Roles of endosomal phosphoinositides in intracellular trafficking

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Endolysosomal trafficking is a tightly regulated process that involves membrane fusion and fission events, which ensure proper delivery and exchange of cargoes within endosomes, lysosomes, and other intracellular organelles or vesicles. It is essential for several cellular functions such as protein and lipid transport, autophagy, and degradation of macromolecules in lysosomes. How it is regulated is a fundamental issue in cellular biology. Our research program intends to use viruses as means to monitor and understand vesicular trafficking in cells. Indeed, viruses have evolved mechanisms to use and even regulate endosomal maturation or endolysosomal trafficking, since it can determine whether the infection is successful or not. Therefore, they can represent powerful and invaluable tools for the study of cellular trafficking pathways. We found that proteins of the Vac14/Fig4/PIKfyve complex, which regulates the synthesis of the phosphoinositide(3,5)bisphosphate at the endolysosomal membrane, are essential for infection by Ebola virus (EboV) and severe acute respiratory syndrome coronavirus (SARS-CoV). How the enzymatic complex synthesizing PI(3,5)P2 is activated and the role of PI(3,5)P2 in endolysosomal trafficking remain unclear. Here, we propose to use EboV and SARS-CoV to answer these questions. The goal of this research program is to understand how this complex and its resulting phosphoinositide are regulated and how they control endolysosomal trafficking in cells, a fundamental cellular process. Hypothesis: The endosomal phosphoinositide PI(3,5)P2 is critical for the trafficking of viral particles to intracellular compartments where viral fusion occurs. Short-term objectives: 1) Examine the effect of genetic and chemical manipulation of PI(3,5)P2 levels 2) Perform a molecular dissection of the Vac14/Fig4/PIKfyve complex 3) Identify PI(3,5)P2 effectors Our long term goal is to gain a molecular understanding of the Vac14/Fig4/PIKfyve interactome and function. To meet our objectives, we will use a combination of genetic, chemical biology, and virological tools. Combining these specific tools with our expertise in molecular/cell biology and viral entry, we hope to fill a gap in the understanding of endolysosomal trafficking and how this fundamental process can be hijacked by viral pathogens.