The development of myofibrils in differentiating muscle cells is one of the most impressive macromolecular assembly processes in nature. It involves the interaction of a great number of different proteins that has to be precisely controlled both in terms of space and time (Franzini-Armstrong and Fischman 1994
; Squire 1997
). Many of these proteins occur in multiple isoforms whose expression patterns are fundamentally different and are therefore thought to fulfill highly specialized roles (Pette and Staron 1997
). The filamins are an example for such a gene family that became evident only rather recently. In the chicken system, the existence of several distinct filamin isoforms was suspected already several years ago, mainly due to differentially reacting antibodies (Gomer and Lazarides 1981
, Gomer and Lazarides 1983
; Price et al. 1994
). Further insights into the differential functions of the avian isoforms has been hindered by the lack of primary sequence information, with only one isoform so far cloned (Barry et al. 1993
). Similar studies in mammalian systems relied for a long time on antibodies reacting with only a single polypeptide that on top could not be identified in striated muscles (Brown and Binder 1993
). Molecular genetic studies pointed at the existence of an additional isoform expressed in human skeletal muscle that was called ABP-L (Maestrini et al. 1993
). Its complete cDNA was cloned recently and the protein was renamed γ-filamin (Xie et al. 1998
). We have succeeded in obtaining an antibody that reacts with both filamin isoforms, which made it feasible to approach the question of the different roles of these proteins in muscle (van der Ven et al. 2000
). Here we used this mAb (called RR90) in combination with an antibody that we could show to be specific for α-filamin, with the idea that a subtraction of both antibody labels might yield firm statements about the distribution of the individual filamin isoforms. Thus, in adult striated muscle fibers, γ-filamin is expressed while nonmuscle α-filamin is limited to blood vessels and connective tissue cells (). This still leaves the interesting question unanswered: why, within the myofibers, γ-filamin shows a dual pattern of distribution? In accordance with cell fractionation studies (Thompson et al. 2000
), we found the majority of filamin in the myofibrils that correspond to the particulate sarcoplasmic fraction; conversely, a small fraction of γ-filamin was located at the sarcolemma (). We propose that this differential targeting, which at the same time implies distinct functions, could be the result of alternatively spliced γ-filamin variants. This idea is supported by a provisional genetic analysis that revealed short alternatively spliced exons for α-filamin (Maestrini et al. 1993
). Definitive proof, however, will require the establishment of the complete genomic structure of the FLNC
gene and the subsequent elucidation of peptide-specific antibodies. In line with this view, quite distinct staining patterns were observed in developing chicken muscle cells. On the one hand, filamin was first located in stress fiber–like structures, and then disappeared for several days, and finally reappeared at the periphery of Z-discs (Gomer and Lazarides 1981
). On the other hand, filamin was detected in the developing chicken heart only from the 14 somite stage onwards; i.e. at a time when functional myofibrils in the beating heart exist already for ~4 h (Price et al. 1994
). It is very likely that the antibodies used in these studies did not reflect the complete spectrum of filamin isoforms that can be expected to be expressed in avian tissues.
The subtractive antibody labeling approach was also used on developing skeletal muscle cells in culture. This clearly demonstrated not only that γ-filamin is expressed within a few hours subsequent to the induction of muscle differentiation (see also van der Ven et al. 2000
), but also that solely this isoform participates in the striking reorganization of the actin cytoskeleton during myofibril formation ().
The specific function of filamin in myofibrils cannot be solely attributed to its well-documented capacity to cross-link actin filaments, particularly since this region shows very little variability between the isoforms. Therefore, different functions must be determined in other, more diverse parts of the molecule. The region that mainly attracted our attention was the unique insertion of 78 amino acids located within Ig-like domain 20. We show here that this insertion, which previously was shown to be specifically expressed in differentiated muscle (Xie et al. 1998
; van der Ven et al. 2000
), is encoded by a single extra exon that is differentially spliced (). Transfection studies and yeast two-hybrid screenings were used to address the specific function of this molecular region. The transfection studies were aimed at elucidating its role for the subcellular targeting of filamin in muscle cells. Previous studies using either the α- or γ-filamin isoforms have emphasized their membrane association (see Introduction). Here, we identify for the first time a myofibrillar targeting signal within γ-filamin that is functional in both cardiac and skeletal muscle cells. Since in both cell types myofibrils were assembled normally in the presence of the overexpressed recombinant protein, it does not seem to exert a dominant negative effect on this process, at least at the time scale of our experiments. We also believe it is justified to assume that this signal must reside in the unique domain 20 insertion because α-filamin (which does not contain this insertion) does not show any specific localization upon overexpression in muscle cells.
The strict targeting of Ig-like domain 20 of γ-filamin urged us to search for a myofibrillar protein that might specifically interact with it. Yeast two-hybrid screens identified the recently discovered protein myotilin (Salmikangas et al. 1999
) as a binding partner for γ-filamin, and three independent experiments (coimmunoprecipitation experiments using recombinant fragments, coimmunoprecipitation from double-transfected yeast cells, and double transfection of nonmuscle cells) confirmed and further confined this interaction ( and ). Since myotilin was previously shown to bind α-actinin, we assume that the function of this Z-disc protein is the indirect anchorage of γ-filamin, which is localized at the periphery of sarcomeric Z-discs (Thompson et al. 2000
; van der Ven et al. 2000
), to α-actinin in the central Z-disc.
In contrast to the overexpression of the myotilin binding part of γ-filamin, the overexpression of the γ-filamin–binding region of myotilin had a dramatic effect on the assembly of myofibrils in differentiating C2C12 myotubes. This implies that the amino terminal part of myotilin that is lacking in this construct and is known to contain an α-actinin binding site (Salmikangas et al. 1999
) plays an important role in the control of myofibrillar assembly. Obviously, the interaction γ-filamin–myotilin–α-actinin is disturbed by overexpression of this truncated myotilin fragment that is still able to associate with γ-filamin. This regulatory role is further strengthened by the observation that overexpression of full-length myotilin does not result in abnormal myofibrillogenesis. Thus, it seems that the α-actinin–binding part of the myotilin molecule is indispensable for the proper function of the protein.
The identification of myotilin as a γ-filamin binding partner described in this report, together with earlier observations that γ-filamin binds γ- and δ-sarcoglycan (Thompson et al. 2000
) is particularly interesting with regard to recent findings on LGMD. Mutations in the genes encoding all three binding partners cause LGMD (Noguchi et al. 1995
; Nigro et al. 1996
; Hauser et al. 2000
). Furthermore, the FLNC
gene itself has been mapped to an LGMD candidate region on chromosome 7 (Speer et al. 1999
). Interestingly, caveolin-1 was recently identified as a binding partner of α-filamin (Stahlhut and van Deurs 2000
). Considering the high degree of similarity of the isoforms of both proteins, the muscle isoform of caveolin (caveolin-3, which in turn is a further LGMD gene), can be assumed to interact in a similar way with muscle filamin, although definitive proof is still lacking.
Until recently, this genetically heterogeneous disease has been attributed essentially to mutations of the genes encoding sarcolemmal proteins such as α-, β-, γ-, and δ-sarcoglycans, caveolin-3, and dysferlin (reviewed in Bushby 1999
; Toniolo and Minetti 1999
). In LGMD 2C and LGMD 2F, which are caused by mutations in the γ- and δ-sarcoglycan genes, respectively, the fraction of γ-filamin that is localized to the sarcolemma is highly increased. Considering these observations, the traditional role of filamins in the submembraneous cytoskeleton and the fact that the vast majority of other identified interactors of filamins until now are mainly membrane-associated proteins, an LGMD model was suggested that favors a cyclic translocation of γ-filamin from membrane attachment sites to intracellular locations. In LGMD 2C and 2F and other muscle diseases characterized by abnormalities in both sarcoglycans this cycling was supposed to be disturbed (Thompson et al. 2000
Our finding that the muscle-specific isoform of filamin specifically binds myotilin, adds an interesting facet to the theme of LGMD: mutations in myotilin, a sarcomeric Z-disc protein that interacts with α-actinin, were suggested to cause LGMD1A (Hauser et al. 2000
). Filamin is therefore the first protein identified that can link membrane-related effects to myofibrillar defects. This finding strongly corroborates the notion that, in LGMD myofibers, large amounts of filamin accumulate at the sarcolemma (Thompson et al. 2000
). LGMD may therefore be caused by two interdependent molecular events: one, the destabilization of membrane attachment complexes and, the other, the weakening of myofibrils and/or myofibrillar connections particularly at the Z-disc. The recently described LGMD 2G-causing mutations in the Z-disc protein telethonin (Moreira et al. 2000
), a protein that has been implied to be involved in signaling events during myofibril development (Mayans et al. 1998
; Mues et al. 1998
), strongly supports this assumption.
The relationship of γ-filamin with both sarcolemmal and myofibrillar proteins, which are involved in LGMD, is highly indicative of an important function for this protein in signaling processes between the sarcolemma and myofibrils. In line with this idea, patients were identified whose disease phenotype showed genetic linkage to chromosome 7q (Speer et al. 1999
), a chromosomal region that also includes the FLNC
gene (Maestrini et al. 1993
; Xie et al. 1998
). This increases the probability that defects in the FLNC
gene itself might cause LGMD in patients who do not have mutations in any of the known LGMD genes. Our work might therefore provide a molecular basis to define a novel signaling pathway from the sarcolemma to the myofibril. Disturbances of this pathway at any point may result in essentially the same clinical manifestation in the form of LGMD.