Henry F. Epstein

A mutant affecting the heavy chain of myosin in Caenorhabditis elegans

Abstract A set of non-complementing, closely linked, ethyl methanesulphonate-induced mutations in Caenorhabditis elegans specifically affects the structure and function of body-wall muscle cells but not the pharyngeal musculature. One of these mutations, e675, is semidominant and results in the production of a new protein of about 203,000 molecular weight in addition to normal myosin at about 210,000 Mr. The abnormal polypeptide chain is structurally very similar to normal myosin heavy chain when maps of iodinated peptides are compared. The E675 mutant shows a clear relation between defective movement, disruption of the body-wall muscle structure, and the molecular defect in the myosin heavy chains. The altered chain is synthesized in heterozygotes, suggesting that the e675 mutation is either in a structural gene for the heavy chain or in a cis acting control element. The hypothesis that there are two classes of myosin heavy chain within the same cells is discussed.

Myosin and paramyosin of Caenorhabditis elegans: Biochemical and structural properties of wild-type and mutant proteins

Myosin and paramyosin have been purified from the nematode, Caenorhabditis elegans. The properties of the myosin in general resemble those of other myosins. The native molecule is a dimer of heavy (210,000 dalton) polypeptide chains and contains 18,000 and 16,000 dalton light chains. When rapidly precipitated from solution, it forms small, bipolar aggregates, about 150 nm long, consistent with the expected molecular structure of a rigid rod with a globular head region at one end. Its ATPase activity is stimulated by Ca2+ and EDTA. The myosin binds to F actin in a polar and ATP-sensitive manner, and the Mg2+-ATPase is activated by either F actin or nematode thin filaments. Dialysis of myosin to low ionic strength produces very long filaments. When a myosin-paramyosin mixture is dialyzed under the same condtions, co-filaments form which consist of a myosin cortex, surrounding a paramyosin core. Some properties of myosin from the mutants E675 and E190, which have functionally and structurally altered body wall muscles, are compared with those of wild-type myosin. These myosins of these results are discussed in terms of the myosin heavy chain composition.

Paramyosin of Caenorhabditis elegans

Abstract Paramyosin has been isolated from the nematode, Caenorhabditis elegans . Its identity has been established by a variety of criteria, including purification, molecular weight, immunological cross reactivity with known paramyosin and formation of characteristic paracrystals. The presence of paramyosin in both pharyngeal and body-wall musculature was shown by a technique that allows analysis by sodium dodecyl sulphate gels of the protein in a single worm. The possibility of defining the role of paramyosin in the structure and function of the invertebrate muscle through the isolation of mutants in this protein is discussed.

Two homogeneous myosins in body-wall muscle of Caenorhabditis elegans

Abstract Myosin purified from the body-wall muscle-defective mutant E675 of the nematode. Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974). When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >80% 210,000 or >80% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains. Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains. E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled E190 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin.

Myosins exist as homodimers of heavy chains: Demonstration with specific antibody purified by nematode mutant myosin affinity chromatography

Abstract The body-walls of Caenorhabditis elegans contain two different myosin heavy chains (Epstein, Waterston and Brenner, 1974) that associate to form at least two species of myosin (Schachat, Harris and Epstein, 1977a). To better define the distribution of these heavy chains in myosin molecules, we have characterized the myosin of C. elegans by immunochemical methods. Specific, precipitating anti-myosin antibody has been prepared in rabbits using highly purified nematode myosin as the immunogen. The difference in reactivity of the anti-myosin antibody with wild-type myosin containing both kinds of heavy chains (designated unc -54 and non- unc -54 heavy chains on the basis of genetic specification) and myosin from the mutant E190 that lacks unc -54 heavy chains Indicates that there are antigenic differences between myosin molecules containing unc -54 heavy chains and myosin molecules containing only non- unc -54 heavy chains. Antibody specific for the unc -54 myosin determinants has been prepared by the immunoadsorption of anti-myosin antibody with E190 myosin. This specific anti- unc -54 myosin antibody precipitates myosin that contains only unc -54 heavy chains. At the limits of resolution of our immunoprecipitation techniques, we could detect no heterodimeric myosin molecules containing both unc -54 and non- unc -54 heavy chains. The body-wall myosins of C. elegans therefore exist only as homodimers of either class of heavy chain. This specific anti- unc -54 myosin antibody promises to be a valuable tool in elucidating the role of two myosins in body-wall muscle and in molecular characterizations of mutant myosins in C. elegans. We report here the use of this antibody to detect antigenic differences between unc -54 myosin from the wild-type and the muscle mutant E675. In conjunction with the original anti-myosin antibody, other studies show that both unc -54 and non- unc -54 myosins exist within the same body-wall muscle cells (Mackenzie, Schachat and Epstein, 1978) and that both myosins are coordinately synthesized during muscle development in C. elegans (Garcea, Schachat and Epstein, 1978). We discuss the implications of the self-association of unc -54 and non- unc -54 myosin heavy chains into homodimeric myosins within the same body-wall muscles with respect to the assembly of thick filaments and their organization into a regular lattice.

Muscle development in Caenorhabditis elegans: Mutants exhibiting retarded sarcomere construction

We have studied the structural changes within the body-wall muscle cells of Caenorhabditis elegans during postmitotic development. In wildtype, the number of sarcomeres progressively increases, and each sarcomere appears to grow in length and depth continuously during this period. In mature wild-type cells, the anterior-most body-wall muscle cells have 6--7 sarcomeres; the rest have 9--10 sarcomeres per cell. Twelve mutants in the unc-52 II gene exhibit markedly retarded sarcomere construction and progressive paralysis. Several unc-52 mutants, such as the severely paralyzed SU200, produced only 2--3 sarcomeres per body-wall muscle cell, while the other mildly paralyzed unc-52 mutants, such as SU250, build 3--4 sarcomeres per muscle cell. Other structures such as the pharynx and even the noncontractile organelles of the body-wall muscle cells do not appear to be structurally or functionally altered. The unc-52 body-wall sarcomeres become moderately disorganized as they are outstripped by cell growth; sufficient order is preserved, however, so that the majority of thick and thin filaments still interdigitate. The myosin heavy chains of SU200 body-wall muscle fail to accumulate normally, while the pharyngeal myosin heavy chains do not appear to be specifically affected. This biochemical result correlates well with the specificity of morphological changes in the mutant. A model is discussed in which the biochemical and morphological deficits are explained by a simple regulatory mechanism.

Immunocytochemical localization of two myosins within the same muscle cells in caenorhabditis elegans

Abstract Nematodes synthesize two major classes of myosin heavy chains. These heavy chains associate to form only homodimeric myosin molecules, and these myosin homodimers are anti-genically different from one another (Schachat, Garcea and Epstein, 1978). The two myosins may be designated unc -54 myosin, since this species is altered in mutants of the unc -54 locus, and non- unc -54 myosin, since this class is not affected in unc -54 mutants. We present here experiments in which specific anti-myosin IgG and anti- unc -54 myosin IgG are used to locate the two myosins within the same body-wall muscle cells of Caenorhabditis elegans. These results are necessary for further evaluation of the possible functions of the two myosin homodimers in the thick filaments of these muscles. Myosin can be localized to all body-wall and pharyngeal muscle cells using anti-myosin antibody. In longitudinal sections of body-wall muscle, the staining with anti-myosin coincides with the birefringence of A bands that contain thick filaments. Anti- unc -54 myosin stains all body-wall A bands uniformly but does not react with the pharynx. This result demonstrates that unc -54 is located exclusively in body-wall muscle cells of the wild-type strain N2. Non- unc -54 myosin is localized with anti-myosin in all body-wall muscle cells of the unc -54 null mutant E190, as expected; however, unc -54 myosin could not be detected by anti- unc -54 myosin antibody in this mutant. Since we can localize unc -54 myosin and non- unc -54 myosin in all body-wall muscle cells of wild-type and E190, respectively, we conclude that the two myosins must be present in the same muscle cells. In addition, since unc -54 myosin is located in all body-wall A bands, at least some sarcomeres must contain both myosins. This conclusion is consistent with the observations of Garcea, Schachat and Epstein (1978) that wild-type and E190 synthesize similar amounts of non- unc -54 myosin. Within the limits of resolution of our methods, unc -54 myosin is distributed throughout body-wall A bands. We conclude, therefore, that the majority of thick filaments within these A bands must contain unc -54 myosin along their entire length. Possible roles for unc -54 and non- unc -54 myosins in the assembly and organization of thick filaments are discussed.

Coordinate synthesis of two myosins in wild-type and mutant nematode muscle during larval development

Abstract In this paper we examine the role of two myosins in body-wall muscle cells of the nematode Caenorhabditis elegans. Large populations of nematodes are synchronized, and the synthesis and accumulation of myosin heavy chains and total protein are followed through postmitotic larval development. Growth is exponential with time for both the wild-type N2 and the body-wall muscledefective mutant E675, with a longer doubling time for the mutant. Utilizing the electrophoretic polymorphism of the E675 myosin heavy chains, we show that distinguishable classes of heavy chains accumulate differentially throughout development. Immunochemical measurements confirm a similar result in N2. Total myosin heavy chain accumulation is also quantitatively similar for the two strains. Myosin heavy chain relative synthetic rates as determined by pulse-labeling are constant throughout development and are equivalent for the two strains. The final fraction of accumulated unc -54 to total heavy chains of approximately 0.63 equals the constant synthetic fraction of approximately 0.62. Since myosin heavy chain accumulation and relative synthesis are equivalent, we conclude that the turnover of heavy chains is also similar in N2 and E675 despite the extensive structural and functional disruption within body-wall muscle cells of the latter strain. Since the accumulated fraction of unc -54 myosin heavy chains reaches a plateau at the constant synthetic fraction, myosin accumulation In the body-wall muscle cells may be attributed to a constant ratio of synthetic rates of the two body-wall myosin species. The coordinate synthesis of two myosins in the same body-wall muscle cells is discussed.

Actin from the nematode, caenorhabditis elegans, is a single electrofocusing species

We have prepared actin from wild type Caenorhabditis elegans animals by three procedures: a purification dependent on the ability of actin to form F-actin, affinity chromatography which preferentially binds G-actin, and co-precipitation of an actin-myosin complex by antimyosin antibodies. Each preparation yields a single electrofucsing species of actin. Comparison of actin from C. elegans embryos and animals reveals that embryos also have the same single electrofocusing species of actin.

Kinetics of azide binding to normal and mutant ferrihemoglobins as evidence for subunit interaction

Abstract The reaction of azide ion with ferrihemoglobin is 30-fold slower than with ferrimyoglobin. The binding of azide ion to two mutant human ferrihemoglobins was investigated to determine whether the lower reaction rate of Hb A is due to subunit interactions. The abnormal hemoglobins studied were Hb MI, in which the proximal heme-linked histidine residue of each α-chain is replaced by tyrosine, and Hb MHP, in which the same change occurs in the β-chains. In these mutant hemoglobins, only the two normal subunits bind azide ion. Hb MI, like myoglobin, reacts rapidly with azide ion, while Hb MHP reacts at the slower rate characteristic of Hb A. In contrast, the four proteins have similar binding affinities for azide ion. The large difference in reaction rate between Hb MI and Hb A shows that the binding kinetics of the β-chain depend on whether the adjacent subunit is normal or mutant. Differences in absorption spectra in the Soret region of the azide derivatives of Hb MI and Hb MHP, as well as differences in their rates of reaction with azide ion, indicate that closely related structural changes in the α and β chains can have dissimilar effects.