This project focuses broadly on understanding this new cytokinesis control system, and will additionally provide broader insight into Ndr-Hippo pathway control of cell morphogenesis. 1. Define mechanisms of the budding yeast ECO pathway’s protection cytokinesis. We will determine how the conserved yeast Ndr pathway functions in cytokinesis, protein trafficking, and growth regulation. We identified a high confidence set of in vivo targets of budding yeast Ndr-hippo signaling in the last project period, among other things revealing a novel checkpoint that enforces the order of key cytokinetic events. Our main near-term effort will be determining how yeast Ndr-hippo signaling impacts trafficking of specific exocytic cargoes, such as hydrolytic enzymes that complete the final steps of cytokinesis. Using molecular genetic analysis and assays that read out the status of distinct Ndr-hippo signaling outputs, we will determine the functional significance of these known and likely interactions. 2. Assess substrate docking properties of human Ndr/Lats kinases. Our discovery that a yeast Ndr/Lats kinase domain associates with a peptide docking motif dramatically enhanced our knowledge of the system’s regulatory targets and raised the possibility that human Ndr/Lats kinases engage in similar substrate interactions. We will assess substrate docking of human Ndr and Lats kinases and the associated Mob1 and Mob2 co-activator proteins. In the last project period we developed rigorous biochemical analysis of Ndr/Lats kinases bound to Mob coactivators, and are among a very small number of groups with this expertise. We have refined and validated an “encoded sequence peptide” phage display method that identifies high specificity / low affinity interactions between folded domains and peptides actually present in proteomes of interest. We will combine this with molecular genetic analysis and a novel peptide footprinting approach to validate interactions and map signaling interactions of human Ndr/Lats pathways. 3. Determine if mechanisms analogous to budding yeast ECO protect late cytokinesis / abscission in animal cells. Our discovery of the budding yeast ECO pathway shows that completion of early cytokinesis triggers proteolytic inactivation of a system that blocks incompatible late cytokinetic processes. While the precise morphology of cell division is different in yeast and animal cells, it has recently become clear that there is striking functional conservation of the machinery that executes the sequential steps of cytokinesis. Intriguingly, recent finding suggest that Ndr kinases play an important role in abscission of human cells. We hypothesize that checkpoint mechanisms similar to ECO ensure that processes of midbody morphogenesis and abscission are forestalled until appropriate completion of cytokinetic furrow ingression. We will assess regulation of late cytokinesis and abscission in two different ways: focusing on Ndr kinase regulation and function, and identifying mechanisms that control Rif1-PP1 mediated activation of ESCRT-mediated abscission processes.
|Effective start/end date
|5/12/20 → 4/30/24
- National Institute of General Medical Sciences (5R01GM137133-04)
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