Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.

In the past decade, membraneless assemblies known as biomolecular condensates have been reported to play key roles in many cellular functions by compartmentalizing specific proteins and nucleic acids in subcellular environments with distinct properties. Furthermore, growing evidence supports the view that biomolecular condensates often form by phase separation, in which a single-phase system demixes into a two-phase system consisting of a condensed phase and a dilute phase of particular biomolecules. Emerging understanding of condensate function in normal and aberrant cellular states, and of the mechanisms of condensate formation, is providing new insights into human disease and revealing novel therapeutic opportunities. In this Perspective, we propose that such insights could enable a previously unexplored drug discovery approach based on identifying condensate-modifying therapeutics (c-mods), and we discuss the strategies, techniques and challenges involved.

Centrosomes must resist microtubule-mediated forces for mitotic chromosome segregation. During mitotic exit, however, centrosomes are deformed and fractured by those same forces, which is a key step in centrosome disassembly. How the functional material properties of centrosomes change throughout the cell cycle, and how they are molecularly tuned, remain unknown. Here, we used optically induced flow perturbations to determine the molecular basis of centrosome strength and ductility in C. elegans embryos. We found that both properties declined sharply at anaphase onset, long before natural disassembly. This mechanical transition required PP2A phosphatase and correlated with inactivation of PLK-1 (Polo kinase) and SPD-2 (Cep192). In vitro, PLK-1 and SPD-2 directly protected centrosome scaffolds from force-induced disassembly. Our results suggest that, before anaphase, PLK-1 and SPD-2 respectively confer strength and ductility to the centrosome scaffold so that it can resist microtubule-pulling forces. In anaphase, centrosomes lose PLK-1 and SPD-2 and transition to a weak, brittle state that enables force-mediated centrosome disassembly.

Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM) from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6), together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.

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.

Centrosomes are major microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM) from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2A ), together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ~25% of PCM persisted from one cell cycle into the next, resulting in cytokinetic furrow ingression errors, excessive centrosome accumulation, and embryonic death. Purified PP2A could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and catastrophic rupture of the PCM scaffold.

One Sentence Summary: Phase separation of C. elegans centrosome proteins drive the formation of micron-sized microtubule organizing centers. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/093054 doi: bioRxiv preprint first posted online Dec. 10, 2016; AbstractCentrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C.elegans proteins. We found that macromolecular crowding drives phase separation of the key PCM scaffold protein SPD-5 into spherical droplets that morphologically and dynamically resemble in vivo PCM. These SPD-5 droplets recruited the microtubule polymerase ZYG-9 (XMAP215 homologue) and the microtubule-stabilizing protein TPXL-1 (TPX2 homologue). Together, these three proteins concentrated tubulin ~4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.

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