# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 5 Membrane Heterogeneity and Nanoscale Topography in Ras Dimerization  
Most reconstituted membrane systems used to date for Ras studies are homogeneous lipid bilayers that lack some key features of actual biological membranes. These key attributes include the heterogeneous distribution of important lipid species (such as PS and PIP2), the exquisite physical structure of the local membrane, and the presence of potential scaffold mechanisms. Lateral segregation of proteins and lipids leads to transient or stable membrane domains common in biological membranes, to which other molecules can dynamically localize, creating a rich and complex environment for biological interactions [155; 156]. We will discuss the potential impact of membrane heterogeneity and nanoscale topography on Ras dimerization in this section, and that of effectors and scaffold proteins next. The spatial distribution of PIP2 on the cell membrane is known to be heterogeneous [157; 158]. PIP2, which makes up >90 % of all PIPs but comprises only \(\sim\)1% of all membrane lipids, supports a wide-ranging spectrum of cellular functions by interacting with hundreds of proteins [159; 160]. It is hypothesized that only a small fraction of PIP2 is freely available in the plasma membrane and that the majority is bound to, and released by, proteins in a dynamic manner [161; 162]. The concentrated patches of PIP2 might in turn attract effector proteins resulting in activation of a variety of downstream pathways [158]. Indeed, new imaging techniques suggest that PIP2 is distributed as clusters and monomers. Using EM and freeze-fracture membrane preparations of live cells, PIP2 was shown to be highly concentrated at the rim of caveolea as well as in clathrin-coated pits [163]. Super resolution microscopy using fluorescently tagged anti-PIP2 antibodies also showed PIP2 clusters of 60-90 nm size in cells [162; 164]. PS accounts for 10-20 % of all phospholipids in a typical cell and is found almost exclusively in the inner cytoplasmic leaflet of the membrane [165]. Using the Lact-C2 biosensor [166], evidence accumulated that a variety of intracellular proteins includingKRas bind to or are influenced by PS in a charge-based manner to maintain their appropriate distribution within the cell [139; 167; 168]. Despite its abundance, PS is also distributed heterogeneously in the membrane. EM images of BHK cells transfected with Lact-C2-GFP showed that PS forms clusters at the inner leaflet of the membrane and that some, like PIP2, are associated with caveolae [169]. PS co-segregates with cholesterol, an important component of ordered membrane domains such as rafts, not only in the membrane but throughout subcellular compartments such as the early endosomes [169; 170; 171]. The spatial distribution of lipids is directly relevant to Ras dimerization and clustering. A recent immuno-EM study showed that different Ras nanoclusters have distinct lipid compositions: PIP2 was found to be enriched in GDP-bound HRas nanoclusters, all HRas and KRas nanoclusters contained PS and PI(3,4,5)P (PIP3). Another common anionic lipid, phosphatidic acid (PA), was found to be enriched in GTP-bound KRas nanoclusters [172]. Depletion of PS showed that clustering of GTP-bound KRas nanoclusters linearly depends on PS membrane content and that PS is vital for nanocluster formation and stabilization [172]. Depletion of PIP2 displaces KRas, but not HRas, from the plasma membrane to the Golgi, and in so doing diminishes KRas signaling [173]. As discussed earlier, the heterogeneous distribution of PS, PIP2, and other anionic lipids (such as PA), for example their clustering within or exclusion from the various Ras-associated nanodomains, will directly impact the dimerization properties of the Ras molecules residing in these domains (Figure 6A). However, the effect of these lipids on Ras dimerization is likely more than what was discussed in the last section. Both PS and PIP2 have been involved in actin dynamics, which has been implicated in the spatiotemporal organization of Ras.