Super-Resolution Imaging and Spatial Analysis of RAS on Intact Plasma Membrane Sheets.

The function of lipid-anchored small GTPases RAS proteins is mostly compartmentalized to the plasma membrane (PM). Complex biophysical interactions between the C-terminal membrane-anchoring domains of RAS isoforms and PM lipids drive spatial segregation of RAS molecules in the formation of nanometer-sized domains, termed as nanoclusters. These RAS/lipid proteolipid nano-assemblies are the main sites for efficient effector recruitment and signal transduction. Here, we describe a super-resolution imaging method to quantify the nanometer-sized nanoclustering of RAS over a length scale between 8 and 240 nm on intact PM sheets of mammalian cells. Detailed molecular spatial distribution parameters, including the extent of nanoclustering, average cluster size, clustered fraction, and population distribution can be obtained by the univariate spatial distribution analysis. Intermolecular associations between different RAS isoforms, RAS and various PM lipids, as well as RAS and diverse effectors can be quantified via bivariate co-localization analysis.