Grant: Discovery Grants Program - Individual

Endolysosomal trafficking is a tightly regulated process which ensures proper delivery and exchange of cargoes within endosomes, lysosomes, and other organelles. It is essential for several cellular functions such as protein and lipid transport, autophagy, degradation of macromolecules in lysosomes, in addition to serves as a portal of entry by different pathogens including viruses such as the Ebola virus (EBOV). Two major determinants of organelle identity are required for the accurate trafficking and fusion of vesicles: small GTPase proteins and lipids, such as phosphoinositides (PI). Among others, the rare PI(3,5)P2 is found on late endosomes/lysosomes and is produced by the phosphorylation of PI(3)P by a protein complex composed of Vac14, the phosphatase Fig4, and the kinase PIKfyve. Interestingly, a reduction in PI(3,5)P2 leads to severe vacuolization in cells, but the identity of the PI(3,5)P2 effectors are still largely elusive. In addition, how the activity of the PIKfyve/Fig4/Vac14 complex is regulated is not well understood. The short-term goal of the proposed research program is to gain a molecular understanding of the PIKfyve/Vac14/Fig4 interactome and functions with a long-term goal of understanding the regulation and functions of endosomal PIs in endolysosomal trafficking and other cellular processes. Using a combination of approaches already established in the lab, the objectives covered in this discovery grant are to: 1) Map the dynamic interactome of the PIKfyve/Vac14/Fig4 complex. Using a rapid proximity labeling method, we will map the interactome of Vac14 and Fig4 upon stimulation with growth factor and EBOV-like particles. We will also combine lipid probes and TurboID to identify effectors of PI(3,5)P2. 2) Characterize PIKfyve/Vac14/Fig4 complex interactions with organelles. Our interactome of the PIKfyve/Vac14/Fig4 revealed interactions/proximity to proteins localized at the mitochondria, endoplasmic reticulum, golgi and nucleus. Using microscopy, we will look at the localisation of the PIKfyve/Vac14/Fig4 complex and various markers of other organelles upon stimulation with growth factors and EBOV-like particles. We will also investigate its nuclear localization upon stresses that are known to lead to nuclear PI(5)P using cellular fractionation and microscopy. 3) Characterize candidate interacting proteins The candidate interacting proteins will be validated using fluorescence microscopy and proximity ligation assay. Genome editing will be performed for 2 candidate genes per year, and the KO cells will be characterized phenotypically. Direct interactions with the PIKfyve/Vac14/Fig4 complex will also be assessed. The proposed research program will identify roles of endosomal PIs and elucidate the mechanisms by which the PIKfyve/Fig4/Vac14 complex is regulated, deepening our understanding of vesicular trafficking and how pathogens use this pathway for infection.