Both caveolae and T-tubules represent membrane systems which are continuous with the plasma membrane but have a distinct composition
Both caveolae and T-tubules represent membrane systems which are continuous with the plasma membrane but have a distinct composition. in the early development of the T-tubule system in muscle. The plasma membrane of mammalian cells is divided into a number of different structural and functional microdomains. Much recent interest has been focused on one such domain, the caveola, a surface invagination with unique morphology which is readily identifiable by electron microscopy (Parton, 1996). Caveolae are extremely abundant in endothelial cells, adipocytes, and smooth muscle cells. In endothelia, caveolae appear to play a major role in transport across the endothelial monolayer (Ghitescu et al., 1986; Schnitzer et al., 1994). Other work has suggested a role for caveolae in signal transduction (Lisanti et al., 1994), in specialized endocytic uptake pathways (Anderson, 1993), and in calcium homeostasis (Fujimoto, 1993; Fujimoto et al., 1992). The diversity of the proposed functions of caveolae raises the question of whether they have a single function, as does a clathrin-coated pit, or whether they are structural units used for many different purposes. Caveolae are enriched in cholesterol (Montesano et al., 1982; Rothberg et al., 1990) and in glycosphingolipids (Parton, 1994), and increasing evidence suggests that caveolae are built up around sphingolipidCcholesterol rafts (Simons and Ikonen, 1996). The plasma membrane of all mammalian cells appears to contain such rafts which, upon detergent treatment, can be isolated as insoluble glycosphingolipid-enriched complexes (DIGs;1 Parton and Simons, 1995). DIGs and caveolae share many features, but caveolae appear to be more restricted in distribution, being undetectable in some cell types (Fra et al., 1994; Gorodinsky and Harris, 1995). Caveolin-1, the major protein of caveolae in mammalian cells (Kurzchalia et al., 1994; Parton, 1996), is a 21-kD IKK epsilon-IN-1 integral membrane protein which has been shown to bind cholesterol and to interact with glycosphingolipids (Fra et al., 1995(Eggenheim, Germany). C2C12 cells were cultured as described previously (Way and Parton, 1995). Transfection was carried out using Lipofectin (and and and show the corresponding phase images for and indicate the sarcolemmal region; indicate labeled caveolae), as shown at higher magnification in the inset. An endothelial cell (is unlabeled. Double arrowheads indicate regions of the T-tubule system which generally show negligible labeling. and represents labeling for caveolin-3 and in indicates an unlabeled clathrin-coated pit. and show two sections of IKK epsilon-IN-1 two C2C12 myotubes at different planes through the cell: (close to the base of the cell; midway IKK epsilon-IN-1 through the cell). Tubules and reticular structures (and and and and show cells double labeled for caveolin-1 and the epitope-tagged caveolin-3. In day 1 myoblasts (and and indicate comparable regions of the two cells which are labeled for caveolin-3 but not caveolin-1). After fusion of myoblasts to form myotubes, the epitope-tagged caveolin-3 is present within the T-tubule system which runs thoughout the cell (and show cells double labeled for caveolin-1 and epitope-tagged caveolin3. While caveolin-1 labeling is present in neighboring, undifferentiated myoblasts, IKK epsilon-IN-1 labeling is very low IKK epsilon-IN-1 in the multinucleate myotube (and and and and and and and with Fig. ?Fig.1212 mark units of a reticulum in the and and em D /em ), with caveolin-1 generally showing a more diffuse labeling pattern. In fused C2C12-CAV3HA cells epitope-tagged caveolin-3 was localized to the T-tubule system running throughout the cytoplasm (Fig. ?(Fig.12,12, em E /em , em F /em , and em H /em ). In contrast, caveolin-1 showed negligible labeling in the differentiated cells (Fig. ?(Fig.1212 em G /em ), suggesting that the expression of caveolin-1 is reduced upon muscle differentiation. It therefore appears that caveolin-3 expressed MYCN in C2C12 cells colocalizes with caveolin-1 in the nondifferentiated state, but, as the cells differentiate, the two markers are separated. Caveolin-1 is sorted away from caveolin-3, which eventually associates with.