Structural and Dynamic Study of Caveolin-1 Membrane Nanodomains in Response to Inflammatory Pathway Signaling

Abstract

Within the plasma membrane, the protein caveolin-1 (cav-1) forms various diffraction-limited microdomains that have yet to been precisely characterized in terms of structure and dynamics. Cav-1 is an important regulator of the GPCR protease activated receptor-1 (PAR-1), notably during the activation of cellular inflammation responses. The regulatory role of cav-1 during inflammatory responses is linked to the differential activation of RhoA and Rac1 signaling pathways and to cytoskeletal rearrangements that result in wide scale plasma membrane reorganization and cell migration. Using single molecule and super-resolution imaging techniques coupled with quantitative spatial clustering analyses, we study the membrane distribution and the dynamics of caveolae and flat cav-1 scaffolds in response to PAR-1 pathway activations. Cav-1 knock-out MEF and iMAEC cells were rescued with cav-1 fusions to SNAP tag and immunolabeled against endogenous PAR-1 for dual color 3D super-resolution imaging of individual cav-1 and PAR-1 proteins. PAR-1 was directly stimulated using site-specific activating peptides to trigger inflammatory response cascades. At the plasma membrane, 3D super-resolution imaging revealed the expected ∼100 nm 3D globular shape of caveolae as well as flat cav-1 rich nanodomains with structures matching the expected size and z-dimensional range of cav-1 scaffolds. Using robust auto- and cross-correlation statistical analyses of individual cav-1 and PAR-1 proteins spatially mapped with a precision <10 nm, we show that the distribution, the number and the size of cav-1 nanodomains change dynamically in response to either pro- or anti- inflammatory signaling as does the interconnectivity between caveolae, cav-1 scaffolds and PAR-1 receptors at the cell surface. We are able to couple the molecular re-organization of diffraction-limited cav-1 nanodomains with broader cytoskeletal and plasma membrane remodeling in response to defined signaling cues during cellular wound healing.