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

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 fro...

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Bibliographic Details
Published in:eLife 2014-01, Vol.3, p.e01434-e01434
Main Authors: Gambin, Yann, Ariotti, Nicholas, McMahon, Kerrie-Ann, Bastiani, Michele, Sierecki, Emma, Kovtun, Oleksiy, Polinkovsky, Mark E, Magenau, Astrid, Jung, Wooram, Okano, Satomi, Zhou, Yong, Leneva, Natalya, Mureev, Sergey, Johnston, Wayne, Gaus, Katharina, Hancock, John F, Collins, Brett M, Alexandrov, Kirill, Parton, Robert G
Format: Article
Language:English
Subjects:
Animals
Biophysics and Structural Biology
Carrier Proteins - analysis
Caveolae
Caveolae - chemistry
Caveolin
Cell Biology
Cell Line
cell-free protein expression
Immunoelectron microscopy
Intracellular Signaling Peptides and Proteins - analysis
Invaginations
Kinases
Labeling
Mammalian cells
Membrane proteins
Membranes
Microscopy
Microscopy, Immunoelectron
Molecular Imaging
Musculoskeletal system
Optical Imaging
Phosphate-Binding Proteins
Protein Binding
Protein Multimerization
Proteins
RNA-Binding Proteins - analysis
Signal transduction
single-molecule
Spectrum analysis
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Summary: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.
ISSN:2050-084X
2050-084X
DOI:10.7554/elife.01434