Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, Ϸ30% of the raft protein exists in cholesterol-dependent nanoclusters, with Ϸ70% distributed as monomers. The cluster͞monomer ratio (number of proteins in clusters͞number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster͞monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0 -7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.lipid rafts ͉ microdomains ͉ modeling T he plasma membrane is a complex, heterogeneous, lipid bilayer that is compartmentalized by combinations of lipid-lipid, lipidprotein, and actin-cytoskeleton interactions. This heterogeneity is biologically important because it offers an attractive mechanism to regulate the lateral segregation and output of membrane-anchored signaling proteins (1). Cholesterol-enriched, liquid-ordered domains (lipid rafts) that phase separate from surrounding liquiddisordered membrane in model in vitro systems have been postulated also to exist in biological membranes and to serve important roles in assembling cell-signaling complexes on the inner surface of the plasma membrane. The validity of this hypothesis continues to be debated (2). With the exception of lipid rafts aggregated by caveolin into caveolae, this uncertainty is due to their lack of detectable ultrastructure, the differences in size estimates reported, and the limitations of the techniques used to characterize these domains (3). Rafts were originally defined operationally, through their insolubility in various detergents and the ability of cholesterol depletion to disrupt their formation (4). However, there are problems with these approaches because deterg...

Ras proteins occupy dynamic plasma membrane nanodomains called nanoclusters. The significance of this spatial organization is unknown. Here we show, using in silico and in vivo analyses of mitogen-activated protein (MAP) kinase signalling, that Ras nanoclusters operate as sensitive switches, converting graded ligand inputs into fixed outputs of activated extracellular signal-regulated kinase (ERK). By generating Ras nanoclusters in direct proportion to ligand input, cells build an analogue-digital-analogue circuit relay that transmits a signal across the plasma membrane with high fidelity. Signal transmission is completely dependent on Ras spatial organization and fails if nanoclustering is abrogated. A requirement for high-fidelity signalling may explain the non-random distribution of other plasma membrane signalling complexes.

The plasma membrane nanoscale distribution of H-ras is regulated by guanine nucleotide binding. To explore the structural basis of H-ras membrane organization, we combined molecular dynamic simulations and mediumthroughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H-ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix a4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP-or GDP-loaded H-ras, demonstrating a critical role for these residues in stabilizing GTP-or GDP-H-ras interactions with the plasma membrane. By a similar analysis, we find that the b2-b3 loop and helix a5 are involved in a novel conformational switch that operates through helix a4 and the HVR to reorient the H-ras G-domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP-H-ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi-neutral interaction partner.

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses Hras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.Ras GTPases operate as plasma membrane-localized molecular switches that regulate multiple signal transduction pathways. The three ubiquitously expressed Ras isoforms, H-, N-, and K-ras, are anchored to the inner surface of the plasma membrane by a C-terminal S-farnesyl cysteine carboxy methylester acting in concert with a second signal. The S-farnesyl cysteine carboxy methylester is generated by a triplet of post-

Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott2Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent proteintagged isoforms of Cdc42, CRIB domain, N-WASP and actin; high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterolsensitive, Cdc42-based recruitment of the actin polymerization machinery.