Structural and Dynamic Study of Caveolin-1 Membrane Microdomains by Single Molecule Imaging

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. Using single molecule and super-resolution imaging techniques we study the membrane distribution and the dynamics of caveolae and cav-1 scaffolds at the nanometer scale in both fixed and live cells. Cav-1 knock out MEF cells were rescued with cav-1 fusions to SNAP, ptagRFP or mCherry tags co-expressed at equimolar amounts with a cav-1-split-GFP fusion for 3D super-resolution imaging and quantitative posttranslational modifications of individual cav-1 proteins with single molecule sensitivity. At the plasma membrane, 3D super-resolution imaging by dSTORM and PALM revealed the expected ∼100 nm globular shape of caveolae as well as single layer cav-1 domains with structures matching the expected size of cav-1 scaffolds. We also show that, in live cells, individual cav-1 can be posttranslationally targeted with synthetic peptides using split-GFP as both a targeting platform and an optical reporter for nanometer precision modifications of cav-1 microdomains. Towards a selective and high precision posttranslational destabilization of cav-1 microdomains using the cell's own machinery, we designed synthetic peptides that specifically induce the ubiquitination of split-GFP via the VHL ubiquitin pathway.By combining structural, dynamic and perturbation studies using molecular tools and ultrasensitive single molecule imaging techniques, we start to unveil the molecular organization of cav-1 microdomains and their dynamic interplay at the plasma membrane of eukaryotic cell.