Abstract
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae. DOI: http://dx.doi.org/10.7554/eLife.01434.001.
Highlights
Caveolae are an abundant feature of the plasma membrane of many vertebrate cells
The surface of adipocytes, endothelial cells, smooth muscle, skeletal muscle and many other cell types is characterized by a dense covering of these small invaginations with a characteristic striated coat, as viewed by electron microscopy, and by the presence of membrane proteins termed caveolins (Peters et al, 1985; Kurzchalia et al, 1992; Rothberg et al, 1992; Way and Parton, 1995; Scherer et al, 1996; Parton and Del Pozo, 2013)
Three caveolins are present in mammalian cells with caveolin-1 (CAV1) and caveolin-3 (CAV3) essential for caveolar formation in nonmuscle and muscle cells respectively
Summary
Caveolae are an abundant feature of the plasma membrane of many vertebrate cells. The surface of adipocytes, endothelial cells, smooth muscle, skeletal muscle and many other cell types is characterized by a dense covering of these small invaginations with a characteristic striated coat, as viewed by electron microscopy, and by the presence of membrane proteins termed caveolins (Peters et al, 1985; Kurzchalia et al, 1992; Rothberg et al, 1992; Way and Parton, 1995; Scherer et al, 1996; Parton and Del Pozo, 2013). Three caveolins are present in mammalian cells with caveolin-1 (CAV1) and caveolin-3 (CAV3) essential for caveolar formation in nonmuscle and muscle cells respectively. Caveolins bind cholesterol and fatty acids and form homo-oligomers required for caveolar formation. 140 CAV1 molecules associate with a single caveola in mammalian cells (Pelkmans and Zerial, 2005) and in a model prokaryotic system upon caveolin expression (Walser et al, 2012). Genetic ablation of caveolins in mice has diverse cellular consequences with impact upon
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