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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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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.
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fig10: 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.

Mentions: The functional significance of the developmentally regulated splicing of the H1 region in filamin-B, as well as the deletion of the 41–amino 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(ΔH1) fused into myotubes within 2 to 3 d after the medium switch, which is 1–2 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[ΔH1] and filamin-Bvar-1[ΔH1]) 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) .


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199218&req=5

fig10: 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.
Mentions: The functional significance of the developmentally regulated splicing of the H1 region in filamin-B, as well as the deletion of the 41–amino 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(ΔH1) fused into myotubes within 2 to 3 d after the medium switch, which is 1–2 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[ΔH1] and filamin-Bvar-1[ΔH1]) 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) .

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.

Show MeSH
Related in: MedlinePlus