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First published online November 30, 2007
Journal of Experimental Biology 210, 4399-4410 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008722
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Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch

Daisuke Funabara1,*, Chieko Hamamoto2,*, Koji Yamamoto1, Akinori Inoue1, Miki Ueda1, Rika Osawa1, Satoshi Kanoh1, David J. Hartshorne3, Suechika Suzuki2 and Shugo Watabe4,{dagger}

1 Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
2 Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293, Japan
3 Muscle Biology Group, University of Arizona, Tucson, AZ 85721, USA
4 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8567, Japan


Figure 1
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  		Fig. 1. A schematic representation of the ABRM twitchin molecule and mutants of the D2 peptide. (A) The motif structure of ABRM twitchin is shown together with the region expressed as various 6xHis-fusion proteins that are used in the present study. The D1 and D2 sites are S1075 and S4316, respectively. (B) Phosphorylation of twitchin D2 peptide mutants by PKA. TWD2-S was phosphorylated, whereas TWD2-A and TWD2-D were not phosphorylated. Phosphorylation of TWD2-S was detected by SDS-gel electrophoresis as described previously (Funabara et al., 2001aGo).

 

Figure 2
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  		Fig. 2. Effects of twitchin D2 peptide mutants on actomyosin Mg2+-ATPase activity. (A) ABRM actomyosin Mg2+-ATPase activity in the presence of 10–4 mol l–1 Ca2+. (B) ABRM actomyosin Mg2+-ATPase activity in the absence of Ca2+. (C) Chicken fast skeletal actomyosin Mg2+-ATPase activity in the presence of 10–4 mol l–1 Ca2+. (D) Actin-activated Mg2+-ATPase activity measured with chicken fast muscle myosin plus chicken F-actin and 10–4 mol l–1 Ca2+. (E) Chicken fast skeletal actomyosin Mg2+-ATPase activity in the presence of 10–4 mol l–1 Ca2+ and 0.4x10–6 mol l–1 ABRM paramyosin. See Materials and methods for details. Values shown are means ± s.d. Numbers above columns are N values. D and A mutants of the TWD2-S are indicated; here TWD2-S is unphosphorylated and Thio-TWD2-S is thiophosphorylated.

 

Figure 3
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  		Fig. 3. Binding of twitchin D2 peptide to actin, myosin and paramyosin. (A,C,E) Typical results of solid-phase binding assays for unphosphorylated TWD2-S and thiophosphorylated TWD2-S (Thio-TWD2-S) peptides against chicken fast skeletal actin, scallop myosin and ABRM paramyosin, respectively. (B,D,F) Relative binding abilities of twitchin D2 peptide to chicken fast skeletal actin, scallop myosin and ABRM paramyosin, respectively. Values are means ± s.d. (N=6).

 

Figure 4
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  		Fig. 4. Identification of a twitchin D2 peptide binding region on actin. (A) SDS-PAGE patterns of the digests of chicken fast skeletal actin by trypsin. Numbers above the gel represent the digestion time (h). M, molecular markers. (B) Isolation of the peptide that reacts to unphosphorylated TWD2-S from the digests of actin by reverse-phase high performance liquid chromatography with a TSKgel ODS-80T column (4.6 mmx15 cm). Numbers in the graph represent the fraction numbers collected in this experiment. Each fraction was subjected to a solid-phase binding assay with TWD2-S. Only Fraction 16 (asterisk) reacted with TWD2-S. The inset shows the results of the colorimetric binding assay. Note the change of color only for fraction 16. (C) Second reverse-phase chromatography for fraction 16. Fraction 16 was separated into three peaks and only fraction 16-2 reacted to TWD2-S. The inset shows the results of the colorimetric binding assay; only fraction 16-2 was positive. (D) The binding region of actin with the D2 peptide. Asterisks represent two aspartic acid residues essential for myosin-driven movement of thick filaments on actin-containing thin filaments in the presence of ATP and Ca2+. The sequence of the isolated peptide that reacted to TWD2-S is shown in red. The peptide synthesized and used for competitive binding assay with TWD2-S in the present study is represented in green. (E) Competitive binding assay between chicken fast skeletal actin and its synthetic peptide AGFAGDDAP, measured by solid-phase binding assays (see Materials and methods). TWD2-S was displaced from actin by increasing concentrations of the synthetic peptide. (F) A structural representation of actin. The TWD2-S binding region is shown in yellow.

 

Figure 5
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  		Fig. 5. The binding between scallop striated adductor myosin and chicken fast skeletal F-actin and the influence of the twitchin D2 peptide under catch conditions. SDS-PAGE results for the mixtures indicated are shown. S, supernatant; P, pellet (see Materials and methods). (A) Results using a molar ratio of peptide:myosin of 50:9. TWD2-S facilitated the binding between actin and myosin, whereas the thiophosphorylated form (thio-TWD2-S) did not. Arrowheads indicate F-actin, which co-sedimented with myosin. (B) The concentration-dependence of F-actin binding with myosin via TWD2-S. Samples for SDS-PAGE contained 10 µg actin (S) or myosin (P). For the actin–myosin coprecipitate, samples applied contained 10 µg myosin.

 

Figure 6
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  		Fig. 6. The specificity of anti-twitchin D2 antibody. (A) Immunoblotting using the anti-twitchin D2 antibody against ABRM myofibrillar proteins. Lane 1, protein-loaded gel stained with Commassie Brilliant Blue; lane 2, antibody loaded onto the PVDF membrane reacted only with twitchin and not to the major components of thick filaments, myosin heavy chain, myorod and paramyosin. (B) Immunoblotting using the anti-twitchin D2 antibody against TWD2-S and its phosphorylated form (P-TWD2-S). Arrowheads indicate twitchin peptide and the positions of molecular mass markers (kDa) are shown. (C) Differences in the reactivity of the anti-twitchin D2 antibody against TWD2-S and P-TWD2-S. Closed and open circles represent TWD2-S and P-TWD2-S, respectively.

 

Figure 7
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  		Fig. 7. Electron microscopic observation on ultrathin sections of ABRM reacted with anti-twitchin D2 peptide antibody in active contraction, catch and relaxation stages. Longitudinal (A,C,E,G) and cross-sectional (B,D,F,H) views for ABRM in active contraction (A,B), ABRM in catch (C,D), ABRM in relaxation (E,F) and ABRM without the antibody (control) (G,H). Bars, 200 nm (A–F), 500 nm (G), 100 nm (H).

 

Figure 8
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  		Fig. 8. Histogram showing frequency distribution of distances between any two gold particles (corresponding to the location of the D2 antibody) on the same thick filament, as seen by electron microscopy, and the statistical significance analysis. (A) Frequency was set up for a total of 155 measurements of not more than 400 nm and analyzed on the assumption of compound normal distributions with parameters indicated by the maximum likelihood method. (B) Estimated slope (36.29) of the regression line through the origin, compared to 36.25 (see text for details). The two values were not statistically different (F-test, P=0.91).

 

Figure 9
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  		Fig. 9. Electron microscopic observation on thick filaments labeled with anti-twitchin D2 peptide antibody. (A) Electron micrographs of ABRM thick filaments labeled with the anti-twitchin D2 peptide antibody and negatively stained. Antibodies conjugated with gold particles, indicating localization of twitchin, are distributed on the surface of the filaments at intervals (upper panel) and at helical turns (lower panel). (B) Electron micrograph of a thick filament treated with low angle rotary shadowing after negative staining. The secondary antibody-conjugated gold particles are localized on globular structures. (C) Stereo views of negatively stained thick filaments. Ultrathin filaments, possibly representing twitchin molecules, expand longitudinally on the thick filament as indicated by the white arrow. Arrowheads indicate location of the antibody-conjugated gold particles. (D) Electron microscopic observation of twitchin molecules by rotary shadowing. Twitchin molecule (left) and after treatment with anti-twitchin kinase domain antibody (right). Twitchin (0.06 mg ml–1) was reacted with the anti-twitchin kinase domain antibody (Funabara et al., 2001aGo) and mixed with 40% glycerol. This preparation, and a sample of twitchin without antibody, were sprayed onto mica and subjected to rotary shadowing using platinum and carbon as described above. (E) Models of the parallel array of twitchin molecules (red) superimposed on the Bear-Selby net pattern (Bear and Selby, 1956Go) and relative to myosin head distribution (blue). Bars, 100 nm (A–C), 50 nm (D). The Bear-Selby net reflects the arrangement of paramyosin molecules in the thick filament. The paramyosin molecules assemble into fibers with an axial repeat of 72.5 nm and staggering of these filaments generates the characteristic `checkerboard' array of nodes. In negatively stained samples the nodes are the gaps between molecules where stain is trapped (Squire, 1981Go; Cohen, 1982Go).

 

Figure 10
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  		Fig. 10. A model of interactions of twitchin with myosin and actin for different stages in the contractile cycle. For explanation, see text. Tropomyosin in thin filaments is not shown.

 




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