The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In C. elegans, PCM assembly requires the coiled-coil protein SPD-5. Here we found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.
A centrosome consists of two barrel-shaped centrioles embedded in a matrix of proteins known as the pericentriolar material (PCM). The PCM serves as a platform for protein complexes that regulate organelle trafficking, protein degradation and spindle assembly. Perhaps most important for cell division, the PCM concentrates tubulin and serves as the primary organizing centre for microtubules in metazoan somatic cells. Thus, similar to other well-described organelles, such as the nucleus and mitochondria, the cell has compartmentalized a multitude of vital biochemical reactions in the PCM. However, unlike these other organelles, the PCM is not membrane bound, but rather a dynamic collection of protein complexes and nucleic acids that constitute the organelle's interior and determine its boundary. How is the complex biochemical machinery necessary for the myriad centrosome functions concentrated and maintained in the PCM? Recent advances in proteomics and RNAi screening have unveiled most of the key PCM components and hinted at their molecular interactions ( table 1). Now we must understand how the interactions between these molecules contribute to the mesoscale organization and the assembly of the centrosome. Among outstanding questions are the intrinsic mechanisms that determine PCM shape and size, and how it functions as a biochemical reaction hub.
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.
Although cytoplasmic interactions between Caenorhabditis elegans centrosome proteins SPD-5, RSA-1, and RSA-2 can be detected in vivo, the key proteins required for centrosome assembly, SPD-2 and SPD-5, exist mostly as monomers and are separated from the centrosome-related kinases PLK-1 and AIR-1 in the cytoplasm.
Centrosomes are major microtubule-organizing centers composed of centrioles surrounded by an extensive proteinacious layer called the pericentriolar material (PCM). In C. elegans embryos, the mitotic PCM expands by Polo-kinase (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-5 4A ) 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-5 4A 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 Polo Kinase 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.peer-reviewed)
Cerebral organoids provide unparalleled access to human brain development in vitro. However, variability induced by current culture methodologies precludes using organoids as robust disease models. To address this, we developed an automated Organoid Culture and Assay (ORCA) system to support longitudinal unbiased phenotyping of organoids at scale across multiple patient lines. We then characterized organoid variability using novel machine learning methods and found that the contribution of donor, clone, and batch is significant and remarkably consistent over gene expression, morphology, and cell-type composition. Next, we performed multi-factorial protocol optimization, producing a directed forebrain protocol compatible with 96-well culture that exhibits low variability while preserving tissue complexity. Finally, we used ORCA to study tuberous sclerosis, a disease with known genetics but poorly representative animal models. For the first time, we report highly reproducible early morphological and molecular signatures of disease in heterozygous TSC+/− forebrain organoids, demonstrating the benefit of a scaled organoid system for phenotype discovery in human disease models.
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