Direct Observation of Plasma Membrane Domains using Super Resolution Microscopy

The composition of the plasma membrane has long been modeled as a mosaic fluid. However, in the last few years there has been evidence that suggests the plasma membrane to be a dynamic and highly compartmentalized structure. This organization in domains results in a differential spatial distribution of signaling proteins on both leaflets of the plasma membrane. It is still debated whether inner and outer leaflet domains are linked. The lateral segregation of membrane proteins plays a role in cell signaling and protein-protein interaction. Thus, it is of high scientific interest to further investigate these domains.The Ras protein resides on the inner leaflet of the plasma membrane. Here we used super-resolution microscopy to study the compartmentalization of H-Ras and its membrane anchor CAAX, fused to the photo convertible dye Dendra2. The signal of single Dendra2 molecules is recorded and statistical analysis is applied to localize these molecules. On the apical membrane of 3T3 fibroblast, domains of 150nm were detected for both the full protein and its membrane anchor. To investigate a possible link between inner and outer leaflet domains, cells where treated with Choleratoxin B (CtxB). This leads to clustering of the outer-leaflet ganglioside GM1. Neither size nor the amount of domains were dependent on incubation with CtxB. However, incubation with CtxB did lead to an increase in H-Ras density inside the domains, indicating a connection between lipid organization on the outside and protein distribution on the inside of the plasmamembrane.