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

Background: Caveolin-1 (Cav1) requires the caveolin scaffolding domain for caveola formation. Results: The Cav1 scaffolding domain and oligomerization domain are tightly juxtaposed to the membrane in caveolae. Conclusion: Concerted membrane association of the oligomerization, scaffolding, and intramembrane domains are critical for caveola biogenesis and membrane deformation. Significance: Understanding the membrane association of Cav1 is critical for dissecting how the protein regulates caveola formation and achieves regulation over cellular signaling.

Highlights d A strategy for improving the ADCC potential of therapeutic antibodies is presented d Temporary inhibition of endocytosis increases tumor cell antigen presentation d Prochlorperazine could be repurposed to enhance the efficacy of anti-tumor mAbs d Potential to reduce heterogeneity in tumor cell responses to many IgG1 antibodies

Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.

No abstract