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Single molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae (602.1)
Author(s) -
Gambin Yann,
Sierecki Emma,
Polinkovsky Mark,
Alexandrov Kirill,
Parton Robert
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.602.1
Subject(s) - caveolae , coat protein , chemistry , molecule , membrane , microbiology and biotechnology , biophysics , computational biology , biology , biochemistry , gene , rna , organic chemistry
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 new methods that allow reconstitution of the cavin complex and performed a quantitative assessment of cavin complex formation. We use single molecule fluorescence for its proven ability to directly observe multiple populations and quantify interactions in complex mixtures. These techniques are especially well suited for the study of coat proteins and have been used to study the mechanisms of clathrin assembly/disassembly. Those studies required labeling of recombinantly expressed purified proteins, which in the case of the cavin complex is difficult. We have taken an alternative approach to obtain the stoichiometry of the cavin complex and the interactions between the members of the cavin family (cavin1, cavin2, cavin3) directly from cell extracts. Instead of relying on organic dyes, we could perform the full quantitative analysis using genetically encoded fluorophores, shortcutting purification steps. 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. We could observe fascinating behavior of the cavin members, with exquisite segregation of interactors and defined stoichiometries in mixed oligomers. 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.