Septins are GTPases involved in cytokinesis. In yeast, they form a ring at the cleavage site. Using FRAP, we show that septins are mobile within the ring at bud emergence and telophase and are immobile during S, G2, and M phases. Immobilization of the septins is dependent on both Cla4, a PAK-like kinase, and Gin4, a septin-dependent kinase that can phosphorylate the septin Shs1/Sep7. Induction of septin ring dynamics in telophase is triggered by the translocation of Rts1, a kinetochore-associated regulatory subunit of PP2A phosphatase, to the bud neck and correlates with Rts1-dependent dephosphorylation of Shs1. In rts1-Delta cells, the actomyosin ring contracts properly but cytokinesis fails. Together our results implicate septins in a late step of cytokinesis and indicate that proper regulation of septin dynamics, possibly through the control of their phosphorylation state, is required for the completion of cytokinesis.
During cytokinesis, furrow ingression and plasma membrane fission irreversibly separate daughter cells. How actomyosin ring assembly and contraction, vesicle fusion, and abscission are spatially coordinated was unknown. We found that during cytokinesis septin rings, located on both sides of the actomyosin ring, acted as barriers to compartmentalize the cortex around the cleavage site. Compartmentalization maintained diffusible cortical factors, such as the exocyst and the polarizome, to the site of cleavage. In turn, such factors were required for actomyosin ring function and membrane abscission. Thus, a specialized cortical compartment ensures the spatial coordination of cytokinetic events.
SummaryCentrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis—a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize in vitro and to spontaneously form cytoplasmic scaffolds in vivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies.
During anaphase, spindle elongation pulls sister chromatids apart until each pair is fully separated. In turn, cytokinesis cleaves the cell between the separated chromosomes. What ensures that cytokinesis proceeds only after that all chromosome arms are pulled out of the cleavage plane was unknown. Here, we show that a signaling pathway, which we call NoCut, delays the completion of cytokinesis in cells with spindle-midzone defects. NoCut depends on the Aurora kinase Ipl1 and the anillin-related proteins Boi1 and Boi2, which localize to the site of cleavage in an Ipl1-dependent manner and act as abscission inhibitors. Inactivation of NoCut leads to premature abscission and chromosome breakage by the cytokinetic machinery and is lethal in cells with spindle-elongation defects. We propose that NoCut monitors clearance of chromatin from the midzone to ensure that cytokinesis completes only after all chromosomes have migrated to the poles.
Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.
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