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Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.

Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V - J. Cell Biol. (2003)

Bottom Line: In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum.Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

ABSTRACT
Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

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Localization of ank1.5 and obscurin in differentiating skeletal muscle cells. Differentiated skeletal muscle cells were stained with rabbit antibodies against the myc epitope to recognize the myc-tagged ank1.5 protein (A) and with monoclonal antibodies against α-actinin, which stain the Z line (B). The alternating pattern of ank1.5 and α-actinin is compatible with the localization of ank1.5 near or at the M line. Cells were stained with rabbit antibodies against the COOH-terminal region of obscurin (D) and with monoclonal antibodies against α-actinin (E). Again, the obscurin staining is compatible with localization near or at the M line. Staining of the cells with antibodies against ank1.5 (G) and against obscurin (H) indicates that the two proteins colocalize. Soluble ank1.5 (ank1.5 ΔTM) missing the transmembrane domain (K) is still able to colocalize with obscurin at the M line (L). A myc-tagged mutant ank1.5, unable to bind to obscurin, presents a diffuse signal (N) distinct from that of obscurin (M). Bar, 5 μm.
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fig5: Localization of ank1.5 and obscurin in differentiating skeletal muscle cells. Differentiated skeletal muscle cells were stained with rabbit antibodies against the myc epitope to recognize the myc-tagged ank1.5 protein (A) and with monoclonal antibodies against α-actinin, which stain the Z line (B). The alternating pattern of ank1.5 and α-actinin is compatible with the localization of ank1.5 near or at the M line. Cells were stained with rabbit antibodies against the COOH-terminal region of obscurin (D) and with monoclonal antibodies against α-actinin (E). Again, the obscurin staining is compatible with localization near or at the M line. Staining of the cells with antibodies against ank1.5 (G) and against obscurin (H) indicates that the two proteins colocalize. Soluble ank1.5 (ank1.5 ΔTM) missing the transmembrane domain (K) is still able to colocalize with obscurin at the M line (L). A myc-tagged mutant ank1.5, unable to bind to obscurin, presents a diffuse signal (N) distinct from that of obscurin (M). Bar, 5 μm.

Mentions: The evidence of a specific interaction between ank1.5 and obscurin obtained in in vitro experiments and in heterologous cells opens the question of whether an interaction between these proteins occurs also in skeletal muscle cells. To this end, cultures of differentiating primary skeletal muscle cells were used as an experimental model. To verify the specific localization of the ank1.5 isoform, differentiating skeletal muscle cells were transfected with a plasmid encoding a myc-tagged ank1.5 cDNA. Immunofluorescence studies using anti-myc antibodies and antibodies against α-actinin yielded two distinct alternating signals, which are compatible with the localization of ank1.5 near or at the M line (Fig. 5, A–C). In the original report on the localization of small muscle–specific ank1 isoforms (Zhou et al., 1997), a signal specific for the endogenous small ank1 isoforms was observed at the Z line and M line in skeletal muscle sections. This apparent discrepancy could be due to the transfection strategy and the use of primary cell cultures in our experimental approach. Alternatively, it could reflect a specific localization of ank1.5 with respect to other small ank1 isoforms, considering that Zhou et al. (1997) used a polyclonal antibody raised against an aa sequence that is present in ank1.5 but also in other small ank isoforms.


Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.

Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V - J. Cell Biol. (2003)

Localization of ank1.5 and obscurin in differentiating skeletal muscle cells. Differentiated skeletal muscle cells were stained with rabbit antibodies against the myc epitope to recognize the myc-tagged ank1.5 protein (A) and with monoclonal antibodies against α-actinin, which stain the Z line (B). The alternating pattern of ank1.5 and α-actinin is compatible with the localization of ank1.5 near or at the M line. Cells were stained with rabbit antibodies against the COOH-terminal region of obscurin (D) and with monoclonal antibodies against α-actinin (E). Again, the obscurin staining is compatible with localization near or at the M line. Staining of the cells with antibodies against ank1.5 (G) and against obscurin (H) indicates that the two proteins colocalize. Soluble ank1.5 (ank1.5 ΔTM) missing the transmembrane domain (K) is still able to colocalize with obscurin at the M line (L). A myc-tagged mutant ank1.5, unable to bind to obscurin, presents a diffuse signal (N) distinct from that of obscurin (M). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172649&req=5

fig5: Localization of ank1.5 and obscurin in differentiating skeletal muscle cells. Differentiated skeletal muscle cells were stained with rabbit antibodies against the myc epitope to recognize the myc-tagged ank1.5 protein (A) and with monoclonal antibodies against α-actinin, which stain the Z line (B). The alternating pattern of ank1.5 and α-actinin is compatible with the localization of ank1.5 near or at the M line. Cells were stained with rabbit antibodies against the COOH-terminal region of obscurin (D) and with monoclonal antibodies against α-actinin (E). Again, the obscurin staining is compatible with localization near or at the M line. Staining of the cells with antibodies against ank1.5 (G) and against obscurin (H) indicates that the two proteins colocalize. Soluble ank1.5 (ank1.5 ΔTM) missing the transmembrane domain (K) is still able to colocalize with obscurin at the M line (L). A myc-tagged mutant ank1.5, unable to bind to obscurin, presents a diffuse signal (N) distinct from that of obscurin (M). Bar, 5 μm.
Mentions: The evidence of a specific interaction between ank1.5 and obscurin obtained in in vitro experiments and in heterologous cells opens the question of whether an interaction between these proteins occurs also in skeletal muscle cells. To this end, cultures of differentiating primary skeletal muscle cells were used as an experimental model. To verify the specific localization of the ank1.5 isoform, differentiating skeletal muscle cells were transfected with a plasmid encoding a myc-tagged ank1.5 cDNA. Immunofluorescence studies using anti-myc antibodies and antibodies against α-actinin yielded two distinct alternating signals, which are compatible with the localization of ank1.5 near or at the M line (Fig. 5, A–C). In the original report on the localization of small muscle–specific ank1 isoforms (Zhou et al., 1997), a signal specific for the endogenous small ank1 isoforms was observed at the Z line and M line in skeletal muscle sections. This apparent discrepancy could be due to the transfection strategy and the use of primary cell cultures in our experimental approach. Alternatively, it could reflect a specific localization of ank1.5 with respect to other small ank1 isoforms, considering that Zhou et al. (1997) used a polyclonal antibody raised against an aa sequence that is present in ank1.5 but also in other small ank isoforms.

Bottom Line: In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum.Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

ABSTRACT
Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

Show MeSH