A strong understanding of cell cycle, the process through which cells grow and divide in a controlled manner to promote the growth of organisms, is vital to our understanding of healthy human development, developmental disorders, and cancers. We recently described a novel protein, WDR73, as a microtubule-interacting protein important during mitosis (cell division). We associated WDR73 gene mutations with Galloway-Mowat Syndrome (GAMOS), a rare, recessive developmental disorder characterized by microcephaly, reduced growth of the cerebellum, intellectual disability, growth retardation, blindness, kidney disease and untimely death. Our research determined that WDR73 is required for normal progression through cell cycle, especially mitosis. During mitosis, WDR73 localizes to the microtubules as they form the mitotic spindles and stays with them as they grow to capture the chromosomes during prometaphase and metaphase. WDR73 remains at the spindles during anaphase (segregation of chromosomes) but also extends into the interpolar and kinetochore microtubules until telophase when it concentrates at the midbody microtubules, where final separation of the daughter cells (cytokinesis) is coordinated. The goal of this proposal is to investigate the function of WDR73 during all phases of cell cycle and neuronal differentiation. Based on preliminary data on WDR73?s interactions with other proteins, we hypothesize that WDR73 scaffolds dynamic protein interactions during multiple phases of cell cycle and sub- phases of mitosis. During mitosis, preliminary data suggest that WDR73 may play a role in the nucleation and stabilization of mitotic microtubules. Our studies of GAMOS patient cells further suggest that WDR73 may be required for cell survival, normal timing of exit from cell cycle, and cell differentiation. We will use cell biological and biochemical approaches to investigate these hypotheses through three Specific Aims: (1) Determine the role of WDR73 in the assembly and remodeling of mitotic and interphase microtubule networks. Live and fixed cell imaging will be used in GAMOS patient cells and in cell lines in which WDR73 will be depleted by RNA interference (gene silencing) to determine how loss of WDR73 function impairs microtubule networks and delays cell cycle. (2) Characterize the WDR73 protein interactome in proliferating and non-proliferating (differentiated) cells. Unbiased co-immunoprecipitation and mass-spectrometry will be used during each phase of cell cycle and sub-phase of mitosis, and in post-mitotic neurons to identify novel WDR73 interacting proteins. (3) Investigate the role of WDR73 in neuronal differentiation and survival. RNA interference will be used to assess the impact of loss of WDR73 protein on proliferation, survival, and differentiation of neural cells. This research is innovative because it aims to characterize the function of a novel protein required for proper progression through cell cycle. It is significant because it will enhance our understanding of the regulation of cell cycle and the associated regulation of microtubule networks necessary to sustain normal development of the brain, cerebellum, and kidneys.
Rare diseases like Galloway-Mowat syndrome (GAMOS) are not rare for those who suffer from them and for their families and caregivers. Until in utero gene replacement therapy or genome editing is safe and approved, the first step in developing rational therapies for individuals with GAMOS caused by recessive WDR73 mutations is to learn as much as we can about the normal function of the WDR73 protein and about cellular pathophysiology associated WDR73 mutations. Involving undergraduate students in biomedical research is critical to our Nation?s ability to advance our public health through recruitment of promising students of diverse backgrounds into biomedical research and medicine, and to produce new generations of well-informed science teachers, business leaders, government officials, parents, and citizens.
