Garcea RL et al. (1978) Worm Breeder's Gazette "muscle development in C elegans: mutants exhibiting retarded sarcomere construction."

Mackenzie JM, Epstein HF, Zengel JM, Schachat FH, Garcea RL

[Worm Breeder's Gazette, 1978]

Garcea RL et al. (1978) Worm Breeder's Gazette "Coordinate Synthesis of Two Myosins in Wild-type and Mutant Nematode Muscle During Larval Development"

Garcea RL, Epstein HF, Schachat FH

[Worm Breeder's Gazette, 1978]

We are studying the role of two myosins in body-wall muscle cells of the nematode, Caenorhabditis opulations of nematodes are synchronized, and the synthesis and accumulation of myosin heavy chains and total protein are followed through postmitotic larval development. For both the wild-type N2 and the body-wall muscle-defective mutant E675, growth is exponential with time. The mutant has a longer protein doubling time than wild-type. Utilizing the electrophoretic polymorphism of the E675 myosin heavy chains, we show distinguishable classes of heavy chains accumulate differentially through development. Immunochemical measurements comfirm a similar result in N2. Total myosin heavy chain accumulation is also quantitatively similar for the two strains relative to total protein. Myosin heavy chain relative synthetic rates as determined by pulse- labelling are constant throughout development and equivalent for two strains. The final fraction of accumulated unc-54 to total heavy chains, about 0.63, equals the constant synthetic fraction, about 0.62.

Garcea RL et al. (1978) Cell "Coordinate synthesis of two myosins in wild-type and mutant nematode muscle during larval development."

Schachat FH, Garcea RL, Epstein HF

[Cell, 1978]

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 muscle-defective 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 chains. 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

Schachat FH et al. (1978) Worm Breeder's Gazette "Myosins exist as homodimers of heavy chains: Demonstration with specific antibody purified by nematode mutant myosin affinity chromatography."

Epstein HF, Schachat FH, Garcea RL

[Worm Breeder's Gazette, 1978]

The body-walls of Caenorhabditis two different myosin heavy chains that associate to form at least two species of myosin. In order 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 chain indicates that there are antigenic differences between myosin molecules containing unc-54 heavy chains and myosin molecules containing only non-unc-54 myosin 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 antiunc-54 myosin antibody precipitates myosin that contains only unc-54 heavy chains. At the limits of resolution of our immunoprecipitation techniques, no heterodimeric myosin molecules that contain both unc-54 and non-unc-54 heavy chains can be detected. Therefore, the body-wall myosins of C. elegans exist only as homodimers of either class of heavy chain. This specific anti-unc-54 myosin antibody promises to be a valuable tool in understanding 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 (See Mackenzie, Schachat and Epstein) and that both myosins are coordinately synthesized during muscle development in C. elegans (See Garcea, Schachat and Epstein).

Schachat FH et al. (1978) Cell "Myosins exist as homodimers of heavy chains: Demonstration with specific antibody purified by nematode mutant myosin affinity chromatography."

Garcea RL, Schachat FH, Epstein HF

[Cell, 1978]

The body-walls of Caenorhabditis elegans contain two different myosin heavy chains that associate to form at least two species of myosin. 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 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 conjuction 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 and that both myosins are coordinately synthesized during muscle development in C. elegans. 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.

Mackenzie JM et al. (1978) Worm Breeder's Gazette

Zengel JM, Mackenzie JM, Epstein HF, Garcea RL

[Worm Breeder's Gazette, 1978]

We have studied the structural changes within the body-wall muscle cells of Caenorhabditis ostmitotic development. In wild-type, the number of sarcomeres progressively increases, and each sarcomere appears to continuously grow in length and depth during this period. At maturity, in wild-type, the anteriormost body-wall muscle cells have 6-7 sarcomeres and 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 produce only 2-3 sarcomeres per body-wall muscle cell while the other less paralyzed unc-52 mutants such as SU250 build 3-4 larger sarcomeres per muscle cell. Other structures such as the pharynx and even the non-contractile 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, however, sufficient order is preserved 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 specifically affected. This biochemical result correlates well with the specificity of morphological changes in the mutant. We are conducting experiments to test whether the unc-52 mutants affect only body-wall myosin heavy chains versus other contractile proteins, and if so, unc-54 or non-unc-54 heavy chains or both. Further experiments are planned to test whether the unc-52 effects are due to a synthetic block or increased turnover of the proteins.

Mackenzie JM et al. (1978) Cell "Muscle development in Caenorhabditis elegans: mutants exhibiting retarded sarcomere construction."

Garcea RL, Zengel JM, Epstein HF, Mackenzie JM

[Cell, 1978]

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.

Schachat FH et al. (1977) "Studies on two body-wall myosins in wild type and mutant nematodes."

Schachat FH, Harris HE, Epstein HF, LaPointe JAW, Garcea RL

[1977]

Native myosin purified from the wild-type, N2, and a body-wall defective mutant, E675, of the nematode contains two myosins, each homogeneous for different heavy chains. These myosins can be resolved from one another on hydroxyapatite and, when cleavaged with CNBr, they yield different peptide-fragments. In E190, one of the homogeneous myosins is absent. e190 and e675 are alleles of the same gene, unc-54. The myosin lacking in E190 is the same one affected in E675. This suggests that unc-54 is the structural gene for a myosin heavy chain. In order to determine the role of these different myosins, we plan to use antibodies to locate the myosins on thick filaments from body-wall muscle. Additionally, we are studying the patterns of synthesis and degradation of the two myosins in the wild-type and muscle-defective mutants in order to discover how the observed stoichiometry is maintained.

Epstein HF et al. (1977) International C. elegans Meeting "biochemical and genetic studies of contractile proteins from normal and mutant nematode strains."

Epstein HF, Garcea RL, Harris HE, Schachat FH, LaPointe JAW, Zengel JM

[International C. elegans Meeting, 1977]

Epstein HF et al. (1977) Worm Breeder's Gazette "biochemical and genetic studies of contractile proteins from normal and mutant nematode strains."

Harris HE, Schachat FH, Garcea RL, Zengel JM, Epstein HF, LaPointe JAW

[Worm Breeder's Gazette, 1977]

Myosin, actin, paramyosin and tropomyosin can be purified from N2 and mutant strains and shown to possess native properties by different functional and structural tests in vitro. One interesting physiological fact obtained from this approach is that, in nematode muscle, Ca++-mediated regulation of contraction is associated with proteins on both the thick and thin filaments. The application of these methods to obtaining purified native myosin from various unc-54 strains has permitted us to clarify several questions concerning the previous observation of two kinds of myosin heavy chains in the body-wall. The two kinds of myosin heavy chain form primarily, if not exclusively, myosin molecules containing two such polypeptides of either one or the other class. The unc-54 affected class of myosin is not required for the formation of bipolar or very long filaments or for complexing with paramyosin cores. Altered unc-54 myosin although presumably disrupting the myofilament lattice in vivo, is capable of normal filament assembly in vitro. Preliminary studies suggest that unc-54 myosin accumulates at a different rate than during development than the other myosins. The two major myosins are antigenically distinct from one another. This fact is permitting us to purify antibodies to unc-54 myosin and to localize this myosin in bodywalls during development. A tentative inference from our studies and those of others is that the formation of a myofilament lattice involves the interaction of many gene products and actions, any one of which may have only a subtle influence. Accordingly, we are in the process of building up a sizeable collection of sarcomere-defective mutants. A wide range of phenotypes including a number of temperature-sensitive mutants has been obtained. We believe that only a rather complete genetic dissection in conjunction with detailed biochemical and structural studies will permit us to provide clearcut answers to such seemingly simple questions as 'why are there two myosins?'.