Purification of Native Myosin Filaments from Muscle Carlos Hidalgo, Raúl Padrón, Rachel Horowitz, Fa-Qing Zhao, Roger Craig  Biophysical Journal  Volume 81, Issue 5, Pages 2817-2826 (November 2001) DOI: 10.1016/S0006-3495(01)75923-1 Copyright © 2001 The Biophysical Society Terms and Conditions

Figure 1 SDS-PAGE analysis of gelsolin and gelsolin fragments. The 10% gel was stained with Coomassie brilliant blue. Lane 1, low molecular weight markers; lane 2, gelsolin peak (∼93kDa, arrow) eluting from DEAE column; lane 3, gelsolin fragments (43 and 45kDa, arrows) resulting from thermolysin proteolytic digestion of gelsolin; lane 4, high molecular weight markers. Biophysical Journal 2001 81, 2817-2826DOI: (10.1016/S0006-3495(01)75923-1) Copyright © 2001 The Biophysical Society Terms and Conditions

Figure 2 Negative staining electron microscopy and image analysis of tarantula thick filament purification. (A—C) Electron micrograph of a filament field (i), a selected thick filament (ii), and its computed Fourier transform (iii). (A) Original filament suspension in relaxing solution, pH 6.5, showing thin filaments and helically ordered thick filaments. (B) Filament suspension after gelsolin fragment treatment. Thin filaments are removed, and thick filaments show excellent helical order. (C) Resuspended pellet after treatment of filament suspension with gelsolin fragments followed by first differential centrifugation. In all micrographs, the appearance of helical order of thick filaments is enhanced by viewing obliquely along the filament length or by turning them horizontally. Optical diffraction patterns are marked with arrows pointing to the approximate position of layer lines 1 (43.5nm), 2 (21.7), 3 (14.5nm), 4 (10.8nm), and 6 (7.25nm). Note that purification of the thick filaments by centrifugation removes most soluble proteins, whose presence would normally aid stain spreading (Gregory and Pirie; 1973; Hayat, 2000) and hence preservation of helical order (see text). To demonstrate the presence of helical order in the purified filaments, soluble protein (such as gelsolin fragments, cytosol (supernatant from high-speed centrifugation of the original suspension) or ultrafiltrate of the original filament suspension) was therefore added to the filament suspension or used to rinse the grid before staining. Scale bar, 50nm. Biophysical Journal 2001 81, 2817-2826DOI: (10.1016/S0006-3495(01)75923-1) Copyright © 2001 The Biophysical Society Terms and Conditions

Figure 3 SDS-PAGE analysis of the purification of thick filaments. The 13% gel was stained with Coomassie brilliant blue. (A) Control, centrifugation without gelsolin treatment: lane 1, high molecular weight markers; lane 2, crude filament suspension; lane 3, filament suspension plus relaxing solution; lane 4, supernatant 1 (from first centrifugation); lane 5, pellet 1; lane 6, supernatant 2 (from second centrifugation); lane 7, pellet 2; lane 8, low molecular weight markers. (B) Centrifugation of filament suspension after gelsolin fragment treatment: lane 1, high molecular weight markers; lane 2, crude filament suspension (arrows indicate myosin, paramyosin, actin, tropomyosin, RLC, and ELC); lane 3, filament suspension after gelsolin fragment treatment; lane 4, gelsolin fragments (some undigested gelsolin is also present); lane 5, supernatant 1; lane 6, pellet 1; lane 7, supernatant 2; lane 8, pellet 2 (arrows indicate nonmyosin components at ∼160, 66, and 16kDa); lane 9, low molecular weight markers. (C) Densitometric analysis of the myosin heavy chain/actin ratio in the original filament suspension (FS) and in pellets 1 (P1) and 2 (P2) after centrifugation with and without gelsolin fragment (GF) treatment. Biophysical Journal 2001 81, 2817-2826DOI: (10.1016/S0006-3495(01)75923-1) Copyright © 2001 The Biophysical Society Terms and Conditions

Figure 4 Cryo-electron microscopy and image analysis of filament suspension from tarantula striated muscle. (A and B) An electron micrograph of a filament field (i), a selected thick filament (ii), its computed Fourier transform (iii). (A) Original filament suspension in relaxing solution, pH 6.5, showing thin filaments and helically ordered thick filaments. This was a rare image where relatively few thin filaments were present. (B) Filament suspension after gelsolin fragment treatment. Thin filaments are removed, and thick filaments show excellent helical order (judged both by eye and by the layer lines visible in the Fourier transform), but background is high. Centrifugation removes the background, and clear helical order is visible by eye, but Fourier transforms are less strong (data not shown). It is possible that, as with negative staining, some soluble protein is required to create a specimen layer over the holes in the support film that is optimal for cryo-preservation of the labile array cross-bridges. Scale bar, 50nm. Biophysical Journal 2001 81, 2817-2826DOI: (10.1016/S0006-3495(01)75923-1) Copyright © 2001 The Biophysical Society Terms and Conditions