ABSTRACT
The lipid second messenger PI(4,5)P(2) modulates actin dynamics, and its local accumulation at plasmalemmal microdomains (rafts) might mediate regulation of protrusive motility. However, how PI(4,5)P(2)-rich rafts regulate surface motility is not well understood. Here, we show that upon signals promoting cell surface motility, PI(4,5)P(2) directs the assembly of dynamic raft-rich plasmalemmal patches, which promote and sustain protrusive motility. The accumulation of PI(4,5)P(2) at rafts, together with Cdc42, promotes patch assembly through N-WASP. The patches exhibit locally regulated PI(4,5)P(2) turnover and reduced diffusion-mediated exchange with their environment. Patches capture microtubules (MTs) through patch IQGAP1, to stabilize MTs at the leading edge. Captured MTs in turn deliver PKA to patches to promote patch clustering through further PI(4,5)P(2) accumulation in response to cAMP. Patch clustering restricts, spatially confines, and polarizes protrusive motility. Thus, PI(4,5)P(2)-dependent raft-rich patches enhance local signaling for motility, and their assembly into clusters is regulated through captured MTs and PKA, coupling local regulation of motility to cell polarity, and organization.
Subject(s)
Cell Movement/physiology , Membrane Microdomains/metabolism , Microtubules/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Pseudopodia/metabolism , Animals , COS Cells , Cell Membrane/metabolism , Cell Polarity/physiology , Cells, Cultured , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinases/metabolism , Green Fluorescent Proteins , Mice , NIH 3T3 Cells , Nerve Tissue Proteins/metabolism , Signal Transduction/physiology , Swiss 3T3 Cells , Wiskott-Aldrich Syndrome Protein, Neuronal , cdc42 GTP-Binding Protein/metabolism , ras GTPase-Activating Proteins/metabolismABSTRACT
The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P(2)-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P(2)-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P(2), Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P(2) in a Ca(2+)/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P(2)-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.
Subject(s)
Actins/metabolism , Cell Movement/physiology , Membrane Microdomains/metabolism , Phosphatidylinositol Phosphates/metabolism , Animals , Cytoskeleton/metabolism , GAP-43 Protein/metabolism , Humans , Models, Biological , Phosphatidylinositol 4,5-DiphosphateABSTRACT
Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes and have been implicated in most signaling processes at the cell surface, but the principles and mechanisms through which lipid rafts influence signaling are not well understood. Recent studies have revealed how lipid rafts are rapidly redistributed and assembled locally in response to extracellular signals, and how components of raft-based signaling domains undergo rapid and regulated rearrangements influencing signal quality, duration, and strength. These findings highlight the exquisitely dynamic properties of signaling domains based on lipid rafts, and suggest that processes of raft trafficking and assembly take central roles in mediating spatial and temporal control of signaling.
