Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

Figures

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  Fig. 1.. Light scattering as a function of the concentrations of CaM2, SkM2, and SmM2 polymerized for overnight with (○) and without (●) ATP. The intersections of the curves for the polymerized myosins and myosin monomers (▼) are the critical polymerization concentrations. Note that the scale of the x axis is different for SmM2 than for CaM2 and SkM2. Data points are the average of at least two independent experiments.

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  Fig. 2.. Electron microscopy field images of filaments of CaM2, SkM2, and unphosphorylated SmM2 polymerized for 4 h in 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, and 10 mM Mops (pH 7.0) in the absence and presence of 1 mM ATP. Filaments were cross-linked for 30 min with 0.1 mM EDC before negative staining electron microscopy. (Scale bars: 100 nm.)

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  Fig. 3.. Electron microscopy of individual filaments of CaM2, SkM2, and SmM2 polymerized for 4 h and prepared for analysis as in Fig. 2. CaM2 and SkM2 formed bipolar filaments with (Upper) and without (Lower) bare zones in both the presence and absence of ATP. In the absence of ATP, SmM2 formed bipolar filaments with (Top) and without (Middle) a bare zone and side-polar filaments (Bottom). In the presence of ATP, SmM2 formed only bipolar filaments. Red arrows identify clusters of four heads. (Scale bar: 100 nm.)

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  Fig. 4.. Characterization by electron microscopy of CaM2, SkM2, and SmM2 filaments after polymerization for 4 h in the presence and absence of ATP. The percent of bipolar filaments with (white bar) and without (black bar) bare zones and side polar filaments (gray bar) were for more than 100 filaments in 20 fields of each of three sets each myosin polymerized as in Figs. 2 and 3. Data were from three separate preparations. Error bars in the figure denote SD. All data expressed as mean ± SD. Statistically significant (P value) was calculated using two tailed Student’s t test. N.S., not significant (P > 0.05); **P ≤ 0.01; ***P ≤ 0.001.

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  Fig. 5.. Electron microscopy field images of CaM2, SkM2, and SmM2 polymerized for 4 s in 150 mM NaCl, 2 mM MgCl2, 1 mM DTT and 10 mM Mops (pH 7.0) without and with 1 mM ATP (SmM2). Filaments were cross-linked for 1 min with 0.1 mM glutaraldehyde before negative staining electron microscopy. M, D, PD, and T stand for folded monomer, folded antiparallel dimer, folded parallel dimer, and folded antiparallel tetramer, respectively. Red arrows indicate a cluster of four heads, green arrows association of folded tetramers with growing filaments, and yellow arrows overlapping of growing filaments. (Scale bar: 100 nm.)

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  Fig. 6.. Folded monomers, dimers, and tetramers of CaM2, SkM2, and SmM2 formed during polymerization for ∼4 s. SkM2 and CaM2 were polymerized in the same buffer as in Fig. 2 without ATP, and SmM2 was polymerized in the same buffer both with and without 1 mM ATP. Images show folded monomers (M), folded dimers (D), and folded and partially unfolded tetramers (T). (Scale bar: 100 nm.)

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  Fig. 7.. Individual polymerizing filaments of CaM2, SkM2, and SmM2. Clusters of four heads (red arrows), three heads (blue arrows), and folded tetramers (yellow arrows) on polymerizing myosin filaments at ∼4 s in the same buffer as in Fig. 2. (Scale bar: 100 nm.)

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  Fig. 8.. Growing filaments overlap during the polymerization of CaM2, SkM2, and SmM2. Samples were taken after polymerization for ∼4 s as in Fig. 5. Overlapping filaments (green arrows) and clusters of four heads (red arrows) are identified. (Scale bar: 100 nm.)

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  Fig. 9.. Depolymerization of SmM2 is the reverse of polymerization. (A) Filaments polymerized without ATP, as in Fig. 2, but overnight to ensure complete polymerization, were a mixture of bipolar filaments without or with bare zones and side-polar filaments. (B) Images of filaments depolymerizing ∼4 s after addition of 50 µM ATP and cross-linked with 0.1 mM glutaraldehyde. 1 and 2 show overlapping filaments (green arrows); 3–6 show folded and partially folded tetramers (yellow arrows) associated with depolymerizing filaments; Individual folded tetramers (T), folded dimers (D), and folded monomers (M) also exist ∼4 s after depolymerization. Occasional folded parallel dimers (PD) were present, but no unfolded monomers were seen. (Scale bars: 100 nm.)