Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

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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.
Funding Information
  • Australian Research Council Future Fellowships (FT0991611)
  • Australian Research Council Future Fellowships (FT110100478)
  • Australian Research Council Future Fellowships (FT100100027)
  • National Health and Medical Research Council (NHMRC) Project Grant (APP1025082)
  • NHMRC Fellowships (569542, 1058565)
  • NHMRC Program Grants (511005, 1037320)
  • ARC Discovery Project Grants (DP120101298)
  • ARC Discovery Project Grants (DP130102396)
  • ARC Discovery Project Grants (DP120101423)
  • Australian Research Council (FT0991611)
  • Australian Research Council (FT110100478)
  • Australian Research Council (FT100100027)
  • National Health and Medical Research Council (APP1025082)
  • National Health and Medical Research Council (569542, 1058565)
  • National Health and Medical Research Council (511005, 1037320)
  • ARC Discovery Project Grants (DP120101298)
  • ARC Discovery Project Grants (DP130102396)
  • ARC Discovery Project Grants (DP120101423)

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