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Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

van der Flier A, Kuikman I, Kramer D, Geerts D, Kreft M, Takafuta T, Shapiro SS, Sonnenberg A - J. Cell Biol. (2002)

Bottom Line: When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes.Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region.These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

View Article: PubMed Central - PubMed

Affiliation: Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdams, Netherlands.

ABSTRACT
Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

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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-Bvar-1 (E), or filamin-Bvar-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-Bvar-1 and filamin-B(ΔH1) are associated with actin stress fibers: they did not accumulate in focal contacts. In contrast, filamin-Bvar-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.
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fig8: 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-Bvar-1 (E), or filamin-Bvar-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-Bvar-1 and filamin-B(ΔH1) are associated with actin stress fibers: they did not accumulate in focal contacts. In contrast, filamin-Bvar-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.

Mentions: The subcellular distribution of the different GFP-tagged filamin-B variants was examined in GD25-β1A mouse fibroblasts stably expressing these proteins. The localization of GFP-tagged filamin-B (Fig. 8, C and D) was identical to that of endogenous filamin-B, as revealed by immunostaining using an antibody against the H1 region of filamin-B (Fig. 8, A and B). Thus, the GFP tag did not interfere with the normal localization of filamin-B. Filamin-B was localized at actin stress fibers but was not appreciably concentrated in focal contacts. The distribution of filamin-B(ΔH1) and filamin-Bvar-1 variants was similar to that of filamin-B (Fig. 8, E and F). Interestingly, filamin-Bvar-1(ΔH1) was associated with a proportion of the peripheral focal contacts positive for vinculin (Fig. 8, G–O, and Fig. 9 E). There was also GFP fluorescence in the nucleus in many of these cells, the significance of which is unknown. As anticipated, the filamin-Bvar-1(ΔH1) variant did not react with anti–filamin-B H1 antibody. However, its presence in focal contacts was revealed by GFP fluorescence, whereas endogenous filamin-B, which does react with this antibody, was found associated with actin stress fibers (Fig. 8, M–O). Neither endogenous filamin-B (Fig. 8 B) nor any of the filamin-B variants (unpublished data) were enriched in lamellipodia. The expression of filamin-Bvar-1(ΔH1) in focal contacts did not have an apparent effect on the composition and localization of these structures. They were confined at the end of actin stress fibers (Fig. 8, J–L, and Fig. 9 F) and in addition to vinculin, they contained talin, phospho-tyrosine, and paxillin (Fig. 9, A, B, and D). We did not detect α-actinin in focal contacts (Fig. 9 C). Lastly, in GD25-β1D cells expressing filamin-Bvar-1(ΔH1) there was also a prominent staining of some focal contacts (unpublished data).


Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.

van der Flier A, Kuikman I, Kramer D, Geerts D, Kreft M, Takafuta T, Shapiro SS, Sonnenberg A - J. Cell Biol. (2002)

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-Bvar-1 (E), or filamin-Bvar-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-Bvar-1 and filamin-B(ΔH1) are associated with actin stress fibers: they did not accumulate in focal contacts. In contrast, filamin-Bvar-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.
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Related In: Results  -  Collection

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fig8: 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-Bvar-1 (E), or filamin-Bvar-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-Bvar-1 and filamin-B(ΔH1) are associated with actin stress fibers: they did not accumulate in focal contacts. In contrast, filamin-Bvar-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.
Mentions: The subcellular distribution of the different GFP-tagged filamin-B variants was examined in GD25-β1A mouse fibroblasts stably expressing these proteins. The localization of GFP-tagged filamin-B (Fig. 8, C and D) was identical to that of endogenous filamin-B, as revealed by immunostaining using an antibody against the H1 region of filamin-B (Fig. 8, A and B). Thus, the GFP tag did not interfere with the normal localization of filamin-B. Filamin-B was localized at actin stress fibers but was not appreciably concentrated in focal contacts. The distribution of filamin-B(ΔH1) and filamin-Bvar-1 variants was similar to that of filamin-B (Fig. 8, E and F). Interestingly, filamin-Bvar-1(ΔH1) was associated with a proportion of the peripheral focal contacts positive for vinculin (Fig. 8, G–O, and Fig. 9 E). There was also GFP fluorescence in the nucleus in many of these cells, the significance of which is unknown. As anticipated, the filamin-Bvar-1(ΔH1) variant did not react with anti–filamin-B H1 antibody. However, its presence in focal contacts was revealed by GFP fluorescence, whereas endogenous filamin-B, which does react with this antibody, was found associated with actin stress fibers (Fig. 8, M–O). Neither endogenous filamin-B (Fig. 8 B) nor any of the filamin-B variants (unpublished data) were enriched in lamellipodia. The expression of filamin-Bvar-1(ΔH1) in focal contacts did not have an apparent effect on the composition and localization of these structures. They were confined at the end of actin stress fibers (Fig. 8, J–L, and Fig. 9 F) and in addition to vinculin, they contained talin, phospho-tyrosine, and paxillin (Fig. 9, A, B, and D). We did not detect α-actinin in focal contacts (Fig. 9 C). Lastly, in GD25-β1D cells expressing filamin-Bvar-1(ΔH1) there was also a prominent staining of some focal contacts (unpublished data).

Bottom Line: When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes.Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region.These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

View Article: PubMed Central - PubMed

Affiliation: Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdams, Netherlands.

ABSTRACT
Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin beta1A and beta1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (DeltaH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin beta subunits. The mere presence of the high-affinity binding site for beta1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(DeltaH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

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