Phosphoinositides and engulfment

Abstract

Cellular engulfment of particles, cells or solutes displaces large domains of plasma membrane into intracellular membranous vacuoles. This transfer of membrane is accompanied by major transitions of the phosphoinositide (PI) species that comprise the cytoplasmic face of membrane bilayers. Mapping of membrane PIs during engulfment reveals distinct patterns of protein and PI distributions associated with each stage of engulfment, which correspond with activities that regulate the actin cytoskeleton, membrane movements and vesicle secretion. Experimental manipulation of PI chemistry during engulfment indicates that PIs integrate organelle identity and orient signal transduction cascades within confined subdomains of membrane. These pathways are exploited by microbial pathogens to direct or redirect the engulfment process.

Figure 1. Phosphoinositide structure and dynamics on membranes

(A) Schematic diagram of the structures of PtdIns, PtdIns(4,5)P₂ and the products of PLC hydrolysis. DAG is indicated by black lines, with acyl chains extending from the cytosolic surface (orange line) into the bilayer. The hydroxyl groups at the 3, 4 and 5 positions of inositol (red arrows) can be phosphorylated by PI kinases and dephosphorylated by PI phosphatases. (B) Phosphoinositide dynamics in ruffles and cups. Surface extensions of plasma membrane are enriched in actin filaments (grey lines), whose polymerization and depolymerization are regulated by PIs. Significant transitions in PI profiles are indicated for the enlarged region of plasma membrane (right box). The red arrows indicate enzyme activation by PIs. (C) PI dynamics on vacuolar membranes (phagosomes and macropinosomes). The sequence of PIs appearing during macropinosome formation include PtdIns(4,5)P₂PIP₃PtdIns(3,4)P₂ and PtdIns3P. The subsequent transition to PI(3,5)P₂ on membranes has not been demonstrated directly. Microbial pathogens drive engulfment or modify vacuole maturation using PI-modifying enzymes. (1) PtdIns4P 5-kinase, (2) PI 3-kinase type I, (3) PLCγ1, (4) OCRL and Inpp5B, (5) PTEN, (6) PtdIns-4,5-bisphosphate 4-phosphatase, Salmonella SopB and Shigella flexneri IpgD, (7) SHIP1 and SHIP2, (8) Inpp4B, (9) PI 3-kinase type III, (10) myotubularin and Legionella SidP, (11) PYKEfyve, (12) Legionella SidF, (13) Legionella SidP.

Figure 2. Cellular movements of engulfment

(A) The cytoplasmic movements of FcR-mediated phagocytosis. The sequence left to right shows the movements of membranes during phagocytosis of a particle (green ovals). Actin filaments (grey) form a contractile cuff which advances over the particle. Membrane from intracellular organelles is inserted into the forming phagosome. Blue lines indicate plasma membrane domains with limited lateral mobility of PIs due to diffusion barriers in cup structure. (B) Stereotypical movements of macropinocytosis. The top row shows a progression, left to right, of side-view projections (x-z) of macropinocytic cup formation (ruffle closure) and membrane scission to form an intracellular macropinosome (cup closure). The second row shows the corresponding images as typically seen by light microscopy (x-y projection). Dotted lines indicate ruffles and cups in plasma membrane. Solid lines denote discrete membrane compartments. Blue lines indicate regions of plasma membrane where lateral mobility of inner leaflet molecules is constrained by diffusion barriers. (C) Dynamics of PIs during macropinosome formation. The top row shows the distributions of PtdIns(4,5)P₂ (red lines) which localizes to plasma membrane and is enriched in ruffles and very early cups. The middle row shows the stages of macropinosome formation which have maximal labeling with probes for PIP₃ (orange), PtdIns(3,4)P₂ (violet) and PtdIns3P (green). The bottom row indicates speculated synthesis of PtdIns or PtdIns(3,5)P₂, which have not been demonstrated by microscopy.