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Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize within centrosomes is critical to understand the centriole duplication process and biochemical and architectural differences between centrosomes of different species. Here, at nanoscale resolution, we dissect centrosomal localization of Plk4 in G1 and S phase in its catalytically active and inhibited state during centriole duplication and amplification. We build a precise distribution map of Plk4 and its receptor Cep152, as well as Cep44, Cep192, and Cep152-anchoring factors Cep57 and Cep63. We find that Cep57, Cep63, Cep44, and Cep192 localize in ninefold symmetry. However, during centriole maturation, Cep152, which we suggest is the major Plk4 receptor, develops a more complex pattern. We propose that the molecular arrangement of Cep152 creates flexibility for Plk4 and procentriole placement during centriole initiation. As a result, procentrioles form at variable positions in relation to the mother centriole microtubule triplets., (© 2023 Sullenberger et al.) more...

Centrioles are precisely built microtubule-based structures that assemble centrosomes and cilia. Aberrations in centriole structure are common in tumors, yet how these aberrations arise is unknown. Analysis of centriole structure is difficult because it requires demanding electron microscopy. Here we employ expansion microscopy to study the origins of centriole structural aberrations in large populations of human cells. We discover that centrioles do not have an elongation monitoring mechanism, which renders them prone to over-elongation, especially during prolonged mitosis induced by various factors, importantly including supernumerary centrioles. We identify that mitotic centriole over-elongation is dependent on mitotic Polo-like kinase 1, which we uncover as a novel regulator of centriole elongation in human cycling cells. While insufficient Plk1 levels lead to the formation of shorter centrioles lacking a full set of microtubule triplets, its overactivity results in over-elongated and structurally aberrant centrioles. Our data help explain the origin of structurally aberrant centrioles and why centriole numerical and structural defects coexist in tumors., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.) more...

The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro , PLK4 can self-assemble into condensates that recruit α- and β-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.) more...

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. more...

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells. more...

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis. more...

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., (2010 Elsevier Ltd. All rights reserved.) more...