Structure and Nucleotide-Dependent Changes of Thick Filaments in Relaxed and Rigor Plaice Fin Muscle M. E. Cantino,* ,1 M. W. K. Chew,† P. K. Luther,† E. Morris,† , ‡ and J. M. Squire† *Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269-2242; †Biological Structure & Function Section, Biomedical Sciences Division, Faculty of Medicine, Imperial College, London SW7, United Kingdom; and ‡Biological Sciences Department, Imperial College, London SW7, United Kingdom Received January 30, 2002, and in revised form March 11, 2002 The myosin crossbridge array, positions of non- crossbridge densities on the backbone, and the A- band “end filaments” have been compared in chem- ically skinned, unfixed, uncryoprotected relaxed, and rigor plaice fin muscles using the freeze-frac- ture, deep-etch, rotary-shadowing technique. The images provide a direct demonstration of the heli- cal packing of the myosin heads in situ in relaxed muscle and show rearrangements of the myosin heads, and possibly of other myosin filament pro- teins, when the heads lose ATP on going into rigor. In the H-zone these changes are consistent with crossbridge changes previously shown by others us- ing freeze-substitution. In addition, new evidence is presented of protein rearrangements in the M-re- gion (bare zone), associated with the transition from the relaxed to the rigor state, including a 27-nm increase in the apparent width of the M-re- gion. This is interpreted as being mostly due to loss or rearrangement of a nonmyosin (M9) protein com- ponent at the M-region edge. The structure and titin periodicity of the end-filaments are described, as are suggestions of titin structure on the myosin fil- ament backbone. © 2002 Elsevier Science (USA) Key Words: actin filaments; contractile mecha- nism; crossbridge cycle; myosin ATPase; myosin fil- ament backbone; myosin crossbridge helix; titin. INTRODUCTION Development of a detailed description of the con- tractile events in muscle at a molecular level de- pends on information about the three-dimensional arrangement of myosin heads around the myosin backbone, both at rest and after release from the myosin backbone in other muscle states (Holmes, 1996; Huxley, 1969; Lymn and Taylor, 1971; Squire, 1997). In addition, there is still a great deal to learn about the association of other filamentous proteins, such as nebulin and titin (Labeit and Kolmerer, 1995; Trinick, 1994), along with nonmyosin pro- teins, such as C-protein and X-protein (Bennett et al., 1986), with actin and myosin filaments in skel- etal muscle. Because of its simple A-band lattice, fish skeletal muscle has been found to be particularly useful in structural studies of the sarcomere (Harford and Squire, 1986; Luther et al., 1981, 1996). Previous elec- tron microscopy studies of fish muscle myosin fila- ments have utilized either single filaments (Kensler and Stewart, 1989; Luther et al., 1996), which show good helical order but are isolated from the myofila- ment lattice, or chemically fixed fiber preparations (Luther et al., 1981, 1996; Varriano-Marston et al., 1984), in which the resting myosin head configuration does not appear to be fully preserved. We have been investigating myosin and actin filament structure in situ in chemically skinned, unfixed, uncryoprotected plaice fin muscles using freeze-fracture, deep-etch, ro- tary-shadowing techniques (Cantino et al., 2000; Cantino and Squire, 1986; Heuser, 1981) applied to plunge-frozen fibers. The present paper continues this study. The data and images presented here provide a direct demonstration of the helical packing of the my- osin heads in situ in relaxed muscle (Cantino et al., 2000; Hudson et al., 1997; Squire et al., 1998). Also presented are details of the structures of end filaments (Craig and Knight, 1983; Funatsu et al., 1993; Kensler and Stewart, 1983; Liversage et al., 2001; Trinick, 1981) together with suggestions of titin structure along the main part of the myosin filament backbone, including the bare zone. Previous studies have indicated that there is a significant rearrangement/disordering of the myosin when the heads lose ATP (Clarke, 1986; Craig and 1 To whom correspondence should be addressed at Department of Physiology and Neurobiology, Unit 2242, University of Con- necticut, Storrs, CT 06269-2242. Fax: (860) 486-6369. E-mail: cantino@uconnvm.uconn.edu. Journal of Structural Biology 137, 164 –175 (2002) doi:10.1006/jsbi.2002.4474 164 1047-8477/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.