The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P2, and promote its retention and clustering. Binding and modulation of PI(4,5)P2 depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P2 modulation. In the neuronlike cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by ΔED mutants. Agents that globally mask PI(4,5)P2 mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(ΔED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P2 modulating proteins, upstream of actin and cell cortex dynamics regulation.
The lipid second messenger PI(4,5)P2 modulates actin dynamics, and its local accumulation at plasmalemmal microdomains (rafts) might mediate regulation of protrusive motility. However, how PI(4,5)P2-rich rafts regulate surface motility is not well understood. Here, we show that upon signals promoting cell surface motility, PI(4,5)P2 directs the assembly of dynamic raft-rich plasmalemmal patches, which promote and sustain protrusive motility. The accumulation of PI(4,5)P2 at rafts, together with Cdc42, promotes patch assembly through N-WASP. The patches exhibit locally regulated PI(4,5)P2 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)P2 accumulation in response to cAMP. Patch clustering restricts, spatially confines, and polarizes protrusive motility. Thus, PI(4,5)P2-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.
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)P2-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)P2-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P2, 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)P2 in a Ca2+/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P2-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.
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