CYTOKINESIS CONTROL · Coordinating chromosome segregation with cell division: the molecular role of mitotic exit network proteins in the regulation of cytokinesis

Final Activity Report Summary - CYTOKINESIS CONTROL (Coordinating chromosome segregation with cell division: the molecular role of mitotic exit network proteins in the regulation of cytokinesis)

Cytokinesis, the final step of the cell cycle, is the process by which one cell is separated into two. This is a highly regulated process that needs to be tightly coordinated with chromosome segregation, both in space and time. This regulation is vital to prevent gain or loss of chromosomes during cell division, a process known to underlie chromosome instability and cancer development. In budding yeast, mitotic exit and cytokinesis are initiated by the conserved phosphatase Cdc14, which counteracts mitotic cyclin dependent kinase (Cdk1) phosphorylation events. Full activation of Cdc14 requires the mitotic exit network (MEN) signaling pathway. MEN activity is under control of the scaffolding protein Nud1 (centriolin in human cells) and spindle position checkpoint (SPOC), a surveillance mechanism that inhibits MEN activation until one set of the duplicate chromosomes is delivered in to the daughter cell body. The regulation of the MEN by the SPOC is essential to allow cytokinesis to occur only after proper chromosome segregation. The MEN is activated at the spindle pole bodies (SPBs, equivalent to the mammalian centrosome) and consists of the GTPase Tem1, the conserved protein kinases Cdc15 and Dbf2. Although down-regulation of mitotic Cdk1 activity by the MEN is a pre-requisite for cytokinesis, isolated evidence suggested a post-mitotic function of MEN in cytokinesis, mostly based on the relocation of MEN components from the SPBs to the site of cell division upon mitotic Cdk1 regulation. This function of the MEN has however remained unexplored, as most of the studies were directed towards the understanding of MEN activation. The aim of the project was therefore to investigate the molecular role of MEN proteins during cytokinesis. To achieve this aim, we used a combination of genetic, microscopic and biochemical approaches. Electron microscopy analysis of MEN mutants revealed malformation of the primary and secondary septa, which separate the mother from the daughter cell compartment. Moreover, MEN deficient cells stayed interconnected indicating a cell separation defect. We systematically analysed components of the late cytokinetic apparatus in MEN deficient cells and found that proteins involved in septum formation are not able to localise properly to the site of cell division upon Cdk1 down-regulation. These included the essential protein Inn1, the SH3-domain protein Cyk3 and the chitin synthase Chs2. Furthermore, MEN mutants forced to exit mitosis showed defects in actin re-polarisation and vesicle trafficking. Using the yeast two-hybrid system, the tandem affinity purification (TAP) strategy and high-throughput genomic screenings, we identified novel MEN and Cdc14 interactors involved in cytokinesis. Among those, we established the conserved F-BAR-family protein Hof1 as a substrate of Dbf2 kinase. The identification of Hof1 phosphorylation sites by mass-spectrometry and analysis of phosphomutants revealed an important stepwise regulation of Hof1 by Dbf2 and other identified mitotic kinases and Cdc14. This regulation proved to be important to relocate Hof1 from the septin scaffolding complex to the actomyosin ring, where Hof1 is needed to promote cytokinesis. Interestingly, the human orthologue of Hof1, PSTPIP, associates with the cleavage furrow and is subject to cell cycle dependent phosphorylation, raising the exciting possibility of functional conservation. Work over the past years has revealed that yeast homologues of MEN in several organisms regulate cytokinesis rather than mitotic exit. This highlights the importance of the work performed by the team, aiming to provide a unified view to explain the molecular function of MEN in the control of cell division.

Data: CORDIS, © European Union