Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.

Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 skeletal myoblasts in culture. Using differentiated C2C12 skeletal myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, alpha-sarcoglycan, and beta-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.

beta-Scruin, a homologue of the actin crosslinking protein scruin, is localized to the acrosomal vesicle of Limulus sperm.

Scruin (alpha-scruin) is an actin bundling protein found in the acrosomal process of Limulus polyhemus sperm. We have cloned and sequenced a second scruin isoform from Limulus, beta-scruin, that is 67% identical to alpha-scruin. Northern and Southern analyses confirm that beta-scruin and alpha-scruin are encoded by distinct genes. The sequence of beta-scruin, like alpha-scruin, is organized into N- and C-terminal superbarrel domains that are characterized by a six-fold repeat of a 50 residue motif. Western analysis using rabbit polyclonal antisera specific for alpha- and beta-scruin indicate that beta-scruin, like alpha-scruin, is found in Limulus sperm but not blood or muscle. Both immunofluorescence microscopy and immunogold-EM localize beta-scruin within the acrosomal vesicle at the anterior of sperm but not in the acrosomal process. The function of beta-scruin in this membrane-bounded compartment that is devoid of actin is unknown. However, the location of beta-scruin together with the fact that it contains two putative beta-superbarrel structural folds, which are known to be catalytic domains in a number of proteins, suggests it may have a possible enzymatic role.

Sequential expression and differential localization of I-, L-, and T-fimbrin during differentiation of the mouse intestine and yolk sac.

During the differentiation of the intestine epithelium, three cytoskeletal proteins, villin, fimbrin, and myosin I, are sequentially expressed and localized to the apical membrane. Recently, we found that in the adult mouse and human, three fimbrin isoforms are expressed in a cell specific manner. I-fimbrin is expressed by intestine and kidney epithelial cells, L-fimbrin is expressed by leukocytes and many tumors, while T-fimbrin is expressed by various cells and tissues. Because non-intestinal isoforms of fimbrin could be expressed early in development, the expression of fimbrin isoforms during days 10.5 to 16.5 of intestine development was investigated. By immunofluorescence microscopy, T-fimbrin was detected in the early stages of intestinal epithelial cell differentiation until day 14.5 and was localized predominantly at the apical surface. L-fimbrin was also detected during this period but it was localized to the basal surface of the epithelium instead of the apical surface. By day 16.5 no L or T-fimbrin was detected in the epithelium. I-fimbrin was first detected at day 14.5 and a brush border-like apical localization pattern was seen by day 16.5. Unlike the intestinal cells, the visceral endoderm expressed I, L, and T-fimbrin throughout the period examined, with the level of I-fimbrin increasing as time progresses. L-fimbrin was more evident at the earlier stage than at the later stage of the development. Collectively, these results suggest that three fimbrin isoforms play different roles during epithelial cell differentiation. T- and I-fimbrin expression could be critical for the formation and extension of the microvilli whereas L-fimbrin may play a role in controlling cell adhesion.

Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages.

The actin-bundling protein fimbrin is homologous to 1-plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069-1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton-X-100 (soluble fraction), Tween 40-deoxy-cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone-extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC-extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic 32PO4 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins.

Expression and localization of villin, fimbrin, and myosin I in differentiating mouse F9 teratocarcinoma cells.

F9 embryonic carcinoma cells are a multipotent cell line which can be induced to differentiate into cells resembling the visceral endoderm, an extraembryonic absorptive epithelium characterized by apical microvilli. We have examined the role of villin, fimbrin, and myosin I, the major actin-binding proteins in the intestinal and visceral yolk sac microvilli, in the development of epithelial polarity and the assembly of the microvillus cytoskeleton in differentiating F9 cells. By immunoblot analysis villin was first detected at 4 days of differentiation. Confocal microscopy localized villin at Day 4 to the apical surface and by Day 6 to the basolateral surfaces as well. In comparison, fimbrin and myosin I were both present in undifferentiated F9 cells and became associated with the apical surface after villin during differentiation to visceral endoderm. The accumulation of villin, fimbrin, and myosin I at the apical surface in differentiating F9 cells correlated with the appearance of microvilli containing organized actin filament bundles. Two mouse villin cDNAs were isolated and characterized to examine villin expression during F9 differentiation. Mouse villin was encoded by two transcripts (3.8 and 3.4 kb) which differ in their 3'-noncoding region. Both villin mRNAs were first detected by Day 4 of differentiation and their appearance coincided with expression of the visceral endoderm marker alpha-fetoprotein. The pattern of expression and order of accumulation of villin, fimbrin, and myosin I in differentiating F9 cells are common to developing gut and yolk sac epithelium. This suggests that microvillus assembly is directed by a sequence of temporally and spatially regulated localizations of these actin-binding proteins.

Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium.

The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8.5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2-3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2-3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.