Article |
Address correspondence to Arnoud Sonnenberg, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, Netherlands. Tel.: (31) 20-512-1942. Fax: (31) 20-512-1944. E-mail: asonn{at}nki.nl
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Abstract |
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Key Words: filamin isoforms; alternative splicing; ß1D integrin; myogenesis; cytoskeleton
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Introduction |
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The first 275 NH2-terminal amino acids of filamin contain an ABD, composed of two calponin homology domains. This domain is followed by 24 repetitive, 100-residue segments, interrupted by two
30amino acid flexible loops (hinge-1 [H1] and -2), which show little homology among the products of the filamin isogenes. The H1 loop, between repeats 15 and 16, is lacking in some splice variants of filamin-B and filamin-C (Xie et al., 1998; Xu et al., 1998). It has been suggested that in the dimeric filamin molecule, the flexible H1 regions are essential for the separation of its ABDs and thus for its ability to promote orthogonal cross-linking of actin filaments (Gorlin et al., 1990). The H2 loop between repeats 23 and 24 is present in all filamin isoforms, and the COOH-terminal repeat 24 is essential in the tailtail dimerization of the filamin molecule (Gorlin et al., 1990). In addition to its ability to cross-link actin filaments, filamin-A serves as a docking site for various transmembrane cell surface molecules such as GP-Ib
, Fc-
RI, tissue factor, and the ß2 and ß1A integrin subunits (Ohta et al., 1991, 1999; Sharma et al., 1995; Meyer et al., 1997; Loo et al., 1998; Pfaff et al.,1998; Calderwood et al., 1999). Filamin-B binds to GP-Ib
(Takafuta et al., 1998; Xu et al., 1998), whereas the muscle-associated filamin-C binds to sarcoglycans (Thompson et al., 2000) and a number of muscle-specific proteins (Faulkner et al., 2000; van der Ven et al., 2000; Takada et al., 2001). Furthermore, intracellular signal transduction proteins, such as the small GTPases RalA, RhoA, Rac-1, Cdc42 (Ohta et al., 1999), the guanidine-exchange factor Trio (Bellanger et al., 2000), the tumor necrosis factor receptorassociated factor-2 (Leonardi et al., 2000), and the membrane-associated proteases, furin and presenilin, bind to filamin (Liu et al., 1997; Zhang et al., 1998). These interactions point to a scaffolding function of filamin, rather than a distinct role as an actin filamentorganizing molecule. Analyses of melanocytic cell lines deficient in filamin-A (Cunningham et al., 1992), as well as of patients with the neuronal migration disorder periventricular heterotopia (Fox et al., 1998; Sheen et al., 2001), indicate a role for filamin-A in cell motility during stabilization of the cell membrane. However, relatively little is known about the specific roles of filamin variants in the properties of cells and the regulation of cellular processes.
Integrins are heterodimeric adhesion receptors that provide a structural link between proteins of the extracellular matrix (e.g., fibronectin, laminin, and collagen) and the intracellular cytoskeleton (Hynes, 1992; van der Flier and Sonnenberg, 2001b). In addition, integrins play a role in several different signaling events (Giancotti and Ruoslahti, 1999). Although the small (2050 amino acids) cytoplasmic tails of the integrin and ß subunits have no intrinsic catalytic domains or obvious proteinprotein interaction domains, studies combining biochemical and genetic approaches, have led to the identification of a range of cytoskeletal, adaptor, and signaling molecules, which interact with the integrin cytoplasmic tails and are therefore implicated in integrin function (Liu et al., 2000; van der Flier and Sonnenberg, 2001b). Cytoskeletal proteins, such as the actin cross-linking proteins talin,
-actinin, and filamin-A (Critchley, 2000), interact with integrin ß subunits. These molecules have been implicated in the linking of actin stress fibers to the cell membrane at specialized structures, known as focal adhesions or focal contacts, which are formed at cellsubstrate contact sites. Binding to ß subunits of this class of proteins was originally shown in biochemical experiments and was recently confirmed in yeast two-hybrid assays (Loo et al., 1998). Another category of proteins that interact with integrins are signaling molecules, including the ARF-GEF protein, cytohesin-1, the WD-repeat proteins Rack1 and WAIT-1, ICAP-1, and integrin-linked kinase (Hannigan et al., 1996; Kolanus et al., 1996; Chang et al., 1997; Liliental and Chang, 1998; Rietzler et al., 1998).
This study originated with the desire to identify cytoplasmic proteins that bind to the integrin ß1D subunit, specifically in cardiac and skeletal muscle. Using the yeast two-hybrid technique, we isolated a new splice variant of filamin-B. Characterization of the interactions in yeast two-hybrid analyses and biochemical assays indicated that this filamin variant has a high binding activity and a broad specificity for integrin ß subunits. Subsequent analysis of filamin mRNA splicing during in vitro myogenesis revealed that the H1 region of filamin-B and filamin-C is removed during the differentiation of myoblasts. Transfection studies with different filamin-B variants tagged with green fluorescent protein (GFP) revealed that the interaction with integrins and the cellular localization of the various filamin variants is different, and they affect myotube morphology and the pace of in vitro myogenesis of C2C12 cells in a variant-specific manner.
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Results |
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Expression and genomic determination of novel filamin-B splice variants
To explore the expression pattern of the transcript for filamin-Bvar-1, cDNAs of multiple human tissues were analyzed in a PCR reaction, using primers that flank repeats 19 and 20 of filamin-B. Fig. 1 A shows that the cDNA encoding the previously reported filamin-B wild-type sequence was amplified from all tissues tested (683-bp product). In addition, a smaller PCR product of 560 bp, corresponding to the filamin-Bvar-1 specific part, was detectable in heart, lung, and skeletal muscle. Nested PCR analysis revealed a weak expression of filamin-Bvar-1 in all tissues tested (unpublished data). A similar splice variant lacking the corresponding region (amino acids 21272167) in human filamin-A, filamin-Avar-1, was also detected by (nested) PCR analysis (unpublished data). Two additional filamin-Bspecific PCR products of 830 (filamin-Bvar-2) and 753 bp (filamin-Bvar-3) were detected in cardiac tissue (Fig. 1 A). Cloning and sequencing of these PCR fragments revealed that they represent two partially overlapping cardiac filamin-B cDNAs (Fig. 1 B). Intriguingly, in filamin-Bvar-2, the insertion of a 147-bp sequence results in a truncated protein with a unique COOH-terminal sequence of 24 amino acids. The insertion in filamin-Bvar-3 of only the first 70 of the 147 bp inserted in filamin-Bvar-2 leads to a frameshift, and as a result, four more amino acids are encoded COOH-terminally in this protein. Hence, both cardiac-specific filamin-B transcripts encode truncated filamin-B proteins that lack the four COOH-terminal repeats, including the 24th dimerization domain.
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Filamin isoforms and their variants determine specificity for association with ß subunits
We next tested the interaction of the COOH-terminal domain, i.e., repeats 1924 of filamin-B and filamin-Bvar-1 with different ß subunits, using the yeast two-hybrid system. In addition, we tested the homologous regions of filamin-A and filamin-Avar-1. The results (Fig. 2
A) show that the COOH-terminal domain of wild-type filamin-B(1924), containing repeats 1924, interacts only with ß1A. In contrast, an equivalent construct encoding filamin-Bvar-1(1924), which lacks amino acids 20822122 that span repeats 19 and 20, and an NH2-terminal deletion mutant of filamin-B, truncated at amino acid 2123 (filamin-B, 20*24), bind not only to ß1A but also to the ß1D, ß3, and ß6 subunits. Similar results were obtained with proteins from the original isolated filamin-Bvar-1 clones that contain amino acids 20272602 (unpublished data). Quantitative ß-galactosidase activity assays indicated that filamin-Bvar-1(1924) bound two to three times more strongly to ß1A than did wild-type filamin-B(1924) (Fig. 2 B). The binding of filamin-Bvar-1(1924) to ß3 was weaker and comparable to that of wild-type filamin-B(1924) to ß1A, and that of filamin-Bvar-1(1924) to ß1D was of intermediate strength.
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Analysis of a series of filamin-B constructs truncated at the NH2 or COOH terminus indicates that repeat 21 is necessary, but not sufficient, for interaction with ß1A (Fig. 2 A). It is possible that the presence of repeat 24 facilitates the dimerization of filamin, and thus of repeat 21, thereby greatly increasing the strength of the binding to ß1A. The interaction of filamin-Bvar-1 with ß3 and ß6 was abolished by the deletion of repeats 2324, whereas it did not affect binding to ß1A or ß1D. Interactions of both ß1A and ß1D with filamin-Bvar-1(19 and 20) could still be demonstrated, although the binding appeared to be weaker than that of a construct containing repeat 21, filamin-Bvar-1(1921). These data suggest that deletion of the 41amino acid region in filamin-B leads to either the removal of inhibitory sequences in repeat 19 or to the introduction of a new binding site for ß1A and ß1D in the remaining part of repeat 20 (amino acids 19952185). The presence of repeat 21 increases binding activity, which is consistent with the data showing that this repeat is necessary for efficient binding of filamin-B to ß1A and contributes to the binding activity of filamin-Bvar-1 (1921) to ß1 integrins. As may be the case for the binding of filamin-B to ß1A, binding of filamin-Bvar-1 to ß3 and ß6 probably requires dimerization mediated by repeat 24.
Biochemical interaction of filamin variants with integrins
To confirm the interactions between the different filamin splice variants and the ß1A and ß1D subunits observed in yeast, we expressed the regions containing repeats 1924 of filamin-Avar-1 or filamin-Bvar-1, or the corresponding regions of wild-type filamin-A(1924) or filamin-B(1924), in COS-7 cells. The proteins were tagged at their NH2 terminus with hemagglutinin A (HA) and expression of equivalent amounts of proteins in COS-7 cells was confirmed by immunoblotting with anti-HA antibody (Fig. 3
A). Binding of the filamin constructs to ß1A and ß1D was tested in a pull-down assay using glutathione-S-transferase (GST) fusion proteins containing the cytoplasmic domains of these integrin subunits, immobilized on glutathione-Sepharose beads (Fig. 3 B). As shown in Fig. 3 C, GSTß1A bound to filamin-Avar-1(1924) and filamin-Bvar-1(1924), but not to the corresponding fragments of the wild-type filamin isoforms (filamin-A, 1924, and filamin-B, 1924). Binding of GSTß1D to the different filamin-A and filamin-B constructs was either undetectable or very weak. The interaction between ß1A and filamin-B(1924) detected in yeast could not be confirmed in the pull-down assay, probably because it is too weak. Interestingly, we found that a truncated filamin-B construct that lacks the first 14 amino acids of repeat 19 (filamin-B, 20092602) could be efficiently precipitated with GSTß1A (unpublished data). Thus, it appears that not only the deletion of COOH-terminal residues of repeat 19, as in the variant-1 protein, but also the deletion of NH2-terminal residues of repeat 19 results in stronger binding of filamin-B to ß1A. Together, these data suggest that repeat 19 contains an inhibitory element for binding of filamin-B to ß subunits.
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Alternative splicing of filamins during in vitro myogenesis
During differentiation of mouse C2C12 myoblasts into myotubes, the expression of ß1D is induced, whereas that of ß1A is downregulated (Belkin et al., 1997; van der Flier et al., 1997). We investigated whether this switch is paralleled by changes in the expression of filamin isoforms and/or their variants. Total RNA was isolated at different time points of myogenic differentiation, and the expression of filamin isoforms was analyzed by RT-PCR using appropriate primers. We studied the splicing of the region encoding the 41 amino acids in filamin-A and filamin-B, as well that of the H1 region of filamin-A, filamin-B, and filamin-C (Fig. 5
A). The latter were included because variants of human filamin-B and filamin-C, lacking the H1 region, have been described previously (Xie et al., 1998; Xu et al., 1998). Fig. 5, BD, shows that C2C12 myoblasts express all three murine filamin isoforms. In addition, whereas the H1 region is present in filamin-A throughout differentiation, this region is absent from the filamin-B and filamin-C isoforms. This deletion appears to precede the switch from ß1A to ß1D that occurs during myogenic differentiation. Interestingly, we detected in C2C12 cells and among several murine and human cDNAs (unpublished data) a third filamin-B transcript that encodes a variant with a shorter H1 (H1s) region (Fig. 5 D). This transcript arises as a result of the usage of intrinsic splice-donor and acceptor sites that are present in the murine and human filamin-B genes (position 5280 and 5312, respectively; GenBank/EMBL/DDBJ accession no. NM_001457). We did not detect the filamin-Avar-1 and filamin-Bvar-1 transcripts.
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GFP COOH-terminal tags do not interfere with filamin dimerization
Before initiating studies to define the cellular localization of the different splice variants of filamin-B, we examined whether the addition of GFP at the COOH terminus of filamin influences the ability of this protein to form dimers. To this end, a filamin-B(1924) construct with GFP at the COOH-terminal end, and a control construct, tagged with HA at the NH2-terminal end, were transiently expressed in CHO cells. After 2 d, cell lysates and intact cells were treated with the chemical cross-linking reagent dithiobis-succinimidyl propionate (DSP) at two concentrations. As shown in Fig. 6
, the addition of increasing amounts of cross-linker led to a shift from monomeric to dimeric tagged filamins, as visualized by immunoblotting with antibodies against HA or GFP. The similar dimerization capacity of HA- and GFP-tagged filamin-B(1924) indicates that the GFP tag had no effect on dimerization. The same samples under reducing conditions, which disrupt the disulfide bond, only contained filamin monomers. Specificity of the cross-linking reaction was checked using the NH2-terminal HA-tagged filamin-Bvar-1(1923) construct, which did not form dimers due to truncation of the COOH-terminal repeat 24, which is required for dimerization.
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Filamin-B variants affect myoblast differentiation in vitro
The functional significance of the developmentally regulated splicing of the H1 region in filamin-B, as well as the deletion of the 41amino acid region, was explored by analyzing the effects of ectopic expression of the four different filamin-B splice variants on myogenesis of C2C12 cells. Myogenic differentiation was induced by switching the culture to a medium containing 2% horse serum (differentiation medium). Interestingly, C2C12 cells expressing filamin-Bvar-1(H1) fused into myotubes within 2 to 3 d after the medium switch, which is 12 d earlier than the fusing of cells from the other transduced cell lines (Fig. 10
A). Furthermore, the myotubes formed by the cells expressing the filamin-B variants lacking the H1 region (filamin-B[
H1] and filamin-Bvar-1[
H1]) were thinner than those formed by the other transduced cell lines or GFP control cells. This difference in morphology was more obvious when the myotubes were stained for MHC (Figs. 11 and 12)
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Immunoblot analysis of MHC demonstrated that the morphological differentiation is accompanied by biochemical changes. The induction of MHC in the filamin-Bvar-1(H1)expressing C2C12 cells was faster than in the other cells, where the induction was similar to that in GFP control cells (Fig. 10 B). Taken together, these results show that expression of the filamin-double variant, filamin-Bvar-1(
H1), accelerates muscle differentiation in vitro.
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Discussion |
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Finally, analysis of the splicing of mRNAs for filamins during in vitro myogenesis of C2C12 cells revealed that the H1 region of filamin-B and filamin-C is removed during myotube formation. The biological consequences of these modifications are discussed below.
Specific interactions of filamin variants
The novel filamin-B variant, filamin-Bvar-1, is the fourth reported ß1D-binding protein identified in a yeast two-hybrid scree; the other three are MIBP, melusin, and skelemin (Reddy et al., 1998; Brancaccio et al., 1999; Li et al., 1999a). The yeast two-hybrid interaction assays indicated that filamin-A does not bind to any integrin ß subunit, whereas filamin-B only interacts with ß1A. On the contrary, the splice variants filamin-Avar-1 and filamin-Bvar-1 bind strongly to ß1A and weakly to ß3 and ß6. Additionally, a moderate binding of filamin-Bvar-1 to ß1D was observed. These results provide evidence that isoforms and variants of filamins specifically bind to different integrin ß subunits, which render the interpretation of biochemical studies with filamins more complicated. Furthermore, subcellular localization and morphological and myogenic effects were found to be determined by the type of the expressed filamin variant.
Previously, it has been shown by coimmunoprecipitation and pull-down assays that various integrin ß subunits have different capacities to bind cytoskeletal proteins. The results of these assays showed that the affinity of ß1D for talin is greater than that of ß1A, which could result in ß1D having a stronger link with the cytoskeleton (Belkin et al., 1997; Pfaff et al., 1998; Zent et al., 2000). In addition, the affinity of the ß7 integrin subunit for filamin appeared to be high, whereas that of ß1A and ß1D was found to be intermediate and low, respectively (Pfaff et al., 1998; Zent et al., 2000). Our data suggest that ß1 integrins hardly interact with filamin-A or filamin-B and that most of their binding activity toward these filamin isoforms is with the variant-1 forms. In agreement with previous findings, we observed that the binding of ß1A to filamin-Avar-1 and filamin-Bvar-1 is stronger than that of ß1D. Loo et al. (1998) has previously reported that filamin-A can interact with the cytoplasmic domain of ß1A. However, their conclusion was based on the binding of ß1A to a construct containing the COOH-terminal 478 amino acids of filamin-A. This fragment corresponds to filamin-A(20*24), whose binding properties resemble those of filamin-Avar-1. However, a longer filamin-A protein, extending 176 amino acids at the NH2 terminus, does not bind to integrin ß subunits. Thus, the apparent discrepancies between these reported data and ours might be due to the fact that those studies were concerned with the interaction of ß1A with a splice variant of filamin-A rather than with wild-type filamin-A. Similarly, the filamin proteins previously identified in pull-down experiments using dimerized integrin cytoplasmic domains presumably do not represent wild-type filamin, but filamin splice variants (Pfaff et al., 1998; Calderwood et al., 1999).
The discrepancy between our pull-down interaction results (Fig. 3) and those of yeast two-hybrid assays (Fig. 2) on the interaction of filamin-B variants with ß1D probably can be reduced to a difference in sensitivity between these two assays; the yeast two-hybrid assay being more sensitive than the pull-down assay. Indeed, we found in the ß-galactosidase assays that binding of ß1D to filamin-Bvar-1 is weaker than that of ß1A. An alternative explanation might be that in the GSTß1D fusion protein, the ß1D cytoplasmic sequences are not readily accessible for binding.
Based on the immunoglobulin-like folding of several filamin repeats that are present in the gelation factor of Dictyostelium (Fucini et al., 1997; McCoy et al., 1999), it is likely that as a result of the 41amino acid deletion, three ß-strands of the two adjacent repeats 19 and 20 (strand G from repeat 19 and strands A and B from repeat 20) were lacking in the filamin-A and filamin-B variants. Removal of this stretch of 41 amino acids probably led to the exposure of one or more cryptic binding sites for integrin ß subunits in the remaining part of repeat 20 of filamin-A and filamin-B. Alternatively, the disruption of repeat 19 may have removed elements inhibiting the binding of filamin-B to ß1A. Finally, together with the deleted 41 amino acids in human filamin-Bvar-1, a conserved cAMP-kinase consensus site (serine 2150) was removed that, when phosphorylated in filamin-A, confers an increased resistance to calpain cleavage at residues 17611762 in the H1 region (Gorlin et al., 1990; Jay et al., 2000). This suggests long-range conformational effects by the 19 and 20 repeat region of filamin and, although serine 2150 is not conserved in murine filamin-B, deletion of this region may have additional effects beyond the modulation of the strength and specificity of filamin-ßintegrin interactions. Interestingly, filamin-C contains an insertion of 82 amino acids, exactly juxtaposed to the homologous variant-1 region, which could give specific features to this filamin variant associated with striated muscle (Thompson et al., 2000). Evidence of alternative splicing of mRNA has not yet been reported for this region (Xie et al., 1998).
Specialized cellular roles and specific cellular localization of filamin-B variants
Several previous reports have indicated complex expression patterns of filamin variants during myogenesis (Gomer and Lazarides, 1981, 1983a,b; Chiang et al., 2000). In this study, we extended those data by the identification of additional filamin-Bvar and H1 splice variants. We demonstrate that removal of the H1 region from filamin-B and filamin-C is induced during myogenesis in vitro. Furthermore, the presence or absence of the H1 region in filamin-B affects the morphology of the formed myotubes. Filamin-B variants lacking H1 form thinner myotubes in contrast to the more pouch-like tubes formed by H1-containing filamins.
Deletion of the H1 region from both filamin-B and filamin-C might determine the type of actin filaments with which these filamin isoforms interact. This would explain why during differentiation of myoblasts into myotubes, a process that is associated with a dramatic reorganization of the cytoskeleton, the splicing of the mRNA for this region is regulated and why forced expression of it has such a dramatic effect on the morphology of the formed myotubes. Parallel bundles of actin filaments might become more tightly packed by the loss of the H1 region of filamin, because the flexibility of the filamin dimer lacking the H1 region is reduced, as has been anticipated by Gorlin et al. (1990). Our observation that filamin-B variants lacking the H1 region show the tendency to be more polarized at the ends of actin stress fibers supports this hypothesis. The identification of shorter filamin-B(H1s) alternative splice variants in several murine and human tissues provides additional possibilities for the modulation of the H1 region, and subsequent effects on the organization of the F-actin network.
Indeed, several studies have shown the effects of differences in the organization of the actin cytoskeleton on myoblast morphology and differentiation. For example, expression of dystrophin, or its homologue utrophin, in dystrophin-deficient mdx mice affects the type of fibers present in muscle (Rafael et al., 2000). Also, RhoA-mediated induction of stress fibers increases myoblast differentiation, in contrast to the inhibitory effects on myogenesis by Rac-1 and Ras (Wei et al., 1998; Gallo et al., 1999).
By transfecting filamin-B variants into cells, it was shown that the colocalization of filamin with ß1-integrincontaining focal adhesions is not solely dependent on the presence of an integrin-binding site on filamin-Bvar-1. Only the filamin-Bvar-1(H1) variant, in which a high affinity binding site for ß1A and ß1D is combined with the absence of the H1 region, is concentrated at the tips of actin stress fibers, where it is colocalized with ß1 integrins. Two previously described chicken filamin variants could resemble filamin-Bvar-1(
H1) (Pavalko et al., 1989; Tachikawa et al., 1997), because of their localization at the ends of stress fibers in focal adhesions or at the dense plaques in smooth muscle cells. It is possible that the effect induced by the loss of the H1 region permits the linkage of filamin-Bvar-1(
H1) with special organized actin stress fibers in focal contacts, thereby facilitating binding of the variant-1 site to integrins. However, there are other possible explanations, e.g., similar to the deletion of the var-1 region, the deletion of the H1 region may have induced a binding site for one or more focal contact proteins that, together with ß1, mediate the localization of filamin-Bvar-1(
H1) in focal contacts.
Our finding that filamin-Bvar-1(H1) is present together with talin in focal contacts raises the question of which of these two proteins is actually associated with ß1. Filamin-Bvar-1(
H1) and talin bind to overlapping sites on the ß1 cytoplasmic domain and, therefore, it seems unlikely that these two proteins simultaneously bind to ß1. We believe it is reasonable to assume that a proportion of ß1 is associated with talin, whereas other ß1 subunits interact with filamin-Bvar-1 (
H1). An alternative possibility, which we consider to be less likely, but nevertheless cannot formally be excluded, is that only filamin-Bvar-1(
H1) is associated with ß1 and talin is retained in focal contacts by an interaction with vinculin.
Previous reports have indicated a critical role for integrins in myogenic differentiation (Menko and Boettiger, 1987; Sastry et al., 1996) and muscle integrity (Volk et al., 1990; Mayer et al., 1997; Hayashi et al., 1998). Conceivably, the acceleration of myogenesis observed in C2C12 cells expressing filamin-Bvar-1(H1) might be due to an altered interaction of this filamin variant with the ß1 integrins. An increased interaction may help to stabilize the expression of integrins at the cell surface by firmly anchoring them to the actin cytoskeleton. For example, the reexpression of filamin-A, in filamin-Adeficient melanoma cells, has been associated with an increase in the surface levels of both the GP-Ib
and ß1 integrins (Meyer et al., 1998). However, we did not detect, by FACS® analysis, any changes in surface expression levels of ß1 integrins upon the ectopic expression of filamin-B variants (unpublished data). This discrepancy could be due to the complete lack of filamin-A in the M2 cells used by Meyer et al. (1998). If cells contain endogenous filamin, the ectopic expression of filamin has no detectable effects. The effects of filamin-Bvar-1(
H1) on myogenesis may also be the consequence of a different localization and/or function of integrins in C2C12 cells. Indeed, as already mentioned, the distribution of filamin-Bvar-1(
H1) is typically polarized in differentiating C2C12 cells and is localized together with ß1 in focal contacts in GD25-ß1A cells. However, whatever the underlying mechanism for the effect of filamin-Bvar-1(
H1) on myogenesis is, the physiological significance of it remains uncertain, because it could not be demonstrated that this variant is expressed in C2C12 cells.
In conclusion, our study provides the first evidence that alternative mRNA splicing controls the cellular localization of filamins and their interaction with integrins. This indicates that individual filamin variants could be specialized in linking integrins or other transmembrane proteins to the actin cytoskeleton, comparable to the filamin-Cspecific (Thompson et al., 2000) and dystrophin-mediated connection with the sarcoglycan adhesion complex in muscle cells. Ultimately, these specific activities of filamin variants modulate cell morphology and differentiation.
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Materials and methods |
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The mouse anti-vinculin (V11F9) (Glukhova et al., 1990) was provided by Dr. M. Glukhova (Institut Curie, Paris, France). The mouse anti-ß1D cytoplasmic domain (2B1) and rabbit antifilamin-B (H1) antibodies have been described previously (van der Flier et al., 1997; Takafuta et al., 1998). Mouse anti-sarcomeric -actinin (EA-53), anti-talin (8d4), and
-actinin (BM-75.2) were purchased from Sigma-Aldrich; mouse anti-paxillin (349) and anti-phosphotyrosine (PY20) were from Transduction Laboratories; mouse antiGal4 binding domain (BD), mouse antiGal4 activation domain (AD), and rabbit anti-HA antibodies (sc-510, sc-1663, and sc-805, respectively) were from Santa Cruz Biotechnology, Inc.; mouse anti-HA (12CA5) were from Boehringer; mouse anti-sarcomeric MHC (MF20) were from the Developmental Studies Hybridoma Bank; and rabbit and mouse anti-GFP (B34) were from CLONTECH Laboratories, Inc. and BabCO. The sheep antimouse and donkey antirabbit HRP-conjugated antibodies were obtained from Amersham Pharmacia Biotech. TO-PRO-3 iodide, Alexa®-568coupled phalloidin, and Texas redconjugated goat antimouse and goat antirabbit were from Molecular Probes.
Yeast two-hybrid screen and plasmid constructs
The ß1D (amino acids 752798) or ß1A cytoplasmic domain (amino acids 752801) were fused to the Gal4 DNA BD in pAS2.1 (CLONTECH Laboratories, Inc.). These constructs were used as bait in yeast two-hybrid screens of a human skeletal muscle and a human keratinocyte library (CLONTECH Laboratories, Inc.; HL4010AB and HL4024AB, respectively) in the yeast Gal4 transcriptional AD expression vector pGAD10 (CLONTECH Laboratories, Inc.). Plasmids were introduced into the yeast host strains PJ694A (a gift from Dr. P. James, University of Wisconsin, Madison, WI; James et al., 1996) or Y190 (CLONTECH Laboratories, Inc.) by transformation. The yeast two-hybrid library screen was performed essentially according to the CLONTECH Laboratory, Inc. two-hybrid manual.
To analyze the proteinprotein interactions of truncated filamin-A and filamin-B with several integrin cytoplasmic tails, mutants, and truncated filamins, pACT (-derived) and pAS (-derived) constructs were cotransformed into the yeast strain PJ694A. Interaction was assayed by selection for growth on plates containing SC-LTHA, a yeast synthetic complete medium lacking the vector markers Leu and Trp, as well as the interaction markers His and Ade, and containing 2 mM 3-aminotriazole (Sigma-Aldrich; A8056) to suppress residual histidine synthesis in the strain PJ694A, as described previously (Geerts et al., 1999). Interaction was scored as the percentage of the plating efficiency on SC-LTHA containing 2 mM 3-aminotriazole compared with the plating efficiency on SC-LT, a medium lacking only the vector markers Leu and Trp, when grown for 5 or 10 d at 30°C. Quantitative determination of ß-galactosidase activities was performed in triplicate using of three independent clones grown in SC-LT with O-nitrophenyl B-D-galactopyranoside (Sigma-Aldrich) as the substrate, according to the manufacturer's recommendations.
All filamin-A and filamin-B deletion constructs (starting at the amino acids as indicated in Figs. 2 and 4; for amino acid alignment see van der Flier and Sonnenberg, 2001a) were generated by PCR using Pwo DNA polymerase (Roche Molecular Biochemicals). Filamin-A and filamin-B deletion constructs were cloned in the modified yeast expression vector, pACT2.4 (Gal-4[AD], a derivative of pACT2.1) or pAS2.1 (Gal4[BD]). The Gal4(BD) ß1A, ß1D, ß2, ß3A cytoplasmic domain and ß1A truncation constructs in the pAS2.1 have been described previously (Wixler et al., 2000). The ß6 cytoplasmic domain construct in pAS2.1 was a gift of S. Spong (Lung Biology Center, University of California San Francisco, San Francisco, CA). The ß1 chimeric cytoplasmic domains ß1A/D792, ß1A/D796, and ß1D/A787 were generated by PCR and cloned into pAS2.1. All plasmid constructs were checked by sequencing, and the expression of fusion proteins in yeast was confirmed by immunoblotting with Gal4(BD)- and (AD)-specific antibodies. None of the filamin constructs used activated the reporter genes autonomously in yeast, nor did they bind to any of several different integrin -subunits tested.
Genomic PCR and RT-PCR analysis
RT-PCR analysis was performed on Human Multiple Tissue cDNA Panel I and II (CLONTECH Laboratories, Inc.) using Taq DNA polymerase and primers deduced from the human filamin-B sequence.
The following primer sets were used to detect the human filamin-Bvar-1 cDNA: either BV1/BV2 or nested PCR using primers BV9/BV11 on the BV9/BV10 PCR products. BV11 specifically amplifies the filamin-Bvar-1 cDNA because it spans the 123-nt deleted sequence with one basepair at its 3' end. Similarly, human filamin-Avar-1 was detected using the nested primers AV1/AV3 on the AV1/AV2 PCR product. Human filamin-Bvar-1 (H1) transcripts with or without the H1 region were detected using primers BV12/BV13. Genomic sequences encoding repeats 19 and 20 of human filamin-B were determined by PCR using Taq DNA polymerase on human genomic DNA isolated from two different cell lines, using a standard procedure and combinations of the primers BV1BV8 as indicated in Fig. 1 C. All BV primers are numbered according to the filamin-B sequence data available from GenBank/EMBL/DDBJ under accession no. NM_001457, except primers BV7 and BV8, which are numbered according to sequence data submitted under accession no. AF353666. Numbering of AV primers is according to the filamin-A sequence available under accession no. NM_001456. BV1: GAAGATGGCACCTGCAAAGTC (62886308); BV2: GACCGGCACCAGGTAGGG (69716954); BV3: GTGCCTGGGGTTTATATCGTC (63246344); BV4: GGTGCGGGTGATGCTCTCT (64376419); BV5: GGAGGGAAGAGTCAAAGAGAG (64046424); BV6: GATCTGGGCAGAGCAGTTC (177159); BV7: GAACTGCTCTGCCCAGATC (159177); BV8: CACGGTGAACTGGAAGGGG (66866668); BV9: TCCAGTCGGAGATTGGTGA (61156133); BV10: GACCGGCACCAGGTAGGG (69716954); BV11: ATATCACTGCTGTTGATTTC/A (65176498/6374); BV12: GTGACCTGCACGGTTCTGA (50715089); BV13: ATCACTGCTGTTGATTTC/AGGC (65136496/63726369); AV1: ACCCGCGATGCAGGCTATG (63866404); AV2: CACGGTGAACTGGAAGGGG (66906672); AV3: CCGACCAGCACGTGCCTG/A (65436771/6675).
Total RNA from C2C12 cells was isolated at various time points after myotube induction, using RNAzolTMB (Tel-Testine), as recommended by the manufacturer. Subsequently, cDNA was prepared from 10 µg RNA using an oligo d(T)15 primer and SuperScriptTM (Life Technologies). The H1 regions of murine filamin-A, filamin-B, and filamin-C (partial sequences are deposited under GenBank/EMBL/DDBJ accession nos. AF353668, AF353669, and AF354670, respectively) were amplified using the primer sets mAH1/mAH2, mBH1/mBH2, and mCH1/mCH2, respectively. mAH1: CCTGATGGCTCAGAGGTAGA (120); mAH2: GTGATCTCCCCTTCTTGATA (344324); mBH1: CTGACGTCATTGAAAATGAAGAT (123); mBH2: AATGGAATCACCAAGTCAAAGG (274253); mCH1: GATGGGGCAGAGCTCGATG (119); mCH2: GTCAGCTCCCCTTTCTGCAC (341322).
For the detection of potential murine filamin-A and filamin-B variant-1 mRNAs (partial sequences are deposited under GenBank/EMBL/DDBJ accession nos. AF353671 and AF353672, respectively), RT-PCR was performed using mAV1/mAV2 and mBV1/mBV2, respectively. The variant-1 products were further amplified by nested PCR using the mAV1/mAV3 and mBV1/mBV3 primer sets on 1 µl of the RT-PCR products. mAV1: GGCTACGGTGGGCTTAGTC (119); mAV2: GAAGGGGCTCCCAGGTACA (453435); mAV3: TGACCAGCATGTGCCTG/A (138154/278); mBV1: AGGCTATGGTGGCATATCCT (120); mBV2: GAACGGGCTTCCTGTTACG (454436); mBV3: GCTGACGAGCATGTGCCTG/A (137155/278). Murine ß1A and ß1D integrin (GenBank/EMBL/DDBJ accession no. Y00769) were analyzed using GGCAACAATGAAGCTATCG (22192237) and CCCTCATTCGGATTGAC (24842465) as a primer set. PCR products were analyzed on 2% agarose gels and the identity of all PCR products was confirmed by cloning and subsequent sequencing.
Cloning of HA-tagged and GFP fusion proteins
Truncated forms of filamin-A and filamin-B were fused to the NH2-terminal HA epitope tag in pcDNA3-HANII, a modified pcDNA3 vector (Invitrogen). pCI-puro-eGFPc was generated by inserting an eGFP PCR product, using pEGFP-N1 (CLONTECH Laboratories, Inc.) as a template, into pCI-puro(XbaI/NotI), a pCI-neoderived (Promega) vector in which the aminoglycoside phosphotransferase gene was replaced by the gene for puromycin-N-acetyl transferase, which induces puromycin resistance. LZRS-eGFPc-IRES-zeo was constructed by subcloning the eGFP fragment from pCI-puro-eGFPc into the retroviral expression vector LZRS-ms-IRES-zeo (Kinsella and Nolan, 1996; van Leeuwen et al., 1997). The four fusion proteins of full-length filamin-B variants and GFP as depicted in Fig. 7 A were generated by a three point ligation into the XbaI/NotI sites of pCI-puro-eGFPc. All four possible combinations between one 5' filamin-B fragment (XbaI/SacII), either containing or not containing the H1 region, and one 3' filamin-B fragment (SacII/NotI), either wild-type or variant-1, were prepared. Retroviral expression constructs were obtained by recloning the above filamin-B variants from pCI-puro-eGFP into the retroviral vector LZRS-eGFPc-ms-IRES-zeo. The 5' filamin-B fragment containing the H1 region was obtained by XbaI/SacII digestion of wild-type filamin-B (Takafuta et al., 1998). A 3' filamin-B fragment containing a 3' NotI site for in-frame fusion with GFP was generated by PCR, using primer set D4/U5 and full-length filamin-B as template. A 5' filamin-B fragment lacking H1 (amino acids 17041727) was generated by fusion PCR. In brief, two PCR products were obtained using D1/U1 and D2/U2 primer pairs. Products were annealed through overlapping sequences (underlined) and reamplified with D1/U2 to yield the H1 deletion. This PCR product was then used to replace the MunI/SacII fragment of wild-type filamin-B. A 3' fragment of filamin-Bvar-1, containing an intrinsic SacII and an in-frame 3' NotI site was generated by fusion PCR. One PCR product was obtained using primer set D4/U4 and wild-type filamin-B as template. The other PCR product was obtained using primer set D5/U5 and filamin-Bvar-1 cDNA as template. The final fusion PCR product was obtained after reannealling the two obtained products and reamplification using primer set D4/U5. The primers used are under GenBank/EMBL/DDBJ accession no. NM_001457. D1: TGCAATTGATGCCCGAGATGC (47514771); U1: ACATAGGCCTCTTCGGTCAC/CATGACAGTGA (53325313/52405230); D2: ACAGCCCCTTCACTGTCATG/GTGACCGAAGAGG (52215240/53135325); U2: GGCCGCGGCCATAGACTTT (61656147); D4: GTCCAGTCGGAGATTGGTGA (61146133); U4: CCACCGCCAAGGATATGCC (62496231); D5: GGCATATCCTTGGCGGTGG (62316249); U5: gcggcggccgcgAGGCACTGTGACATGAAAAGG (79377917) (NotI site is in bold).
Binding to GST fusion proteins
For pull-down assays, ß1A and ß1D cytoplasmic domains were fused with GST in the bacterial expression vector pRP261, a derivative of the pGEX-3X vector. The recombinant proteins were expressed in Escherichia coli strain BL21 (DE3) and purified from bacterial lysates by the use of glutathione-Sepharose 4B beads. Constructs encoding NH2-terminally truncated filamins were transfected in COS-7 cells using the DEAE-dextran method (two 10-cm Petri dishes with 106 seeded cells were used for three pull-down assays). 48 h after transfection, cells were lysed for 20 min in 1 ml ice cold lysis buffer (10 mM Pipes, pH 6.8, 50 mM NaCl, 150 mM sucrose, 0.5% Triton X-100, 1 mM EDTA, 10 µg/ml calpeptin, 1 mM PMSF, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 0.1 U/ml aprotinin). Lysates were cleared by centrifugation at 15,000 g for 10 min at 4°C, and subsequently diluted 10 times in dilution buffer (10 mM Pipes, pH 6.8, 50 mM NaCl, 150 mM sucrose, 3 mM MgCl2). Diluted lysates were incubated overnight at 4°C with 15 µl glutathione-Sepharose 4B beads containing 50 µg GST fusion proteins. The beads were washed with the same lysis buffer and centrifuged through a sucrose cushion (10 mM Pipes, pH 6.8, 50 mM NaCl, 800 mM sucrose, 1 mM MgCl2), and proteins were resolved by SDS-PAGE and immunoblotted with anti-HA antibody. For GST precipitations of full-length COOH-terminally GFP-tagged filamins, stable retrovirally infected C2C12 or GD25-ß1A cells were lysed and incubated with the various GST fusion proteins. Expression of the full-length GFP-tagged filamin-B variants was increased by preincubating the cells overnight with 5 mM sodium butyrate. GST fusion protein loading of the beads was checked by Coomassie brilliant blue staining, and the filamin fusion proteins were visualized by immunoblotting, as described previously (Geerts et al., 1999).
Chemical cross-linking of the HA- and eGFP-tagged filamin-B truncations and immunoblotting
CHO cells were transfected with NH2-terminal HA-tagged or COOH-terminal GFP-tagged truncated constructs of full-length filamin-B (FLN-B[1924] 20092602 or FLN-Bvar-1[1923] 20272047), using a cationic lipid-based DNA delivery protocol (Lipofectamin; Life Technologies). After a 3-h incubation, the lipofectamin reagent was replaced by fresh growth medium. 2 d later, transfected cells were lysed in 0.5 ml lysis buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1% Nonidet P-40) for 5 min on ice and subsequently subjected to cross-linkage for 1 h on ice by adding different concentrations (0, 0.25, or 1 mM) of DSP (Pierce Chemical Co.; 25 mM stock in DMSO). Alternatively, the intact cell monolayer was treated with 1 mM of the cell-permeable cross-linker, dissolved in phosphate-buffered saline, for 1 h. Subsequently, the cross-linking reaction in the cell lysate was stopped by adding 25 µl of 1 M Tris-HCl (pH 6.8), whereas the intact treated cells were washed twice with PBS and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40. All cell lysates were cleared by centrifugation at 14,000 rpm for 10 min and 1/12 portions of the total cell lysates were separated by SDS-PAGE on 7% gels under both nonreducing and reducing (5% ß-mercaptoethanol) conditions and analyzed by immunoblotting.
Immunofluorescence and flow cytometry
Bulk populations of sorted cells expressing GFP fusion proteins were grown on coverslips and, in one step, fixed and permeabilized in 3% paraformaldehyde, 2% Triton X-100 in PBS for 10 min at room temperature. Cells were blocked in PBS, 2% BSA for 1 h, and incubated with primary antibodies (optionally in the presence of Alexa®568-labeled phalloidin or TO-PRO-3) in the same buffer for 1 h at room temperature. After washing in PBS, cells were incubated in the presence of FITC- or Texas redconjugated secondary antibodies for 1 h. Preparations were then washed in PBS, mounted in Vectashield (Vector Laboratories), and analyzed with a confocal Leica TCS NT microscope.
For flow cytometry and cell sorting, cultured cells were trypsinized, washed twice in PBS, 2% FCS, and sorted on a FACStar Plus® (Becton Dickinson) by their level of GFP expression.
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Footnotes |
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Dirk Geerts' present address is Department of Human Genetics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, Netherlands.
* Abbreviations used in this paper: ABD, actin-binding domain; AD, activation domain; BD, binding domain; DSP, dithiobis-succinimidyl propionate; F-actin, filamentous actin; FLN-B, full-length filamin-B; GFP, green fluorescent protein; GST, glutathione-S-transferase; H1, hinge-1; H1s, shorter H1; HA, hemagglutinin A; MHC, myosin heavy chain.
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Acknowledgments |
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A. van der Flier was supported by a Yamanouchi Studentship (Yamanouchi Research Institute, Oxford, UK) and a grant from The Netherlands Heart Foundation.
Submitted: 9 March 2001
Revised: 7 December 2001
Accepted: 8 December 2001
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References |
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