Worm Breeder's Gazette 15(3): 21 (June 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Biology, Williams College|
|2||Present address: 135 Stone Hill Rd., Williamstown, MA 01267|
Unc-45(ts) mutations cause a temperature dependent moderate disorganization of muscle thick-filaments, resulting in slow movement at 25oC. Genetic results suggest a role for UNC-45 in thick filament organization, and the gene sequence shows the protein to be a member of the tetratricopeptide repeat (TPR) family (Venolia et al, manuscript submitted). There are additional recessive lethal alleles of unc-45, and muscles of homozygous unc-45(let) worms are severely disorganized. Thus, we assume that the ts alleles produce a partially active protein. If the ts Unc worms are starved at 25oC, they begin moving like wild-type worms. When such worms are refed, they slow down again. We have examined this phenomenon in some detail, eliminating various explanations, and we offer a hypothesis to explain it. We first determined that the rescue is dependent on muscle myosin by observing double mutants for unc-45(ts) and unc-54(e190), which eliminates the major myosin, MHC B. These worms gained no movement advantage from starvation. We then developed a protocol for analyzing the kinetics of this process. To eliminate considerations of drag on the worm being affected by the amount of bacteria on the surface of the plate, all measures of movement ability were made in liquid media. Wild type and starved worms flip-flop vigorously in liquid, while Unc worms trace the same movements more slowly. Thus, flip-flops/time could be quantified. The 5 known ts alleles were compared for their starvation rescue, and it was determined that m94 and e286 were still mildly slow when starved, while su2002 and b131 were least paralyzed when fed, so the strain with the most striking change upon starvation was r450. This strain was used for all further experiments. Synchronized populations, raised at 25oC were tested for starvation rescue and it was determined that all stages of the life cycle are rescuable, with wild-type movement achieved in 12-24 hours and persisting for up to 1 week. The rescue was definitely not dependent upon passage through the dauer phase. Similarly, loss of rescue on refeeding occurs at all stages, requiring 1-2 days (longer for older worms). Electron microscopic studies of the rescued worms indicate a slight, but statistically significant increase in number of thick filaments (5 vs. 7/cm2 of muscle cell, at a fixed magnification in electron micrographs, compared with roughly 12/cm2 in wild-type) and we observed that the muscle is spread more thinly over a somewhat smaller circumference in the starved worms. These changes could conceivably account for the improved movement in the starved worms, but they would indicate a considerable redundancy in muscle structure in the wild-type worm. We examined the proteins in starved and fed worms by SDS-polyacrylamide gels of crude extracts. Two starvation induced changes were noted. A 200kd protein, which could be MHC, increased 2-fold, and a 170kd protein decreased 2-fold. One hypothesis to explain these observations is as follows. At the restrictive temperature, unc-45(ts) alleles are functioning properly in their MHC A interactions (if necessary), but are directly or indirectly converting MHC B to an aberrant form. The aberrant MHC B can be assembled into thick filaments, but is also a monkey wrench in the mature thick filament, preventing proper contraction. During starvation, the majority of thick filaments are destroyed and reformed. We propose that a stress protein is induced during starvation, which can refold unc-45 to its wild-type structure, and thus all MHC B made during starvation is wild-type. Upon refeeding, the stress-protein disappears, and UNC-45 proceeds to create aberrant MHC B, which disturbs the function of the already formed filaments. Because unc-45(ts) alleles behave recessively, we must postulate that a half dose of unc-45 is enough to prevent significant formation of aberrant MHC B. ***** Lee Venolia 135 Stone Hill Road Williamstown, MA 01267 phone (413)458-4485