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Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D. M. III, I. Ortiz, G. C. Berliner, and H. F. Epstein, 1983, Cell, 34:477-490). Biochemical and electron microscope studies of KCI-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.The body wall muscle cells of the nematode Caenorhabditis elegans contain two chemically and genetically distinct isoforms of myosin heavy chain, A and B. Immunocytochemical localization with both affinity purified and specific monoclonal antibodies shows that the two isoforms coexist in the same muscle cells and sarcomeres (13,17). All isolated thick filaments react with the monoclonal antibodies of either specificity, indicating that the isoforms are contained within the same filaments. However, the A form is localized to the central 1.8 #m of the 9.7-#m-long filaments, whereas the B form is located in the polar region, but is absent in the central 0.9 #m of the filament (17). Fig. 1 is a schematic diagram showing locations of the A and B heavy chains and examples of thick filaments that were reacted with the specific antibodies. The surface of nematode thick filaments is differentiated by myosin isoform content into five zones.The locations of the myosin heavy chain isoforms in this model explain several independent observations regarding nematode body wall myosins. Previous studies of nematode myosins indicate that native molecules are homodimers of either heavy chain isoform; no heterodimers are detected (21,22). The different locations of the heavy chain types imply that the myosin molecules within thick filaments are primarily homodimers. The ratio of B to A heavy chains appears constant, whereas their amounts increase 40-fold during the post-embryonic stages of the nematode life cycle (4). This fixed ratio, ~4:1 (28), is consistent with the distributions of the two forms in the thick filaments shown in Fig. 1.The different locations of myosins A and B have important structural correlations. The central regions that contain only myosin A are the regions in which myosin molecules pack in a bipolar fash...

Abstract. The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2 % with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C elegans. M YOSIN-CONTAINING filaments have been a major focus of research for over 30 years (11). The demonstration that myosin was contained in one set of filaments was critical to the foundation of the sliding-filament theory of muscle contraction (15). The isolation of natural filaments containing myosin and the formation of synthetic filaments from purified myosin were key confirmations of the theory and raised the important question as to whether the intrinsic self-assembly of myosin molecules explained the formation of thick filaments in vivo (14).The discovery that additional, nonmyosin proteins are components of natural thick filaments in both vertebrate and invertebrate muscles suggests that the assembly of these filaments may be more complex than simple self-assembly of myosin. For example, recent studies of rabbit skeletal muscles indicate that epitopes of the C, H, and X proteins are localized at very specific and distinct positions near the surface of specific thick filaments (1). Studies of various invertebrate muscles show that paramyosin is a major protein component of the backbones of the thick filaments (30). In other invertebrate muscles, paramyosin may be present only as a relatively minor protein (3,12,25). The diversity of nonmyosin protein components of thick filaments, their locations, and their relative amounts supports the alternative hypothesis that the assembly of thick filaments, in general, may require a sequence of specific reactions involving bot...

A spectrum of thick filament-related structures exhibiting novel structural features is isolated in addition to the normal thick filaments from unc-15 and unc-82 mutants of Caenorhabditis elegans. Many assemblages have multiple myosin-coated filaments extending from both ends of central domains exhibiting paracrystalline paramyosin. The filament ends resemble the polar core structures of native thick filaments. Assemblages with filaments at only one end and short thick filaments that branch are also present. This spectrum of novel structures accumulates at high levels in specific mutants due to alterations in paramyosin or other interacting proteins. The multifilament structures are either alternative assemblages of thick filament proteins and substructures or usually transient nucleation centres active in the assembly of thick filaments which are favoured under mutant conditions.