BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I

Functional analysis of a novel de novo variant in PPP5C associated with microcephaly, seizures, and developmental delay

We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI = 1, o/e = 0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the proband\u27s phenotypes

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Journal clubs in the time of preprints

Early-career researchers can learn about peer review by discussing preprints at journal clubs and sending feedback to the authors

Protease dead separase inhibits chromosome segregation and RAB-11 vesicle trafficking

<p>Separase cleaves cohesin to allow chromosome segregation. Separase also regulates cortical granule exocytosis and vesicle trafficking during cytokinesis, both of which involve RAB-11. We investigated whether separase regulates exocytosis through a proteolytic or non-proteolytic mechanism. In <i>C. elegans</i>, protease-dead separase (SEP-1^PD::GFP) is dominant negative. Consistent with its role in cohesin cleavage, SEP-1^PD::GFP causes chromosome segregation defects. As expected, partial depletion of cohesin rescues this defect, confirming that SEP-1^PD::GFP acts through a substrate trapping mechanism. SEP-1^PD::GFP causes cytokinetic defects that are synergistically exacerbated by depletion of the t-SNARE SYX-4. Furthermore, SEP-1^PD::GFP delays furrow ingression, causes an accumulation of RAB-11 vesicles at the cleavage furrow site and delays the exocytosis of cortical granules during anaphase I. Depletion of <i>syx-4</i> further enhanced RAB-11::mCherry and SEP-1^PD::GFP plasma membrane accumulation during cytokinesis, while depletion of cohesin had no effect. In contrast, centriole disengagement appears normal in SEP-1^PD::GFP embryos, indicating that chromosome segregation and vesicle trafficking are more sensitive to inhibition by the inactive protease. These findings suggest that separase cleaves an unknown substrate to promote the exocytosis of RAB-11 vesicles and paves the way for biochemical identification of substrates.</p

A Role for Separase in the Regulation of RAB-11-Positive Vesicles at the Cleavage Furrow and Midbody

SummaryCell division requires coordinated regulation of chromosome segregation and cytokinesis. Although much is known about the function of the protease separase in promoting sister chromosome separation, the role of separase during cytokinesis is unclear. We show that separase localizes to the ingressing furrow and midbody during cytokinesis in the C. elegans embryo. Loss of separase function during the early mitotic divisions causes cytokinesis failure that is not due to eggshell defects or chromosome nondisjunction. Moreover, depletion of separase causes the accumulation of RAB-11-positive vesicles at the cleavage furrow and midbody that is not a consequence of chromosome nondisjunction, but is mimicked by depletion of vesicle fusion machinery. Collectively, these data indicate that separase is required for cytokinesis by regulating the incorporation of RAB-11-positive vesicles into the plasma membrane at the cleavage furrow and midbody