Membrane structure of caveolae and isolated caveolin-rich vesicles

Abstract

Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80-120 nm with an open porus of 30-50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30-50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles.

Cellular spelunking: exploring adipocyte caveolae

“…As in our previous Western blotting experiments (34), we see no mass spectrometrybased evidence for the caveolae localization of adipocyte proteins involved in insulin's actions, namely the insulin receptor and one of its downstream targets, Glut4. Indeed, the protein composition of adipocyte caveolae obtained rapidly and in reasonable yield is quite simple and in agreement with a previous freeze-fracture study of cultured adipocytes that concluded that there were relatively few proteins in them (42). However, we cannot rule out the possibility that some caveolae-localized proteins escaped our scrutiny and/or that the caveolae population is heterogeneous, as has been suggested (43).…”

Immunopurification and Characterization of Rat Adipocyte Caveolae Suggest Their Dissociation from Insulin Signaling

“…We find caveolae purified by this method to be devoid of GLUT4, the insulin receptor, and flotillin. Indeed, and in agreement with previous freeze fracture studies (28), adipocyte caveolae have a relatively limited protein composition. In addition to the caveolins only two major protein components of caveolae were identified: (a) the semicarbazide-sensitive amine oxidase (SSAO), a very abundant adipocyte protein (29) of unknown physiological significance (30); and (b) the scavenger receptor CD36, which plays an important role in mammalian fatty acid/lipid metabolism (31,32).…”

“…4). The range of vesicle diameter, 30 -130 nm, is broader than observed for native caveolae (70 -80 nm) but corresponds well to that observed for isolated caveolae obtained by coating with cationized silica and/or Triton X-100 extraction (25,26,28).…”

“…Analysis by electron microscopy of the structures recovered from 7C8 beads showed them to be vesicular (Fig. 4) and identical to caveolae isolated by other investigators using independent methodology (28). These structures have a thickening of the membrane which may correspond to caveolae coat, although the characteristic striations of caveolae which can be revealed by rapid freeze etching studies (55) were absent.…”

Membrane lipid domains and membrane lipid domain preparations: are they the same thing?

“…Caveolins (at least caveolin-1 and caveolin-3) are necessary for the formation of caveolae (as derived from experiments with knockout mice) (122,123). These and other evidence (124,125) suggest that caveolin-1 is an essential component of caveolae, even if its presence on the cytoplasmic face of caveolae was somehow challenged by theˆnding that caveolin-1 is rather present in intramembrane particles interacting with caveolae surface (126). Caveolin-1 is also found at many other intracellular locations, including the trans-Golgi network and in other organelles (127,128), and caveolin-1 antibodies also bind to ‰at membrane portions (129,130).…”