), phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.

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Centrosomes were first described by Edouard Van Beneden and named and linked to chromosome segregation by Theodor Boveri around 1870. In the 1960-1980s, electron microscopy studies have revealed the remarkable ultrastructure of a centriole -- a nine-fold symmetrical microtubular assembly that resides within a centrosome and organizes it. Less than two decades ago, proteomics and genomic screens conducted in multiple species identified hundreds of centriole and centrosome core proteins and revealed the evolutionarily conserved nature of the centriole assembly pathway. And now, super resolution microscopy approaches and improvements in cryo-tomography are bringing an unparalleled nanoscale-detailed picture of the centriole and centrosome architecture. In this chapter, we summarize the current knowledge about the architecture of human centrioles. We discuss the structured organization of centrosome components in interphase, focusing on localization/function relationship. We discuss the process of centrosome maturation and mitotic spindle pole assembly in centriolar and acentriolar cells, emphasizing recent literature.

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Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.

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Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.

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Pharmacological inhibition of Drosophila Polo kinase with BI2536 has allowed us to re-examine the requirements for Polo during Drosophila male gametogenesis. BI2536-treated spermatocytes persisted in a pro-metaphase state without dividing and had condensed chromosomes that did not separate. Centrosomes failed to recruit γ-tubulin and centrosomin (Cnn) and were not associated with microtubule arrays that were abnormal and did not form proper bipolar spindles. Centrioles, which usually separate during the anaphase of the first meiosis, remained held together in a V-shaped configuration suggesting that Polo kinase regulates the proteolysis that breaks centriole linkage to ensure their disengagement. Despite these defects spermatid differentiation proceeds, leading to axoneme formation.

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