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The pathway of myofibrillogenesis determines the interrelationship between myosin and paramyosin synthesis in Caenorhabditis elegans

Glenn E. White^*, Christine M. Petry and Fred Schachat

Department of Cell Biology – Box 3011, Duke University Medical School, Durham, NC 27710, USA
^* Present address: Department of Biology, University of North Carolina – Asheville, Asheville, NC 28804, USA

View larger version (24K): [in a new window]   Fig. 1. Accumulation of myofibrillar proteins in the wild-type and thick filament null mutants. The accumulation of contractile proteins in myofibrillar preparations from animals continuously labeled for 3 days with low specific activity 35S-labeled bacteria was quantified following autoradiography of 8% SDS–PAGE gels. (A) the autoradiogram of the wild-type N2 (lane a), the myosin B null CB190 (lane b) and the paramyosin null CB1214 (lane c). (B) Graph of the relative rates of accumulation of myosin and paramyosin normalized to actin reveals that paramyosin accumulation is reduced by 33% in the myosin null, and myosin accumulation is reduced by 28% in the paramyosin null. Quantification is based on four independent determinations on each strain, and values are expressed as means ± S.D.

View larger version (24K): [in a new window]   Fig. 2. Synthesis of myofibrillar proteins in the wild-type (N2) and thick filament null mutants. Synchronized animals at larval stage 3 were pulse-labeled for 2 h with high specific activity 35S-labeled bacteria and the relative synthetic rates of myosin and paramyosin determined by normalization to actin. As is evident in the autoradiogram (A), compared with N2 (lane a), there is a marked reduction in the rates of synthesis of myosin in both the myosin B null CB190 (lane b) and the paramyosin null CB1214 (lane c). The results of the densitometric analysis based on four independent determinations on each strain are presented in (B). Values are means ± S.E.M.

View larger version (31K): [in a new window]   Fig. 3. Myosin B accumulation is differentially affected in the paramyosin null mutant. The Neville gel system (A) was used to quantify the relative abundance of myosin A and B, the two body-wall muscle myosins, in wild-type and paramyosin null CB1214 homogenates, and actin levels were determined by electrophoresis of identical loads on the Laemmli gel system (B). Gels were silver stained, and actin from the Laemmli gel was used as the internal standard for determining the relative accumulation of myosins A and B (C).

View larger version (42K): [in a new window]   Fig. 4. Steady-state myosin A, myosin B and actin mRNA levels in N2 and CB1214. Total RNA was prepared and hybridized with gene-specific riboprobes (A), and steady-state mRNA levels were determined by normalization to total actin mRNA (B). The results demonstrate that in CB1214, the steady-state level of myosin B mRNA is approximately 60% that in the wild-type N2. The blot for the myosin A probe has a significantly longer exposure time due to the lower amount of myosin A in the muscle (Honda and Epstein, 1990). Mean values are based on four independent determinations.

View larger version (21K): [in a new window]   Fig. 5. Decrease in myosin B expression in paramyosin missense mutants. The Neville gel system was used to analyze body-wall muscle myosin expression in myofibrillar preparations (A) from the wild-type (N2), the paramyosin null CB1214 and three paramyosin missense mutations, CB1215, HE2000 and CB73. The fraction of myosin B (myosin B divided by the sum of myosins A and B) in the paramyosin mutants is reduced when compared with the wild-type (B).

View larger version (17K): [in a new window]   Fig. 6. The relationship between the ordered pathway of thick filament assembly, structure and expression. The ordered assembly pathway for C. elegans thick filaments presented by Epstein et al. (1985, 1986) and the structural and synthetic effects of mutants is presented diagrammatically. The relative positions of myosin A and B are indicated on the upper surface, and the position of paramyosin and the filagenins are indicated by exposing the core in the lower half of the filament schematic. The diagram is a modification based on Epstein et al. (1985) and Hoppe and Waterston (2000) that includes the observations on expression presented in the present study.

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