Hotspots of GPI-anchored proteins and integrin nanoclusters function as nucleation sites for cell adhesion.
Recruitment of receptor proteins to lipid rafts has been proposed as an important mechanism to regulate their cellular function. In particular, rafts have been implicated in regulation of integrin-mediated cell adhesion, although the underlying mechanism remains elusive. We used single-molecule near-field optical microscopy (NSOM) with localization accuracy of approximately 3 nm, to capture the spatio-functional relationship between the integrin LFA-1 and raft components (GPI-APs) on immune cells. Dual color nanoscale imaging revealed the existence of a nanodomain GPI-AP subpopulation that further concentrated in regions smaller than 250 nm, suggesting a hierarchical prearrangement of GPI-APs on resting monocytes. We previously demonstrated that in quiescent monocytes, LFA-1 preorganizes in nanoclusters. We now show that integrin nanoclusters are spatially different but reside proximal to GPI-AP nanodomains, forming hotspots on the cell surface. Ligand-mediated integrin activation resulted in an interconversion from monomers to nanodomains of GPI-APs and the generation of nascent adhesion sites where integrin and GPI-APs colocalized at the nanoscale. Cholesterol depletion significantly affected the reciprocal distribution pattern of LFA-1 and GPI-APs in the resting state, and LFA-1 adhesion to its ligand. As such, our data demonstrate the existence of nanoplatforms as essential intermediates in nascent cell adhesion. Since raft association with a variety of membrane proteins other than LFA-1 has been documented, we propose that hotspots regions enriched with raft components and functional receptors may constitute a prototype of nanoscale inter-receptor assembly and correspond to a generic mechanism to offer cells with privileged areas for rapid cellular function and responses to the outside world.
BioNanoPhotonics group, IBEC-Institute for Bioengineering of Catalonia and CIBER-bbn, Baldiri Reixac 15-21, 08028 Barcelona, Spain.
Thomas S
TS
van Zanten
Alessandra
A
Cambi
Marjolein
M
Koopman
Ben
B
Joosten
Carl G
CG
Figdor
Maria F
MF
Garcia-Parajo
D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000172:deficiency; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D008958:Models, Molecular; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism*; D049329:Nanostructures / Q000737:chemistry*
D017261:Glycosylphosphatidylinositols; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1; D002784:Cholesterol