Regulation and Function of Phosphoinositide Lipid Signals

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Organelles are the “organs” of cells. There are many types of organelles, each with their own unique biochemical and functional properties. For example, the endoplasmic reticulum (ER) is a labyrinth of membrane tubules where protein synthesis occurs, whereas lysosomes are small, round organelles packed with digestive enzymes that eliminate unwanted materials like damaged proteins and microbes. How organelles form, or change, is a key question in cell biology. The phosphoinositide (PIP) lipids are architects of organelle identity. There are seven PIP species that are differentially distributed among organelles. Each PIP species binds a unique set of proteins, which decorates the host organelle with specific molecular properties. In order to understand how PIPs define organelle identity, we need to understand A) how the enzymes that synthesize and degrade PIPs are regulated and B) how PIPs and their effector proteins work. Phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is a specific PIP that is synthesized by the lipid kinase PIKfyve. PI(3,5)P2 governs lysosome properties, which is important for cells to clear unwanted material. My NSERC-funded research has focused on understanding how PIKfyve is regulated and how it functions. We showed that inhibition of PIKfyve impairs immune function, including neutrophil migration towards microbes (chemotaxis) and the engulfment and digestion of microbes (phagocytosis and phagosome maturation). In addition, we discovered that lysosomes fail to separate (fission) from other lysosomes after fusion during PIKfyve ablation, causing their coalescence. This separation defect may occur because PIKfyve modulates protein machinery that generate force and/or deforms membranes that elicit fission. With NSERC support: i) We will use unbiased methods based on lysosome isolation, molecular tagging, and mass spectrometry to understand how the composition of lysosomes change during PIKfyve ablation. These data may identify complexes involved in membrane fission. ii) We will study the mechanisms by which PIKfyve governs phagocytosis and cell migration. We will use immune and cancer cells to test the hypothesis that PIKfyve coordinates the assembly and disassembly of the actin cytoskeleton to mold cell shape and generate forces necessary for these processes. iii) We will study how PIKfyve modulates the assembly of actin on lysosomes and if this interfaces with the ER to demarcate fission sites. ER contact sites with other organelles is emerging as a mediator of organelle fission. In all, this research will provide new insight into how PIKfyve governs lysosome function and interfaces with the cell's force generating machinery. This may then aid researchers better understand deleterious effects caused by PIKfyve loss. In turn, this may provide the Canadian pharmaceutical industry with novel strategies to treat conditions like Charcot-Marie Tooth neurodegenerative disease caused by PI(3,5)P2 malfunction.