Different obscurin isoforms localize to distinct sites at sarcomeres

Abstract

We used four antibodies to regions of obscurin isoforms A and B, encoded by the obscurin gene, to investigate the location of these proteins in skeletal myofibers at resting and stretched lengths. Obscurin A ( approximately 800 kDa) which was recognized by antibodies generated to the N-terminal, Rho-GEF, and the non-modular C-terminal domain that lacks the kinase-like domains, localizes at the level of the M-band. Obscurin B ( approximately 900 kDa) which has the N-terminal, Rho-GEF, and the C-terminal kinase-like domains, localizes at the level of the A/I junction. Additional isoforms, which lack one or more of these epitopes, are present at the Z-disk and Z/I junction.

Figure 1. Obscurin isoforms

A.Antibodies were prepared to distinct regions of obscurin: the N-terminal Ig domain, and the Rho-GEF domain, the non-modular C-terminus of obscurin A and to Ob68 adjacent to the first kinase-like domain (SKII) in the giant kinase isoform, obscurin B.

Figure 2. Localization of obscurin isoforms in muscle at resting length

(A-F) Double immufluorescence experiments with antibodies to Z-disk epitopes of titin (B, C, red) and α-actinin (E, F, green) to mark Z-disks, and antibodies to the N-terminus (A, C, green) and Rho-GEF domain of obscurin (D, F, red) show that the latter primarily label at the level of the M-band. (G-L) Similarly, with the two C-terminal antibodies, to the non-modular domain (G, I, red) and to Ob68 SKII (J, L, red). Both label primarily at the level of the Z-disk and M-band, identified by comparison with α-actinin (H, I; K, L green). Immunodepletion experiments are shown in the lower left corners of the larger panels (A, D, G, J) Average sarcomere lengths are indicated. Bars, 5 μm.

Figure 3. Localization of obscurin isoforms in stretched muscle

As in Figure 2, but muscle was stretched to 1.3 - 1.7 L_o before immunolabeling. (A-F) In addition to M-bands, antibodies to the N-terminus obscurin label the I-band near the A/I junction (A, C, D, F; green; compared to titin at the Z-disk: B, C, E, F; red). (G-L) Antibodies to the Rho-GEF domain also react with structures at both the M-band and A/I junction (G, I, J, L; red; compared to α-actinin H, I, K, L; green). (M-R) The non-modular C-terminus of obscurin is at the level of the Z-disk and M-band (M, O; red; compared to α-actinin: N, O; green) at ˜1.3 L_o. Greater stretching to ˜1.7L_o produces splitting of the C-terminal antibody labeling of the Z-disk (P, R; red; compared to α-actinin: Q, R; green). (S-X) Antibodies to Ob68 label at the level of the M-band and at two locations within the I-band, near the A/I junction, and at the Z/I interface (S, U, V, X; red, compared to α-actinin: T, U, W, X; green). Average sarcomere lengths are indicated. Bars, 1 μm.

Figure 4. Immunoblotting of obscurins A and B

In immunoblots of extracts of adult quadriceps, antibodies to the Rho-GEF domain of obscurin and the N-terminal Ig domain recognize the 800kDa isoform (large arrow), likely to be obscurin A, and a larger isoform that is present at much lower levels (small arrow), likely to be obscurin B. Antibodies to the C-terminus of obscurin A recognize the obscurin A isoform, but not the obscurin B isoform. Molecular masses are indicated.

Figure 5. Schematic representation of localization of distinct obscurin isoforms

The sarcomeric locations of each of the isoforms of obscurin we have identified are indicated. An additional isoform that has not been well characterized is likely to be located at the level of the Z-disk. See text for discussion.