Search

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

The remarkable order of striated muscle is the result of a complex series of protein interactions at different levels of organization. Within muscle, the thick filament and its major protein myosin are classical examples of functioning protein machines. Our understanding of the structure and assembly of thick filaments and their organization into the regular arrays of the A-band has recently been enhanced by the application of biochemical, genetic, and structural approaches. Detailed studies of the thick filament backbone have shown that the myosins are organized into a tubular structure. Additional protein machines and specific myosin rod sequences have been identified that play significant roles in thick filament structure, assembly, and organization. These include intrinsic filament components, cross-linking molecules of the M-band and constituents of the membrane-cytoskeleton system. Muscle organization is directed by the multistep actions of protein machines that take advantage of well-established self-assembly relationships., (Copyright 1999 John Wiley & Sons, Inc.)

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, beta-filagenin, and then identified a gene that encodes a novel protein of 201-amino acid residues from databases using these sequences. beta-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four beta-strands but no alpha-helices. Western blotting using an affinity-purified antibody showed that beta-filagenin was associated with the cores. beta-Filagenin was localized by immunofluorescence microscopy to the A bands of body-wall muscles, but not the pharynx. beta-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, beta-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that beta-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.

The analysis of both naturally occurring and experimentally induced mutants has greatly advanced our understanding of muscle development. Molecular biological techniques have led to the isolation of genes associated with inherited human diseases that affect muscle tissues. Analysis of the encoded proteins in conjunction with the mutant phenotypes can provide powerful insights into the function of the protein in normal muscle development. Systematic searches for muscle mutations have been made in experimental systems, most notably the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. In addition, known muscle protein genes from other organisms have been used to isolate homologs from genetically manipulatable organisms, allowing mutant analysis and the study of protein function in vivo. Mutations in transcription factor genes that affect mesoderm development have been isolated and genetic lesions affecting myofibril assembly have been identified. Genetic experiments inducing mutations and rescuing them by transgenic methods have uncovered functions of myofibrillar protein isoforms. Some isoforms perform muscle-specific functions, whereas others appear to be replaceable by alternative isoforms. Mutant analysis has also uncovered a relationship between proteins at the cell membrane and the assembly and alignment of the myofibrillar apparatus. We discuss examples of each of these genetic approaches as well as the developmental and evolutionary implications of the results.

The challenge presented by myofibril assembly in striated muscle is to understand the molecular mechanisms by which its protein components are arranged at each level of organization. Recent advances in the genetics and cell biology of muscle development have shown that in vivo assembly of the myofilaments requires a complex array of structural and associated proteins and that organization of whole sarcomeres occurs initially at the cell membrane. These studies have been complemented by in vitro analyses of the renaturation, polymerization, and three-dimensional structure of the purified proteins.

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.

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 (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. 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.