# Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation
## 5 Membrane Heterogeneity and Nanoscale Topography in Ras Dimerization
Actin and its binding proteins form a dynamic network inside the cell that undergoes continuous remodeling. PIP2 is important for the  
Figure 6: Membrane heterogeneity and topography in Ras dimerization. (**A**) Heterogeneous distribution of anionic lipids such as PS and PIP2 yields membrane regions with lipid compositions favoring some but not other Ras conformations, which could directly impact Ras dimerization. (**B**) Example mechanisms that couple actin polymerization with the local enrichment of PS (left) or PIP2 (right), yielding membrane patches of anionic lipids with relatively low mobility, which could entrap membrane proteins such as Ras. (**C**) Local enrichment of lipids could induce membrane curvature, potentially impacting Ras dimerization. (**D**) Endocytic and pre-endocytic compartments are examples of enriched anionic lipids coupled with actin polymerization and membrane curvature. There is evidence that Ras immobilization occurs in the same membrane nanodomains that internalize Ras, making some (pre-)endocytic compartments candidate sites for Ras dimerization and clustering. Parts of the figure were based on refs [155; 174; 175]. regulation of the actin cytoskeleton by serving as a platform for protein recruitment, and changes in PIP2 concentration or clustering have a significant impact on actin dynamics. An increase in PIP2 leads to actin filament assembly [176; 177] whereas a reduction in PIP2 results in actin cytoskeleton defects [178]. PS also directly interacts with actin-binding proteins such as talin and spectrin [179; 180]. Hotspots of actin dynamics, some referred to as 'actin asters', have been implicated in the organization of membrane nanoclusters of Glycosphosphatidylinositol-anchored proteins (GPI-APs) [181; 182]. The long, saturated acyl-chains of PS likely serve as the molecular link between lipid-tethered GPI-AP clusters and the actin cytoskeleton [183]. These mechanisms yield patches of anionic lipids with the ability to trap other membrane proteins including Ras (Figure 6B). An additional aspect of the local enrichment of these anionic lipids is their connection with membrane curvature, which has also been shown to influence the activity of many signaling molecules [184]. Biological membranes are not flat but covered in pits, protrusions, ruffles, and other types of structures. The generation and maintenance of these structures are fundamental to cell morphology and numerous other functions [185]. PIP2 directly interacts with Bin-Amphiphysin-Rvs (BAR) domain proteins to induce membrane curvature [186], and membrane curvature can be induced by the proximity of anionic phospholipids alone (Figure 6C) [187; 188; 157]. It was hypothesized that cholesterol associates with PS in membrane nanodomains and sufficiently separates the headgroups of PS to limit spontaneous curvature [187]. PS also plays an important role in the formation and stability of caveolae [189; 190], which may indirectly regulate Ras nanoclustering [142]. A few recent studies investigated the relationship between the spatiotemporal organization of Ras proteins and membrane topography [191; 192; 193]. One study investigated the recruitment of the fluorescently labelled minimal membrane anchor of the NRas (tN-Ras) isoform from solution to fluorescently labelled liposomes of different diameters [191], suggesting that membrane curvature is a novel modulator of NRas lipid anchor and palmitoyl chain partitioning. A certain synergy may exist between bilayer lipid shape and membrane curvature in regulation of the recruitment of lipidated proteins to the membrane in general [192]. By growing cells on substrates with artificially engineered nanobars to create membrane curvatures, another recent study revealed that tH-Ras was concentrated at the curved ends of positively curved membranes compared to mutant KRas [193]. Immuno-EM confirmed enrichment and elevated nanocluster formation of tH-Ras in membrane regions with high curvature, whereas KRas and tK-Ras showed the opposite.