Direct mapping of nanoscale compositional connectivity on intact cell membranes

Abstract

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterolmediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains <120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (<150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation. cholera toxin | membrane heterogeneity | near-field scanning optical microscopy | raft ganglioside GM1 | single-molecule detection

Biomolecule-Conjugated Quantum Dot Nanosensors as Probes for Cellular Dynamic Events in Living Cells

“…This experimental finding was verified using three cell lines with a wide range of EGFR expression levels and membrane cholesterol concentrations, suggesting that these results may represent a general behavior of unliganded and activated receptors in live cells. Reigada et al recently applied near-field scanning optical microscopy to fixed monocytes and found that raftophilic proteins did not physically intermix at the nanoscale with CTxB-GM1 nanodomains but converged within a characteristic distance [62]. Our result of unliganded EGFR agrees with this finding but on live cells without CTxB tightening.…”

“…From the comparison of cross-correlation shown in Figures 15c and 14, we could remove the aggregation model, and we concluded that our data was better described by the segregation model with a segregation distance <100 nm. It was discovered that the raft lipid species GM1 can be tightened by pentameric cholera toxin-β (CTxB), which initiates a minimum raft coalescence to form the GM1 nanodomains [62]. The plasma membranes in our study were in their native state without perturbations from either intense laser spot or cross linking reagent.…”

Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

“…Although the first suggestions of protein clustering and membrane heterogeneity were highlighted in the 1970s in the classical article by Singer and Nicholson (2), it is only in the past decade that strong evidence of their existence has emerged. Domains ranging in size from a few tens of nanometers to~100-200 nm in diameter have been found in the past decade, both through indirect biochemical assays (3) and using sophisticated microscopy techniques including fluorescence recovery after photobleaching (4,5), fluorescence resonance energy transfer (6), fluorescence correlation spectroscopy (7-9), single-particle tracking (10)(11)(12), single-molecule microscopy (13)(14)(15), and electron microscopy (16). However, a common view on the nature and biological role of such membrane domains is still lacking.…”

Fish oil increases raft size and membrane order of B cells accompanied by differential effects on function

“…In vivo, FO also enhanced GM1 surface levels, which could be due to targeting of GM1 biosynthesis and/or traffi cking to the plasma membrane. Although the GM1 molecules were poised to form rafts in the absence of cross-linking, they were not in a state where large-scale phase separation could be observed ( 38,39 ).…”