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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00806

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A caveolin-3 mutant that causes limb girdle muscular dystrophy type 1C disrupts Src localization and activity and induces apoptosis in skeletal myotubes

¹ Department of Neurology and Neurological Science, Stanford University School of Medicine, Stanford, California, 94305-5235, USA
² Neurology Service and GRECC, Veterans Affairs Palo Alto Health Care System, Palo Alto, California, 94304, USA

View larger version (8K): [in a new window]   Fig. 1. Schematic of caveolin-3 protein. This protein has a caveolin signature sequence (conserved in all caveolin proteins), a scaffolding domain known to bind various signaling proteins and a hydrophobic intramembrane domain. The bracket within the scaffolding domain indicates the site of the TFT mutation known to cause LGMD-1C.

View larger version (69K): [in a new window]   Fig. 2. Localization of wild-type and mutant caveolin-3 protein in myoblasts and myotubes. (A) Myoblasts were stably transfected with wild-type caveolin-3 (WT) or with the TFT mutant caveolin-3 (TFT). Caveolin-3 localization was assessed immunocytochemically in myoblasts or in myotubes. In myoblasts, the TFT mutant protein was exclusively perinuclear, whereas wild-type caveolin-3 was also membrane-associated and cytosolic. In control myotubes (derived from myoblasts transfected with vector alone), endogenous caveolin-3 was strongly expressed at the plasma membrane (arrows) with very limited intracellular and perinuclear expression. In contrast, in myotubes expressing the TFT mutation, caveolin-3 was most abundantly expressed in the perinuclear region (arrows), with only small amounts of protein present at the plasma membrane. (B) To test for any effects of the TFT mutation on the biochemical differentiation of myoblasts, cultures were analyzed by western blotting for the expression of the differentiation-specific protein, skeletal muscle slow myosin heavy chain (sMyHC). Control myoblasts (vector alone) and myoblasts expressing the TFT mutation were analyzed before differentiation with the cells maintained in GM, and on days 1, 2 and 3 of differentiation after the cells have been switched to DM. No differences in the induction of myosin expression were observed between the cell populations.

View larger version (31K): [in a new window]   Fig. 3. Normal localization and expression patterns of caveolin-1 in the presence of the TFT mutant caveolin-3. (A) Myoblasts stably expressing the TFT mutation or vector alone (Control) were analyzed for localization of caveolin-1. Caveolin-1 localized almost exclusively to the plasma membrane and no differences between cell populations were observed. (B) The level of caveolin-1 and caveolin-3 expression was assessed by western blot analysis in myoblasts maintained in GM or undergoing differentiation in DM. Expression was analyzed 1, 2 and 3 days after the initiation of differentiation. In control cells and in cells expressing the TFT mutation, caveolin-1 was highly expressed in myoblasts and declined during myogenic differentiation. Expression of caveolin-3 increased with differentiation in control cells but was expressed at high levels throughout differentiation in cells constitutively expressing the TFT mutation. Actin was used as the loading control in all experiments.

View larger version (33K): [in a new window]   Fig. 4. Diminished incorporation of caveolin-3 into lipid rafts in myotubes expressing the TFT mutant caveolin-3. (A) Extracts of myotubes expressing vector alone (Control) or the TFT mutation were subjected to sucrose density gradient fractionation. 50 µl samples from each fraction (1-12) were analyzed by western blotting for expression of caveolin-3. In control myotubes, caveolin-3 was detected exclusively in the lipid raft (LR) fraction. In myotubes expressing the mutant caveolin-3, most of the caveolin-3 (endogenous plus mutant) was instead found in the non-lipid raft (NLR) fraction with only a small proportion present in the LR fraction. (B) Extracts of myoblasts expressing wild-type caveolin-3 or the TFT mutation were subjected to sucrose density gradient fractionation and analyzed for caveolin-3 expression as in A. Wild-type caveolin-3 preferentially localized to the LR fractions, while mutant protein was completely excluded from lipid rafts.

View larger version (29K): [in a new window]   Fig. 5. Reduced albumin uptake in myotubes expressing the TFT mutant caveolin-3. (A) Control myotubes and myotubes expressing the TFT mutation were incubated with Texas Red-conjugated albumin (20 µg/ml) for 30 minutes and then analyzed by fluorescence microscopy for cellular uptake of Texas Red. In control myotubes, Texas Red was observed in foci throughout the cytosol (arrows) and surrounding the nucleus (nuc). In contrast, only occasional Texas Red-positive foci were observed in the cytosol of myotubes expressing the caveolin-3 mutant. (B) Control myotubes and myotubes expressing the TFT mutation were incubated with Texas Red-conjugated albumin as described above, and total cell lysates were analyzed by western blotting using an antibody against Texas Red. The levels of Texas Red were substantially lower in myotubes expressing mutant caveolin-3 compared with control myotubes. Actin was used as the loading control.

View larger version (32K): [in a new window]   Fig. 6. Effects of the TFT caveolin-3 mutation on caveolin-3 binding to Src and targeting of Src to lipid rafts. (A) Equal amounts of protein from control myotubes and myotubes expressing the TFT mutation were used for immunoprecipitation of Src. The total immunoprecipitated samples were then analyzed by western blotting for levels of Src and caveolin-3. Even though the total levels of Src were similar in both cell populations, there was a decrease in associated caveolin-3 in myotubes expressing the TFT mutation compared to control myotubes. (B) In order to test for the effects of the TFT mutation on the targeting of Src to lipid rafts, extracts from control myotubes or myotubes expressing the TFT mutation were subjected to sucrose density gradient fractionation (SDGF) as in Fig. 4. For ease of analysis, fractions 4 and 5 were pooled from each gradient and referred to as the lipid raft (LR) fraction. An equal amount of protein (10 µg) from the LR fraction of each cell population was analyzed by western blotting for levels of Src and caveolin-3. Both Src and caveolin-3 levels were reduced in LR fractions from myotubes expressing the mutant caveolin-3.

View larger version (32K): [in a new window]   Fig. 7. Failed targeting of Src to the nucleus and altered Src activation in myotubes expressing the TFT mutant caveolin-3. (A) Control myotubes and myotubes expressing the TFT mutation were analyzed immunocytochemically for the cellular distribution of Src. While Src was present diffusely in the membrane and cytoplasm and concentrated in nuclei of control myotubes, it was not targeted to these cellular compartments but was instead retained in a perinuclear distribution in myotubes expressing the mutant caveolin-3. (B) The nuclear fraction from control myotubes and myotubes expressing the TFT mutation were analyzed by western blotting for expression of Src. There was clearly diminished Src targeting to the nuclei of mutant myotubes. Actin was used as the loading control. (C) Equal amounts of protein from total cell lysates of control myotubes and myotubes expressing the TFT mutation were analyzed by western blotting for phosphorylation of Src at tyrosine 418 (pSrcY418) and total Src. Hyperphosphorylation of Src at this residue was observed in myotubes expressing the TFT mutation compared with control myotubes, while total cellular Src did not differ between the two cell populations.

View larger version (11K): [in a new window]   Fig. 8. The TFT mutant caveolin-3 causes an increased incidence of apoptosis in myotubes. Control myotubes and myotubes expressing the TFT mutation were stained at day 1, day 3 and day 6 of differentiation using the TUNEL assay. The percentages of myotubes containing TUNEL-positive nuclei were determined. At least four non-overlapping fields were counted for each culture, and four separate cultures for each cell population were analyzed. At day 3 and day 6 of differentiation, there were significantly more apoptotic TFT-expressing myotubes than apoptotic control myotubes. Data are presented as mean±s.d. At each time point, one-way analysis of variance was used to compare the data for each mutant to the control data (*P<0.05, **P<0.005).

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