JMB—MS 661 Cust. Ref. No. PEW 077/95 [SGML] J. Mol. Biol. (1995) 251, 191–196 COMMUNICATION Steric-blocking by Tropomyosin Visualized in Relaxed Vertebrate Muscle Thin Filaments William Lehman 1 *, Peter Vibert 2 , Pedro Uman 3 and Roger Craig 3 1 Department of Physiology Although widely accepted, the steric-blocking model of vertebrate skeletal muscle regulation has not been confirmed. Previous attempts to directly Boston University School of visualize tropomyosin in relaxed skeletal muscle and demonstrate that Medicine, 80 East Concord it interferes with the crossbridge–thin filament contractile cycle were Street, Boston, Massachusetts 02118, USA unsuccessful. In the work reported here, tropomyosin was resolved in electron micrographs of native thin filaments isolated from relaxed vertebrate 2 Rosenstiel Basic Medical striated muscle. Three-dimensional helical reconstructions of these filaments Research Center, Brandeis showed continuous narrow strands of density, representing tropomyosin, University, Waltham which followed the outer domains of successive actin monomers. The results Massachusetts 02554, USA obtained from fitting the atomic model of filamentous actin to these reconstructions illustrate, and are consistent with, the mechanism of 3 Department of Cell Biology steric-blocking, since tropomyosin was found to be positioned on the actin University of Massachusetts surface of thin filaments over clusters of identifiable amino acids required Medical School, Worcester for myosin crossbridge docking. Massachusetts 01655, USA  1995 Academic Press Limited Keywords: actin; tropomyosin; thin filaments; muscle regulation; electron *Corresponding author microscopy X-ray diffraction studies performed over 20 years ago on vertebrate skeletal muscle suggested that, during activation, tropomyosin movement occurred on thin filaments (Huxley, 1972; Haselgrove, 1972; Parry & Squire, 1973) prior to the onset of cross- bridge cycling (Kress et al ., 1986). This Ca 2+ -induced movement was thought to expose the blocked myosin-binding sites on actin, allowing crossbridge interaction and contraction to proceed. Over the intervening years, attempts to directly visualize tropomyosin movement in vertebrate skeletal muscle by electron microscopy failed to provide definitive evidence for the steric model of muscle regulation. Although tropomyosin was visualized, and its position determined, in filaments in the high Ca 2+ ‘‘on’’-state, its location in the ‘‘off’’-state was not ascertained, possibly because of disorder (Milligan et al ., 1990). This uncertainty contributed to skeptic- ism about the steric model. Recent studies on native thin filaments of Limulus muscle, however, resolved tropomyosin not only in the on-state but also in the off-state (Lehman et al ., 1994), and this finding led us to reinvestigate the structure of vertebrate muscle filaments in the off-state. Preparations of native thin filaments isolated by newly developed methods were particularly well suited for electron microscopy and three-dimensional reconstruction, and enabled us to resolve tropomyosin in frog thin filaments in the off-state. Fitting the known atomic structure of the actin molecule (Lorenz et al ., 1993) into its position within the actin–tropomyosin envelope of our maps demonstrated that tropomyosin is positioned on the surface of actin over several clusters of amino acids involved in myosin binding (Rayment et al ., 1993). By defining the off-state in near-atomic terms, we have thus been able to provide support for the steric blocking model. Negatively stained native thin filaments (Fig- ure 1A) isolated from frog thigh muscle under relaxing conditions exhibit the characteristic double- helical array of actin subunits. In addition, they frequently display elongated strands of tropomyosin aligned with the long-pitch actin helices, as well as periodic bulges repeating at 40 nm intervals, which presumably arise from troponin (Vibert et al ., 1993; Lehman et al ., 1994). The bulges, however, are not as large as those detected in arthropods (Bullard et al ., 1988; Lehman et al ., 1994), probably because the mass of troponin in vertebrates is smaller. Density maps were calculated from the averaged Abbreviations used: F-actin, filamentous actin; S.D., standard deviation; HRP, horseradish peroxidase; EGTA, ethyleneglycol bis (-aminoethylether)-N,N'-tetraacetic acid; RPM, revolutions per minute. This paper is dedicated to Drs H. E. Huxley and A. G. Szent-Gyo ¨rgyi on the occasion of their seventieth birthdays. 0022–2836/95/320191–06 $12.00/0  1995 Academic Press Limited