In striated muscle, the specialized linkages between the subsarcolemmal cytoskeleton and the extracellular matrix are crucial to the transduction of contractile force, the organization of myofibrils, and the integrity of the membrane during contraction. Despite the importance of these connections, the molecular mechanisms that regulate membrane anchorage complexes remain poorly defined. In this study, we have identified CAP as an FLNc-associated protein that participates in linking the cytoskeleton to the sarcolemma in muscle cells.
Costameres were originally described as electron-dense plaques rich in the focal adhesion protein vinculin (Koteliansky and Gneushev, 1983
; Pardo et al., 1983a
; Shear and Bloch, 1985
; Bloch et al., 2002
). Indeed, these structures share many of the features of focal adhesions, and they are considered a striated muscle-specific elaboration of focal adhesions (Ervasti, 2003
; Samarel, 2005
). Our previous studies identified CAP as a component of the cell–ECM adhesion complex in fibroblasts. CAP interacts with both paxillin and vinculin, and the latter is crucial in anchoring CAP at adhesion sites (Zhang et al., 2006
). CAP was also reported to localize to cell–cell junctions in epithelial cells (Mandai et al., 1999
). These findings prompted us to explore whether CAP is also localized at membrane anchorage structures in muscle cells, including costameres, myotendinous junctions (MTJs), and intercalated discs.
During myoblast differentiation, CAP localization changes from focal adhesions to pre- and nascent costameres. Similar observations have been reported from studies of ponsin (another name of one CAP isoform) in human skeletal muscle cells (Gehmlich et al., 2007
). In a recent study using an in vitro myoblast differentiation model, several focal adhesion components, including paxillin, vinculin, focal adhesion kinase (FAK), and integrin, were found to redistribute to costameres upon muscle differentiation (Quach and Rando, 2006
). In addition, focal adhesion signaling through FAK is essential for costamerogenesis and myofibrillogenesis. The similar redistribution of CAP, together with the significant up-regulation of the CAP protein, suggests a potential role for CAP in the formation of costameres during myogenesis. Moreover, the increased expression of certain isoforms of CAP suggests different isoforms may have specific functions in mature muscle cells that have yet to be defined.
Using two independent approaches, we identified a novel interaction between CAP and the muscle-specific filamin FLNc. This interaction is mediated through the direct binding of the N-terminal part of CAP to the second repeat of FLNc. The N-terminal domain of FLNc binds to actin and the last repeat at the C terminus mediates its homodimerization. FLNc dimers cross-link F-actin to form parallel bundles or orthogonal networks. Our previous studies revealed that CAP binds to F-actin in an in vitro cosedimentation assay (Zhang et al., 2006
). Therefore, we propose the existence of a CAP–FLNc–actin tertiary structure, where CAP helps to cross-link and stabilize the F-actin network. Indeed, cooverexpression of CAP and FLNc induced the formation of an intensive network of actin fibers, as shown in A.
Filamins play an essential role in the modulation of cell shape and motility, and loss of function mutations in FLNa produce defects in neuronal migration (Fox et al., 1998
). Although overlapping functions are suggested among filamin family members, few studies have examined the role of FLNc in the regulation of cell morphology and motility. FLNc has been suggested to be involved in migfillin-mediated cell shape regulation (Tu et al., 2003
). Here, we show that overexpression of FLNc enhances cell spreading on fibronectin and that this function of FLNc is inhibited by CAP. Our recent study showed that CAP negatively regulates cell spreading via stabilizing cell–ECM adhesion structures (Zhang et al., 2006
). We demonstrate here that CAP causes an accumulation of FLNc at adhesion sites, which may restrict the availability of FLNc at the periphery to dynamically regulate actin structures required for cell motility. The exact signaling pathway involved in the coordination between CAP and FLNc in modulating adhesion-mediated cytoskeleton rearrangement requires further study.
We propose a model in which CAP operates as a bifunctional adapter protein that binds to both filamin via N-terminal sequences, and cell adhesion proteins such as vinculin and paxillin via its C-terminal SH3 domains. Therefore, CAP may be able to reorganize the actin cytoskeleton by bringing filamin cross-linked actin to cell–ECM adhesion sites. In muscle cells, costameres contain two separate but synergistic cell—ECM-interacting complexes: the DGC and the vinculin–talin–integrin anchorage system (Anastasi et al., 2003
). FLNc was initially identified as a binding partner of δ- and γ-sarcoglycans in the DGC complex (Thompson et al., 2000
). In mdx
mice, where the dystroglycan-associated protein dystrophin is deleted, the whole DGC complex is destabilized and degraded (Ervasti and Campbell, 1991
; Ohlendieck and Campbell, 1991
). Interestingly, membrane associated FLNc is greatly increased in mdx
muscles despite an 80% decrease of sarcoglycans, suggesting another interaction/signal that regulates the localization of FLNc on the membrane.
Identification of the interaction between CAP and FLNc could potentially function as this second link of FLNc to the plasma membrane. CAP is a component of the integrin–focal adhesion complex through its binding to vinculin. Our data demonstrate that FLNc is recruited to cell–ECM adhesions by overexpression of CAP. Moreover, membrane staining of CAP is significantly increased in mdx
muscles, suggesting that CAP may be responsible for the elevated FLNc on the membrane. Previous studies have shown an increase of α7β1 integrin in DMD patients and mdx
mice, and overexpression of α7β1 integrin may compensate for the absence of the DGC complex and reduce the development of severe muscle disease in transgenic mice (Hodges et al., 1997
; Burkin et al., 2001
). By recruiting FLNc to the integrin–vinculin complex, CAP may function as an additional link that brings the myofibril cytoskeleton to the sarcolemma.
Another interesting finding from this study is that CAP is highly expressed in oxidative muscle fibers. Skeletal fibers are generally classified into type I and type II species that display marked differences in contraction, endurance, and metabolism. Type I fibers are rich in mitochondria that provide a slow but stable and long-lasting supply of ATP via oxidative metabolism. While type IIb fibers have the lowest content of mitochondria, these muscles have high levels of glycolytic enzymes, providing a rapid source of ATP independent of oxygen; therefore, they are more susceptible to fatigue. The metabolic and contractile properties of type IIa and IIx fibers lie in between. The expression of the CAP gene is transcriptionally induced by peroxisome proliferator-activated receptor (PPARγ) activators (Ribon et al., 1998a
; Baumann et al., 2000a
). Interestingly, both PPARγ and its coactivator PGC-1α induce a type I gene expression profile and a transition of muscle fibers from type IIb to type IIa and type I in transgenic mice (Lin et al., 2002
; Wang et al., 2004
). Moreover, PGC-1β drives the formation of oxidative IIx fibers (Arany et al., 2007
). Therefore, the transcriptional regulation by PPARγ may account for the enriched expression of CAP in type I and IIa oxidative fibers, consistent with a role for CAP in the regulation of insulin-mediated glucose metabolism (Baumann et al., 2000b
; Lesniewski et al., 2007
). Interestingly, fiber type analyses in mdx
mice have shown that the diaphragm muscle in these mice responds to progressive degeneration with a transition to a slower twitch phenotype. By 24 mo, there was a sevenfold increase in the slow twitch type I fiber and the type IIb/x fibers were almost gone in the mdx
diaphragm. These changes were associated with reduced power output and marked increase in muscle endurance, both of which help to preserve the contractility and survival of the muscles (Petrof et al., 1993
). Therefore, the enrichment of CAP in oxidative muscles and the increased expression and membrane localization in mdx
muscles suggest that CAP may provide a link between the regulation of muscle structural integrity and metabolism that together contribute to the endurance and strength of skeletal muscle.
In summary, the identification of the interaction between CAP and filamin C furthers our understanding of the composition and regulation of membrane linkage complexes in skeletal muscle. Increased expression of CAP during myotube formation and its function in the regulation of FLNc localization and actin rearrangement suggest a dynamic role of CAP in the formation and maintenance of muscle structural integrity under normal and disease conditions. Further studies on the potential involvement of CAP in various myopathies may help us to better understand the underlying molecular mechanisms and contribute to novel approaches for treatment.