Identification of three human filamin-B variants and analysis of the genomic organization of the FLN-B gene. (A) Tissue expression of filamin-B variants. PCR was performed on a human multiple tissue cDNA panel, using primers BV1 and BV2 designed to amplify repeats 19 and 20 of filamin-B. The 683-bp band, representing wild-type filamin-B, is present in all samples tested. The 560-bp product marked by one asterisk represents filamin-B_var-1. The cardiac splice variants (filamin-B_var-2, 830 bp, and filamin-B_var-3, 753 bp) are indicated by two asterisks. (B) The partial cDNA sequences and deduced amino acid sequence of the human filamin-B variants. The translation of the two cardiac variants (B_var-2 and B_var-3), as well as wild-type and filamin-B_var-1 is indicated in single letter code. Dots indicate the presence of nucleotides, whereas slashes indicate deletion of the corresponding nucleotides. The putative cAMP-kinase sequence is underlined. The nucleotide sequence data are available from GenBank/EMBL/DDBJ under accession no. AF353666. (C) Schematic diagram representing the genomic organization of the region of FLN-B encoding the repeats 19 and 20 and the composition of the cDNA splice variants. Arrows indicate positions of primers used for cloning. (D) Boundaries at the exon–intron junctions of the FLN-B gene segment. Sizes of exons and introns are indicated as are the consensus splice donor and acceptor sequences; GT/AG of each exon–intron border are underlined. Exons are numbered according to Chakarova et al. (2000). (Footnotes a and b) Nucleotides are numbered according to GenBank/EMBL/DDBJ accession nos. NM_001457 and AF353667 (Fig. 1 D), respectively.

Binding of filamin isoforms and variants to GST–β1A and –β1D fusion proteins. (A) Detection of the expressed products from HA-tagged filamin-B cDNA constructs (filamin repeats 19–24) in lysates of COS-7 cells, transiently transfected with the indicated constructs. The upper band represents nonspecific reactivity of the antibody with an endogenous protein. (B) Expression of recombinant GST–β1A and –β1D fusion proteins. The GST fusion proteins containing the cytoplasmic domains of β1A and β1D were purified on glutathione-Sepharose 4B beads and analyzed by immunoblotting with antibodies against GST, β1A, and β1D subunits. (C) Pull-down assay with GST or GST–β1A and –β1D fusion proteins, immobilized on glutathione beads (A) containing HA-tagged fusion proteins as indicated. (Top) Immunoblot detection of bound filamin-A_var-1 and filamin-B_var-1 to GST–β1A and very weak binding to GST–β1D. No binding was detected to filamin-A and filamin-B. (Bottom) Coomassie blue–stained polyvinylidene difluoride membrane showing the amount of GST fusion proteins used for each pull-down assay.

Expression of filamin isoforms and variants during in vitro myogenesis. (A) Schematic representation of the organization of the filamin domains and the regions analyzed by RT-PCR. (B) RT-PCR analysis of alternative splicing of the H1 region of filamin-A, filamin-B, and filamin-C in mouse C2C12 myoblasts and murine tissues, as indicated. Splicing is analyzed at the indicated time points after induction of differentiation. The H1 region of filamin-A is not spliced, whereas in filamin-B and filamin-C the H1 region is removed during myogenesis. Filamin-B(H1s) is detected in C2C12 cells and adult tissues. The switching from the β1A to the β1D variant during C2C12 differentiation is shown. (C) Immunoblot of the protein expression levels of filamin-B β1D, and MHC during myogenesis. Filamin-B expression decreases, while the myogenic differentiation markers β1D and MHC are both induced. (D) Amino acid alignment of the human and murine H1 and variable-1 region. The partial murine filamin sequences are available from GenBank/EMBL/DDBJ under accession nos. AF353668–353670.

Expression of filamin-B variants in human tissues, and characterization of the binding of full-length filamin-B variants to integrin cytoplasmic domains. (A) Schematic presentation of COOH-terminal GFP-tagged filamin-B variant constructs. The internal deletions of filamin-B are indicated by a single line. (B) Detection by PCR amplification of filamin-B_var-1 and filamin-B_var-1(ΔH1) transcripts. PCR was performed on cDNAs from different human tissues, using primers BV12 and BV13 (A) designed to specifically amplify filamin-B_var-1(±ΔH1) cDNAs. cDNAs encoding filamin-B variants with and without the var-1 region were used as positive and negative controls, respectively. As a further negative control, cDNA for filamin-A was used. A PCR product for filamin-B_var-1 was amplified from most tissues. Heart, spleen, and thymus contained transcripts for filamin-B_var-1(ΔH1). A third PCR product (indicated by an asterisk) that migrated slightly faster than the filamin-B_var-1 variant and is present in heart, kidney, liver, lung, and colon, corresponds to filamin-B_var-1(H1s). The authenticity of the PCR products has been confirmed by sequencing. (C) Expression of GFP-tagged filamin-B variants in C2C12 myoblasts. Stably transduced C2C12 cells were obtained as described in the Materials and methods. Equal amounts of total cells lysed in boiling sample buffer were analyzed by immunoblotting. Filamin-B–GFP was detected by mouse anti-GFP antibody. The upper bands represent the full-length filamin–GFP fusion proteins whereas some fainter lower bands represent COOH-terminal proteolytic products. (D) Pull-down assay of full-length filamin-B variants with GST or GST–β1A and –β1D fusion proteins immobilized on glutathione beads containing GFP-tagged fusion proteins as indicated. Immunoblotting showed binding of filamin-B_var-1 and filamin-B_var-1(ΔH1) to GST–β1A, weak binding to GST–β1D, whereas filamin-B and filamin-B(ΔH1) did not bind.

Localization of filamin-B_var-1(ΔH1) in focal contacts. Confocal microscopy of GD25-β1A cells showing the green fluorescence of filamin-B_var-1(ΔH1) compared with red talin (A), phosphotyrosine (B), α-actinin (C), paxillin (D), vinculin (E), and F-actin (F). Insets in C and D are at higher magnifications. Filamin-B_var-1(ΔH1) is found at the end of actin stress fibers and is colocalized (yellow) with talin, phosphotyrosine, paxillin, and vinculin in focal contacts. Bars: (A–D) 10 μm; (E and F) 5 μm.

Localization of GFP or GFP fusions of filamin-B or filamin- B_var-1(ΔH1) in undifferentiated and differentiated C2C12 cells. (A–C) Confocal microscopy of undifferentiated C2C12 myoblasts showing the green fluorescence of GFP and two GFP fusion proteins (filamin-B and filamin-B_var-1[ΔH1]) compared with red F-actin. (D–I) Triple stainings of differentiated myotubes. (D–F) Localization of GFP and GFP fusions (green). (G–I) Same field showing the distribution of sarcomeric MHC (red) and nuclei (blue).

Characterization of the interaction of filamin isoforms and variants with the cytoplasmic domains of different integrin β subunits in yeast. (A) Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (pAS2.1) and filamin-A and filamin-B deletion constructs (pACT). The NH₂- and COOH-terminal positions of the different filamin constructs are indicated. Numbers in parentheses refer to the repeats present in the different constructs. The partially truncated repeat 20 is indicated by an asterisk. Interactions were scored (+) when the plating efficiencies on selective SC-LTHA plates were greater than 80% of those on nonselective SC-LT plates at 5 d of growth, (±) when they were greater than 80% at 10 d of growth, and (−) when no colonies were detected after 10 d of growth. (B) β-Galactosidase activity of the indicated cotransformants was measured by a liquid culture assay with O-nitrophenyl B-d-galactopyranoside as substrate. Controls were used: negative interaction control, empty vectors pAS/pACT; positive interaction control, p53/pSV-40 large T and the complete Gal4 domain pCL1/pAS (171 ± 6 β-galactosidase units; unpublished data). Data are shown as mean ± SD (n = 3).

Characterization of the interaction between mutants of the β1A and β1D cytoplasmic domain with filamin-A and filamin-B splice variants by yeast two-hybrid analysis. Cotransformation of yeast host strain PJ69–4A with the listed combinations of β integrin cytoplasmic domains (in pAS) and filamin (in pACT). Interaction was scored as in Fig. 2. The numbers represent the amino acid positions in β1A and β1D. The conserved NPXY motifs are underlined and the amino acids in β1D that were swapped in the different chimeras are indicated by shading. The common membrane proximal region, which is shared between β1A and β1D, is boxed.

The GFP tag on the COOH-terminal part of filamin-B does not interfere with dimerization in vivo. CHO cells were transfected with either the NH₂-terminal HA- or the COOH-terminal GFP-tagged FLN-B(19–24) or with the NH₂-terminal HA-tagged FLN-B_var-1(19–23) construct lacking the last repeat. 2 d after transfection, filamin dimers in either cell lysates (lysate) or intact cells (cells) were stabilized by chemical cross-linking at two different concentrations of DSP for 1 h, and dimerization of the epitope-tagged filamins was analyzed by immunoblotting under nonreducing conditions (NR) using anti-HA and anti-GFP antibodies. The specificity of the cross-linkage was checked by including NH₂-terminal HA-tagged FLN-B_var-1(19–23), in which the truncation of COOH-terminal repeat 24 abolished dimerization. In addition, cross-linked samples were separated under reducing (R), DSP disrupting conditions resulting in the presence of filamin-B monomers. Closed arrowheads indicate HA-tagged products, and open arrowheads indicate GFP-tagged fusion proteins. A single arrow indicates a filamin monomer, and an arrow doublet indicates dimers.

Expression and localization of filamin-B variants in GD25–β1A mouse fibroblasts. GD25–β1A cells (A and B) and GD25–β1A cells stably expressing GFP-tagged filamin-B (C and D), filamin-B_var-1 (E), or filamin-B_var-1(ΔH1) (F–O), were analyzed for endogenous filamin-B (A, B, and M) and vinculin (A, D–G, and I) expression using indirect immunofluorescence microscopy, staining of F-actin (B, C, J, and L) with Alexa^®568-phalloidin, and GFP fluorescence (C–F, H, I, K, and N). Filamin-B and the variants filamin-B_var-1 and filamin-B(ΔH1) are associated with actin stress fibers: they did not accumulate in focal contacts. In contrast, filamin-B_var-1(ΔH1) is concentrated in focal contacts (G–I) at the end of actin stress fibers (J–L, N, and O). Note that the fluorescence pattern of filamin-B–GFP is similar to that of endogenous filamin-B detected with a polyclonal antibody specific for the H1 region of this filamin isoform. This antibody does not react with filamin-B(ΔH1) and therefore in cells that express this variant, it does not react with focal contacts. Filamin-B is not present in membrane ruffles (B, arrow). Bars, 10 μm.

Effects of the ectopic expression of filamin-B variants on myogenesis. C2C12 myoblasts stably expressing GFP and GFP fusions of the indicated filamin-B variants were grown to confluence and then switched to differentiation medium. (A) The phase contrast micrographs show the morphological appearance of the cells in culture, 4 d after the switch to differentiation medium. (B) Western blot analysis of the expression of sarcomeric MHC in differentiating C2C12 cells. Cell lysates were prepared, separated by SDS-PAGE (5%), and subjected to immunoblot- ting with antibodies against sarco- meric MHC.

Effects of filamin-B variants on myogenesis. Confocal microscopy of C2C12 myoblasts stably expressing GFP fusions of filamin-B variants, 6 d after induction of differentiation. Sarcomeric MHC was immunolabeled in cells to facilitate myotube identification (A–D). The pouch-like myotubes of filamin-B and filamin-B_var-1 expressing cells and the thinner myotubes in cells expressing the ΔH1 variants are clearly detectable. (E) Staining of differentiated myotubes with anti-sarcomeric α-actinin. (F) Same field showing GFP fluorescence of filamin-B at Z- and M-lines (F). Arrowheads indicate Z-lines. Bars: (A–D) 100 μm; (E and F) 10 μm.