The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin.

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.

ZNF692 regulates nucleolar morphology by interacting with NPM1 and modifying its self-assembly properties.

The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.

Organization and Function of Non-dynamic Biomolecular Condensates.

Cells compartmentalize biochemical reactions using organelles. Organelles can be either membrane-bound compartments or supramolecular assemblies of protein and ribonucleic acid known as 'biomolecular condensates'. Biomolecular condensates, such as nucleoli and germ granules, have been described as liquid like, as they have the ability to fuse, flow, and undergo fission. Recent experiments have revealed that some liquid-like condensates can mature over time to form stable gels. In other cases, biomolecular condensates solidify into amyloid-like fibers. Here we discuss the assembly, organization, and physiological roles of these more stable condensates in cells, focusing on Balbiani bodies, centrosomes, nuclear pores, and amyloid bodies. We discuss how the material properties of these condensates can be explained by the principles of liquid-liquid phase separation and maturation.

FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing.

BACKGROUND: Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-ß family growth factors. RESULTS: To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC". This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-ß family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. CONCLUSIONS: FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.

Meeting report - building a centrosome.

Located in the 16th century Wiston House in West Sussex, UK, the 'Building a Centrosome' Workshop was organised by The Company of Biologists and chaired by Fanni Gergely and David Glover (University of Cambridge). Held in March 2013, the Workshop gathered together many of the leaders in the field of centrosome biology, as well as postdocs and students who were given the opportunity to meet and interact with many of the scientists who inspired their early careers. The diverse range of speakers provided a multi-disciplinary forum for the exchange of ideas, and gave fresh impetus to tackling outstanding questions related to centrosome biology. Here, we provide an overview of the meeting and highlight the main themes that were discussed.

Native molecular architectures of centrosomes in C. elegans embryos.

Centrosomes organize microtubules that are essential for mitotic divisions in animal cells. They consist of centrioles surrounded by Pericentriolar Material (PCM). Questions related to mechanisms of centriole assembly, PCM organization, and microtubule formation remain unanswered, in part due to limited availability of molecular-resolution structural analyses in situ. Here, we use cryo-electron tomography to visualize centrosomes across the cell cycle in cells isolated from C. elegans embryos. We describe a pseudo-timeline of centriole assembly and identify distinct structural features including a cartwheel in daughter centrioles, and incomplete microtubule doublets surrounded by a star-shaped density in mother centrioles. We find that centriole and PCM microtubules differ in protofilament number (13 versus 11) indicating distinct nucleation mechanisms. This difference could be explained by atypical γ-tubulin ring complexes with 11-fold symmetry identified at the minus ends of short PCM microtubules. We further characterize a porous and disordered network that forms the interconnected PCM. Thus, our work builds a three-dimensional structural atlas that helps explain how centrosomes assemble, grow, and achieve function.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.

Visualization of Balbiani Body disassembly during human primordial follicle activation.

Dormant human oocytes contain a perinuclear super-organelle, called the Balbiani Body, which is not present in mature oocytes. Here, we use confocal imaging to visualize two Balbiani Body markers-mitochondria and the DEAD-box helicase DDX4-in preantral follicles isolated from a 20-year-old female patient. In primordial follicles, mitochondria were concentrated in a ring near the oocyte nucleus, while DDX4 formed adjacent micron-scale spherical condensates. In primary and secondary follicles, the mitochondria were dispersed throughout the oocyte cytoplasm, and large DDX4 condensates were not visible. Our data suggest that the Balbiani Body breaks down during the primordial to primary follicle transition, thus releasing mitochondria and soluble DDX4 protein into the oocyte cytoplasm.

Cryo-Electron Tomography of Reconstituted Biomolecular Condensates.

The assembly of membraneless compartments by phase separation has recently been recognized as a mechanism for spatial and temporal organization of biomolecules within the cell. The functions of such mesoscale assemblies, termed biomolecular condensates, depend on networks of multivalent interactions between proteins, their structured and disordered domains, and commonly also include nucleic acids. Cryo-electron tomography is an ideal tool to investigate the three-dimensional architecture of such pleomorphic interaction networks at nanometer resolution and thus form inferences about function. However, preparation of suitable cryo-electron microscopy samples of condensates may be prone to protein denaturation, low retention of material on the sample carrier, and contamination associated with cryo-sample preparation and transfers. Here, we describe a series of protocols designed to obtain high-quality cryo-electron tomography data of biomolecular condensates reconstituted in vitro. These include critical screening by light microscopy, cryo-fixation by plunge freezing, sample loading into an electron microscope operated at liquid nitrogen temperature, data collection, processing of the data into three-dimensional tomograms, and their interpretation.

A central helical hairpin in SPD-5 enables centrosome strength and assembly.

Centrosomes organize microtubules for mitotic spindle assembly and positioning. Forces mediated by these microtubules create tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. How PCM resists these stresses is unclear at the molecular level. Here, we use cross-linking mass spectrometry (XL-MS) to map interactions underlying multimerization of SPD-5, an essential PCM scaffold component in C. elegans . We identified an interaction hotspot in an alpha helical hairpin motif in SPD-5 (a.a. 541-677). XL-MS data, ab initio structural predictions, and mass photometry suggest that this region dimerizes to form a tetrameric coiled-coil. Mutating a helical section (a.a. 610-640) or a single residue (R592) inhibited PCM assembly in embryos. This phenotype was rescued by eliminating microtubule pulling forces, revealing that PCM assembly and material strength are interrelated. We propose that interactions mediated by the helical hairpin strongly bond SPD-5 molecules to each other, thus enabling PCM to assemble fully and withstand stresses generated by microtubules.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

During mitotic spindle assembly, microtubules generate tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. The molecular interactions that enable PCM to assemble rapidly and resist external forces are unknown. Here we use cross-linking mass spectrometry to identify interactions underlying supramolecular assembly of SPD-5, the main PCM scaffold protein in C. elegans . Crosslinks map primarily to alpha helices within the phospho-regulated region (PReM), a long C-terminal coiled-coil, and a series of four N-terminal coiled-coils. PLK-1 phosphorylation of SPD-5 creates new homotypic contacts, including two between PReM and the CM2-like domain, and eliminates numerous contacts in disordered linker regions, thus favoring coiled-coil-specific interactions. Mutations within these interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces. Thus, PCM assembly and strength are interdependent. In vitro , self-assembly of SPD-5 scales with coiled-coil content, although there is a defined hierarchy of association. We propose that multivalent interactions among coiled-coil regions of SPD-5 build the PCM scaffold and contribute sufficient strength to resist microtubule-mediated forces.

Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia.

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

ZNF692 organizes a hub specialized in 40S ribosomal subunit maturation enhancing translation in rapidly proliferating cells.

Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.

The material state of centrosomes: lattice, liquid, or gel?

Centrosomes are micron-scale structures that nucleate microtubule arrays for chromosome segregation and mitotic spindle positioning. For these jobs, centrosomes must be dynamic enough to grow, yet stable enough to resist microtubule-mediated forces. How do centrosomes achieve such seemingly contradictory features? While much is understood about the molecular parts of centrosomes, very little is known about their functional material properties. Two prevalent hypotheses pose that the centrosome is either a liquid droplet or a solid lattice. However, many material states exist between a pure Newtonian liquid and a crystalline solid, and it is not clear where centrosomes lie along this spectrum. Furthermore, broad terms like "liquid" or "solid" do not reveal functional properties like strength, ductility, elasticity, and toughness, which are more relevant to understand how centrosomes resist forces. This review covers recent findings and new rheology techniques that reveal the material characteristics of centrosomes and how they are regulated.

Regulated changes in material properties underlie centrosome disassembly during mitotic exit.

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.

Phase separation of BuGZ promotes Aurora A activation and spindle assembly.

The spindle matrix has been proposed to facilitate mitotic spindle assembly. In this issue, Huang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706103) show that the spindle matrix protein BuGZ is sufficient to form micron-scale compartments that recruit and activate Aurora A, a critical kinase for spindle assembly.

Assembly of Mitotic Structures through Phase Separation.

Cells compartmentalize biochemical reactions using organelles, which can be membrane enclosed or built entirely of proteins and ribonucleic acids. Recent studies indicate that many organelles that lack membranes have liquid-like properties, including the ability to flow, fuse, and undergo rapid internal rearrangement. The assembly of these "biomolecular condensates" has been described as liquid-liquid phase separation, whereby their constituent components demix from the cytoplasm, similar to water separating from oil. Other studies suggest that protein phase separation followed by maturation, where intramolecular connections strengthen over time, can lead to gel- or glass-like states. This review discusses how the principles of phase separation might help to understand the assembly and behavior of organelles that operate in mitosis, when the cell assembles the mitotic spindle to segregate chromosomes. Special attention is given to the mitotic pericentriolar material of centrosomes and the spindle matrix.

Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit.

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.

A User's Guide for Phase Separation Assays with Purified Proteins.

The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.

Polo-like kinase phosphorylation determines Caenorhabditis elegans centrosome size and density by biasing SPD-5 toward an assembly-competent conformation.

Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM). In Caenorhabditis elegans embryos, the mitotic PCM expands by Polo-like kinase 1 (PLK-1) phosphorylation-accelerated assembly of SPD-5 molecules into supramolecular scaffolds. However, how PLK-1 phosphorylation regulates SPD-5 assembly is not known. We found that a mutant version of SPD-5 that is insensitive to PLK-1 phosphorylation (SPD-54A) could localize to PCM but was unable to rescue the reduction in PCM size and density when wild-type SPD-5 levels were decreased. In vitro, purified SPD-54A self-assembled into functional supramolecular scaffolds over long time scales, suggesting that phosphorylation only controls the rate of SPD-5 scaffold assembly. Furthermore, the SPD-5 scaffold, once assembled, remained intact and supported microtubule nucleation in the absence of PLK-1 activity in vivo We conclude that PLK-1 is required for rapid assembly of the PCM scaffold but not for scaffold maintenance or function. Based on this idea, we developed a theoretical model that adequately predicted PCM growth rates in different mutant conditions in vivo We propose that PLK-1 phosphorylation-dependent conversion of SPD-5 into an assembly-competent form underlies PCM formation in vivo and that the rate of this conversion determines final PCM size and density.