Clustering of Rac1: Selective Lipid Sorting Drives Signaling.

The ability of lipid-anchored small GTPases to form nanometer-scale lipid domains on the cell plasma membrane (PM) is precipitating exciting new insights into membrane-anchored protein regulation. A recent article by Remorino et al. demonstrates that Rac1, similar to Ras, forms nanoclusters on the PM. The implications of these findings are discussed herein.

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Rac1 Nanoscale Organization on the Plasma Membrane Is Driven by Lipid Binding Specificity Encoded in the Membrane Anchor.

Rac1 is a small guanine nucleotide binding protein that cycles between an inactive GDP-bound and active GTP-bound state to regulate cell motility and migration. Rac1 signaling is initiated from the plasma membrane (PM). Here, we used high-resolution spatial mapping and manipulation of PM lipid composition to define Rac1 nanoscale organization. We found that Rac1 proteins in the GTP- and GDP-bound states assemble into nonoverlapping nanoclusters; thus, Rac1 proteins undergo nucleotide-dependent segregation. Rac1 also selectively interacts with phosphatidic acid (PA) and phosphoinositol (3,4,5)-trisphosphate (PIP3), resulting in nanoclusters enriched in these lipids. These lipids are structurally important because depleting the PM of PA or PIP3 impairs both Rac1 PM binding and Rac1 nanoclustering. Lipid binding specificity of Rac1 is encoded in the amino acid sequence of the polybasic domain (PBD) of the C-terminal membrane anchor. Point mutations within the PBD, including arginine-to-lysine substitutions, profoundly alter Rac1 lipid binding specificity without changing the electrostatics of the protein and result in impaired macropinocytosis and decreased cell spreading. We propose that Rac1 nanoclusters act as lipid-based signaling platforms emulating the spatiotemporal organization of Ras proteins and show that the Rac1 PBD-prenyl anchor has a biological function that extends beyond simple electrostatic engagement with the PM.