Direct mapping of nanoscale compositional connectivity on intact cell membranes.
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 cholesterol-mediated 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.
van Zanten
Thomas S
TS
BioNanoPhotonics group, Institute for Bioengineering of Catalonia (IBEC) and CIBER-bbn, Baldiri Reixac 15-21, 08028 Barcelona, Spain.
Gómez
Jordi
J
Manzo
Carlo
C
Cambi
Alessandra
A
Buceta
Javier
J
Reigada
Ramon
R
Garcia-Parajo
Maria F
MF
D015703:Antigens, CD / Q000737:chemistry; D018958:CD55 Antigens / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002772:Cholera Toxin / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D003198:Computer Simulation; D005677:G(M1) Ganglioside / Q000737:chemistry*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D009010:Monte Carlo Method; D036103:Nanotechnology / Q000379:methods; D011990:Receptors, Transferrin / Q000737:chemistry
D015703:Antigens, CD; D018958:CD55 Antigens; C068126:CD71 antigen; D017261:Glycosylphosphatidylinositols; D011990:Receptors, Transferrin; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D002784:Cholesterol