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 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.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
Yann
Y
Gambin
Nicholas
N
Ariotti
Kerrie-Ann
KA
McMahon
Michele
M
Bastiani
Emma
E
Sierecki
Oleksiy
O
Kovtun
Mark E
ME
Polinkovsky
Astrid
A
Magenau
Wooram
W
Jung
Satomi
S
Okano
Yong
Y
Zhou
Natalya
N
Leneva
Sergey
S
Mureev
Wayne
W
Johnston
Katharina
K
Gaus
John F
JF
Hancock
Brett M
BM
Collins
Kirill
K
Alexandrov
Robert G
RG
Parton
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D021941:Caveolae / Q000737:chemistry*; D002460:Cell Line; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis*; D016253:Microscopy, Immunoelectron; D057054:Molecular Imaging; D061848:Optical Imaging; D028044:Phosphate-Binding Proteins; D011485:Protein Binding; D055503:Protein Multimerization; D016601:RNA-Binding Proteins / Q000032:analysis*
C497835:CAVIN1 protein, human; C112303:CAVIN2 protein, human; C505360:CAVIN3 protein, human; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D028044:Phosphate-Binding Proteins; D016601:RNA-Binding Proteins
caveolae; cell-free protein expression; single-molecule