The ESCRT (Endosomal Sorting Complex Required for Transport) pathway mediates a series of key membrane fission events, including endosomal vesicle formation (to create multivesicular bodies), retrovirus budding, and the abscission step of cytokinesis. In addition, we recently demonstrated that the late-acting ESCRT-III/VPS4 proteins also function early in mitosis, and that their depletion gives rise to aberrant centrosome numbers, spindle assembly, and metaphase chromosome alignment. Thus, the ESCRT pathway functions at multiple stages of cell division. In unpublished studies, we have shown that ESCRT factors bind other proteins with known mitotic/centrosomal roles, including CC2D1A/B, CEP290, and the cohesin complex. Depletion of these factors also induces centrosomal defects, implying that these factors may help mediate mitotic ESCRT functions. I now propose to investigate the mechanism(s) by which late-acting ESCRT-III/VPS4 proteins function in mitosis and centrosome maintenance. Specifically, I propose to test the hypothesis that ESCRT-III/VPS4 proteins and their interaction partners maintain proper centrosome numbers by regulating the timing and/or extent to which the cohesin complex controls centrosome disengagement and replication.
Aim 1 : Mechanistic investigations of ESCRT-III/VPS4 centrosome regulation. I will: a) determine where ESCRT-III proteins function, b) define the mechanism of centrosome amplification (e.g., templated duplication, fragmentation, or de novo synthesis), and c) determine if ESCRT-III/VPS4 centrosomal functions depend on the cohesin complex.
Aim 2 : Biochemistry of ESCRT-III interaction partners. I will: a) characterize ESCRT- III/binding partner protein-protein interactions, b) determine whether these interactions are direct and can be recapitulated with recombinant proteins, c) map binding sites, and d) attempt to rescue ESCRT-III centrosomal defects by expressing exogenous wild-type and non-binding mutants. These experiments will reveal whether ESCRT-III proteins exert their centrosomal functions through interaction partners, and will guide mechanistic studies. In summary, ESCRT factors and binding partners function during both mitosis and cytokinesis. Their depletion alters centrosome numbers and induces aneuploidy, which are hallmarks of cancerous transformations. My proposed research will characterize the mitotic and centrosomal functions of ESCRT-III/VPS4 proteins, with a particular emphasis on understanding the mechanisms by which these proteins function in cell division.
Project Narrative Proteins of the ESCRT pathway are required to help separate the two daughter cells during cell division (a process called cytokinesis). Surprisingly, we have recently shown that ESCRT proteins are also required for earlier stages of cell division (mitosis), particularly in helping to maintain the spindle, a machine that functions in chromosome alignment and separation. Here, we propose studies aimed at understanding precisely how the ESCRT machinery helps to maintain proper spindle morphology, with the overall goal of understanding how mitosis and cytokinesis are normally coordinated and how these processes can go awry in disease states such as cancer.
