Function of Myotubularin Phosphoinositide Phosphatase in Morphogenesis

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DESCRIPTION (provided by applicant): Cell shape results from dynamic spatial regulation, in part conveyed through localized regulation of specific phosphoinositide phosphates (PIPs) that define and control membrane compartment identities. Importantly, mutations in phosphoinositide regulators are associated with morphology defects that contribute to human degenerative diseases and cancers, although the cellular bases of the developmental pathologies are not understood. Myotubularin encodes a conserved phosphoinositide 3-phosphate phosphatase, a negative regulator of PI(3)P and PI(3,5)P2 lipids with known roles on endosomes and lysosomes. A confounding aspect of myotubularin function in metazoans is the existence of six divergent protein families, representing both catalytically active and inactive forms. Human mutations in myotubularin sub-family members MTM1 and MTMR2 are associated with myotubular myopathy and neuropathy and sterility of Charcot-Marie Tooth disease, respectively, suggesting differential regulation, pools of phosphoinositide substrates and/or roles for other mediating factors. The Drosophila genome encodes for one homolog for each of the six human myotubularin families, representing an ideal system for the dissection of distinct phosphoinositide pathways through developmental-genetic analyses. We identified the Drosophila myotubularin homolog of human MTMR2 and MTM1 as necessary for cellular morphogenesis, with defects in late endosomes and cytoskeletal regulation associated with myotubularin loss of function. Our Specific Aims are designed to elucidate pathways that regulate and respond to myotubularin-dependent functions for cellular morphogenesis, relying on genetic manipulations in Drosophila cells and animals. We will perform kinetic cell-based assays to characterize the myotubularin-dependent cellular processes and to identify the in vivo phosphoinositide pools required for cellular elongation. To elucidate factors required for myotubularin regulation and responses, we will conduct genome-wide RNAi screens of myotubularin morphology phenotypes. Finally, we will determine the myotubularin protein localization dynamics and loss-of-function requirements in Drosophila development. This research will provide insights on phosphoinositide pathways important for cell spatial regulation and disease.