Forever seeking closure: the roles of phosphoinositide metabolism during phagocytic and macropinocytic cup closure

Macro-endocytosis is the bulk uptake of extracellular material and encompasses macropinocytosis and phagocytosis. Macropinocytosis is the non-specific bulk uptake of extracellular fluid, and phagocytosis is the receptor-mediated uptake of solid particles. Both are complex and are regulated by the phosphoinositide signalling lipids (PIPs). However, how PIPs regulate the final stages of engulfment – cup closure – is poorly understood.
Cup closure requires controlled regulation of PIPs and Filamentous-actin (F-actin). PI(4,5)P2, a known F-actin regulator is hydrolysed by mammalian inositol 5-phosphatase Oculocerebrorenal syndrome of Lowe (OCRL). In this thesis I use the amoeba and model phagocyte Dictyostelium discoideum to investigate how the OCRL homolog, Dictyostelium discoideum 5-phosphatase 4 (Dd5p4) regulates engulfment. Dd5p4- mutants displayed specific defects during cup closure with phagosomes unable to move through the actin cortex. Hydrolysis of PI(4,5)P2 at the centre of phagocytic and to a lesser extent at macropinocytic cups was largely orchestrated by Dd5p4. Lattice light-sheet microscopy (LLSM) showed that Dd5p4- macropinosomes can be internalised by squeezing through small cortex openings, but then acquired F-actin ‘comets’ containing F-actin, the Actin related protein 2/3 complex (Arp2/3 complex), the Wiskott-Aldrich Syndrome protein (WASP), PI(3,4,5)P3, and PI(3,4)P2.
LLSM also illuminated the presence of thin membrane tubules that briefly connected newly-formed macropinosomes to the cell-surface PM. Like clathrin-coated pits (CCPs), these also required WASP to be efficiently resolved, indicating a new general role for WASP-generated actin in driving the final stages of endosomal formation.