Abstract
Caveolins, major components of small plasma membrane invaginations called caveolae, play a role in signaling, particularly in mechanosignaling. These proteins are known to interact with a variety of effector molecules, including G-protein-coupled receptors, Src family kinases, ion channels, endothelial nitric oxide synthase (eNOS), adenylyl cyclases, protein kinase A (PKA), and mitogen-activated PKs (MAPKs). There is, however, speculation on the relevance of these interactions and the mechanisms by which caveolins may control intracellular signaling. This chapter introduces a method of isolation of giant plasma membrane-derived vesicles (GPMVs), which possess full complexity of membrane they originate from, thus comprising an excellent platform to revisit some of the previously described interactions in a cleaner environment and possibly identifying new binding partners. It is also a powerful technique for studying membrane mechanics, as it was previously used to demonstrate the role of caveolae in mechanoprotection.
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References
Martell JD, Deerinck TJ, Lam SS et al (2017) Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. Nat Protoc 12:1792–1816
Belkin M, Hardy WG (1961) Relation between water permeability and integrity of sulfhydryl groups in malignant and normal cells. J Biophys Biochem Cytol 9:733–745
Baumgart T, Hammond AT, Sengupta P et al (2007) Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc Natl Acad Sci U S A 104:3165–3170
Levental I, Byfield FJ, Chowdhury P et al (2009) Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles. Biochem J 424:163–167
Kaiser HJ, Lingwood D, Levental I et al (2009) Order of lipid phases in model and plasma membranes. Proc Natl Acad Sci U S A 106:16645–16650
Veatch SL, Cicuta P, Sengupta P et al (2008) Critical fluctuations in plasma membrane vesicles. ACS Chem Biol 3:287–293
Levental KR, Levental I (2015) Giant plasma membrane vesicles: models for understanding membrane organization. Curr Top Membr 75:25–57
Levental I, Lingwood D, Grzybek M et al (2010) Palmitoylation regulates raft affinity for the majority of integral raft proteins. Proc Natl Acad Sci U S A 107:22050–22054
Levental I, Grzybek M, Simons K (2009) Raft domains of variable properties and compositions in plasma membrane vesicles. Proc Natl Acad Sci U S A 108:11411–11416
Sezgin E, Kaiser HJ, Levental I (2012) Elucidating membrane structure and protein behaviour using giant plasma membrane vesicles. Nat Protoc 7:1042–1051
Parton RG, Hanzal-Bayer M, Hancock JF (2006) Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci 119:787–796
Galbiati F, Razani B, Lisanti MP (2001) Caveolae and caveolin-3 in muscular dystrophy. Cell 106:403–411
Razani B, Lisanti MP (2001) Caveolins and caveolae: molecular and functional relationships. Exp Cell Res 271:36–44
Sinha B, Koster D, Ruez R et al (2011) Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144:402–413
Dewulf M, Koster DV, Sinha B et al (2019) Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells. Nat Commun 10:1974
Fridolfsson HN, Roth DM, Insel PA et al (2014) Regulation of intracellular signaling and function by caveolin. FASEB J 28:3823–3831
Lamaze C, Tardif N, Dewulf M et al (2017) The caveolae dress code: structure and signalling. Curr Opin Cell Biol 47:117–125
Collins BM, Davis MJ, Hancock JF et al (2012) Structure-based reassessment of the caveolin signaling model: do caveolae regulate signaling through caveolin-protein interactions? Dev Cell 23:11–20
Acknowledgments
The authors would like to thank Christophe Lamaze and Patricia Bassereau and all the people from the Membrane mechanics and dynamics of intracellular signaling laboratory and Membranes and Cellular Functions laboratory. The facilities as well as scientific and technical assistance from staff in the PICT-IBiSA/Nikon Imaging Centre at Institut Curie-CNRS and the France-BioImaging infrastructure (No. ANR-10-INSB-04) are acknowledged. This work was supported by institutional grants from the Curie Institute, INSERM, and CNRS, and by specific grants from Association Française contre les Myopathies (AFM): 22337 (to J.P.) and CAV-STRESS-MUS (14266 to C.M.B.). J.P. was funded by Polish Ministry of Science and Higher Education Mobility Plus program (1668/MOB/V/2017/0) and Labex CelTisPhyBio. The Lamaze team, the PICT-IBiSA/Nikon Imaging Centre at Institut Curie-CNRS, and the France-BioImaging infrastructure are members of Labex CelTisPhyBio (No. ANR-10-LBX-0038) and of IDEX PSL (No. ANR-10-IDEX-0001-02 PSL).
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Podkalicka, J., Blouin, C.M. (2020). GPMVs as a Tool to Study Caveolin-Interacting Partners. In: Blouin, C. (eds) Caveolae. Methods in Molecular Biology, vol 2169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0732-9_8
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DOI: https://doi.org/10.1007/978-1-0716-0732-9_8
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