Caveolin-3 associates with developing T-tubules during muscle differentiation

Abstract

Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the alpha 1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3-labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.

Figure 1

Immunolocalization of caveolin-1 and caveolin-3 in adult mouse skeletal muscle. 0.5 μm frozen sections of mouse skeletal muscle (A– D) were labeled with antibodies to caveolin-3 (A) or caveolin-1 (C). B and D show the corresponding phase images for A and C, respectively. (A) Caveolin-3 labels the periphery of the fiber with negligible internal staining. Note the regular meshwork of labeled elements at the ends of the muscle fiber. (B) In contrast to caveolin-3, antibodies to caveolin-1 specifically label the endothelial cells of muscle capillaries rather than the muscle fibers. Bars, 5 μm.

Figure 2

Immunoelectron microscopic localization of caveolin-3 in adult mouse skeletal muscle. Ultrathin frozen sections of mouse skeletal muscle were labeled with antibodies to caveolin-3. Specific labeling is associated with sarcolemmal caveolae (small arrowheads indicate the sarcolemmal region; large arrowheads indicate labeled caveolae), as shown at higher magnification in the inset. An endothelial cell (e) in B is unlabeled. Double arrowheads indicate regions of the T-tubule system which generally show negligible labeling. m, mitochondria; sl, sarcolemma; n, nucleus; z, Z-line. Bars: (A and B) 200 nm; (inset) 100 nm.

Figure 3

Immunoelectron microscopic localization of caveolin-3 and caveolin-1 in adult cardiac tissue. Ultrathin frozen sections of mouse atrium were labeled with antibodies to caveolin-3 and caveolin-1. Small gold in A represents labeling for caveolin-3 and in B, indicates labeling for caveolin-1. Caveolin-1 labeling (large arrows) is only detectable on caveolae of endothelial cells (e), whereas labeling for caveolin-3 is only evident within the cardiac muscle cells, showing the specificity of the two antibodies. In each case, labeling is associated with uncoated plasma membrane invaginations with the characteristics of caveolae. The arrowhead in A indicates an unlabeled clathrin-coated pit. p, plasma membrane. Bars, 100 nm.

Figure 4

Immunolocalization of caveolin-3 during muscle development. 0.5 μm frozen sections of mouse skeletal muscle were fixed by immersion at various stages of embryonic development (embryonic day 16, A; embryonic day 18, B), immediately after birth (C) or 3 d after birth (D). All sections were labeled at the same time, and images were prepared with the same exposure and development times. Specific labeling is associated with the periphery of the muscle cells at all stages. In addition, internal labeling is evident from embryonic day 16 up to birth. Labeling is often evident as punctate dots of labeling aligned along the longitudinal axis of the muscle cells (A, arrows). In the embryonic day 18, muscle tubular structures, which apparently lead from the sarcolemma, are labeled (B, arrows). In the newborn muscle and especially 3 d after birth, the internal labeling is decreased. Bars, 5 μm.

Figure 5

Immunolocalization of caveolin-3 in C2C12 cells. C2C12 cells were maintained in culture in differentiating medium for 6–8 d after reaching confluency. Cells were fixed with paraformaldehyde and processed for immunofluorescent localization of caveolin-3. Cells were viewed by confocal microscopy. A and B show two sections of two C2C12 myotubes at different planes through the cell: (A close to the base of the cell; B midway through the cell). Tubules and reticular structures (inset) run throughout the entire depth of the cell and are mainly orientated in the longitudinal direction. Specific labeling is associated with the differentiated C2C12 cells, whereas the underlying layer of undifferentiated cells (in the plane of the section shown in A) show low labeling. Bars, 5 μm.

Figure 12

Immunolocalization of epitope-tagged caveolin-3 and endogenous caveolin-1 in C2C12 cells. C2C12 cells expressing caveolin-3 with a COOH-terminal HA tag were fixed after 1 d (A and B), 2 d (C and D), or after 6–8 d, 4–6 d after the cells reached confluency and differentiation medium was added), (E–H). The cells were then labeled with antibodies to caveolin-1 (A, C, and G) or to the HA tag (B, D, E, F, and H). A–D and G and H show cells double labeled for caveolin-1 and the epitope-tagged caveolin-3. In day 1 myoblasts (A and B), the endogenous caveolin-1 and expressed caveolin-3 colocalize (arrows). At later times (C and D), some cells clearly show a different labeling pattern for the two caveolin proteins (arrows 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 (E, F, and inset, arrows). G and H show cells double labeled for caveolin-1 and epitope-tagged caveolin3. While caveolin-1 labeling is present in neighboring, undifferentiated myoblasts, labeling is very low in the multinucleate myotube (arrows). Weak staining represented background labeling as shown by peptide inhibition (not shown). n, nucleus. Bars: (A–D) 2 μm (same magnification); (E–H) 5 μm (E and F, G and H, same magnifications).

Figure 6

Caveolin-3 colocalizes with a T-tubule marker in C2C12 cells. C2C12 cells were cultured as described in the legend to Fig. 5. Cells were fixed with paraformaldehyde and double labeled for caveolin-3 (rhodamine, A, C, E, and G) and for the T-tubule specific marker α1DHPR (FITC, B, D, F, and H). Caveolin-3 colocalizes with α1-DHPR in many tubular/reticular structures throughout the cell (arrows). Note that not all caveolin-3 positive structures are α1DHPR positive (arrowheads). Note the variation in the labeling for α1-DHPR (for example see the relatively low level of labeling of the cell in F) but the clear colocalization with caveolin-3. Bars: (A–D) 5 μm; (E and F) 2.5 μm.

Figure 7

Immunolocalization of caveolin-3 during differentiation of primary muscle cells in culture. Mouse muscle cells from embryonic day 18 were cultured for various periods at 37°C before fixation and labeling for caveolin-3, as described in Materials and Methods. Cells were fixed 1 day after plating (A) or at various times after adding differentiation medium; 1 d (B and C), 5 d (D), 11 d (E and F), and 24 d (G). Cells were viewed by confocal (A–E and G) or conventional microscopy (F). Specific labeling is associated with the perinuclear and peripheral regions of day 0 myoblasts (A–C). From day 5 onwards, labeling is apparent within the putative T-tubule reticulum of fused myotubes (e.g., compare F with Fig. 12 F). At later stages, an increasing number of cells show surface labeling but low intracellular labeling (G). n, nuclei. Bars: (A–C) 5 μm; (D– G) 10 μm.

Figure 8

Immunoelectron microscopic localization of caveolin-3 in C2C12 cells. C2C12 cells were cultured as described in the legend to Fig. 5 and then fixed with a glutaraldehyde-containing fixative. The cells were then processed and immunolabeled for caveolin-3 followed by protein A–gold. Note the specific labeling (gold particles indicated by arrowheads) of extensive regular interconnected arrays of labeled membranes (asterisks). These structures are made up of unit structures with similar dimensions to caveolae (asterisks mark units of a reticulum in the inset). In the less compact arrays, (shown at higher magnification in B), the labeling is stronger, apparently due to greater access to caveolin-3 epitopes, and the individual caveolae-like elements are clearly evident (arrows). Note the similarity of these structures to caveolae clusters of nonmuscle cells. Bars, 200 nm.

Figure 9

Immunoelectron microscopic localization of caveolin-3 in C2C12 cells. C2C12 cells were cultured and processed for frozen sectioning as described in the legend to Fig. 8, except that the cells were fixed in paraformaldehyde. Sections were immunolabeled with affinity-purified antibodies to caveolin-3. Specific immunolabeling for caveolin-3 is associated with 50–60 nm budding profiles with characteristic caveolar morphology (arrowheads). The caveolin-3 labeled elements form complex, extended arrays which penetrate the center of the muscle cell. The complex, clustered arrangement of these structures, as viewed in thin sections, suggests that in three dimensions, the caveolae form large clusters resembling “bunches of grapes.” In some regions, reticular elements are evident (Fig. 4 B, asterisk) which resemble forming T-tubules, as described in early morphological studies (Ishikawa, 1968), but are less compact and regular than those seen in glutaraldehyde-fixed cells. Note that the caveolin-3 labeling is typically associated with the bud-like profiles rather than the tubular interconnecting regions. The double arrowhead indicates a clathrin-coated pit. Bars, 200 nm.

Figure 10

Cholera toxin peroxidase labeling of differentiating C2C12 cells. C2C12 cells cultured as described in the legend to Fig. 5 were incubated with CT-B-peroxidase for 2 h at 4°C and then fixed and processed for embedding in epon. Semi-thick sections (∼200 nm) were cut parallel to the substratum. Peroxidase-labeled elements are seen within the depths of the cell (arrows). The labeled structures are composed of individual bud-like elements of ∼60 nm diam, which form chains or reticula of interconnected structures (insets). Bars, 200 nm.

Figure 11

Immunoelectron microscopic localization of caveolin-3 in cholera toxin surface-labeled C2C12 cells. C2C12 cells cultured as described in the legend to Fig. 5 were incubated with CT-B for 2 h at 4°C before fixation with paraformaldehyde. Ultrathin frozen sections were immunolabeled with antibodies to cholera toxin, followed by 15 nm protein A–gold (arrowheads) and to caveolin-3 detected with 10 nm protein A–gold. Cholera toxin labeling is evident on the cell surface and in the extensive caveolin-3–positive tubulovesicular elements. pm, plasma membrane. Bars, 200 nm.