Worm Breeder's Gazette 9(2): 107
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.
We have been investigating the movement of C. elegans myosin in an in vitro system originally developed by M. Sheetz and J. Spudich ( Nature 303:31 1983). The myosin motility assay utilizes the actin cables that run longitudinally in the giant internodal cell of the algae Nitella axillaris. A 1mm by 4cm cell of Nitella is pinned and cut open longitudinally, exposing polar actin cables that are attached to the cytoplasmic face of rows of chloroplast. The actin cables are about 0.1 m in diameter and are utilized by the Nitella for organelle transport. Myosin is bound to polymer beads of 0.2 m or 0.9 m in diameter and added to the dissected Nitella under experimental assay conditions. The movement of the myosin beads is then followed under a 50X light microscope and velocities are measured by utilizing a time lapse video recorder. In non-nematode systems the velocity of the myosin on Nitella actin cables has been similar to the velocity of myosin movement along actin in the muscle sarcomere. Our initial studies have been on actin-free crude actomyosin preparations of N2, S95, and e190. Care was taken to insure the regulatory light chains did not disassociate during the purification. The maximum observed velocity for N2 in the presence of 1mM ATP and the absence of calcium was 1.9 m/sec. Previously recorded rabbit skeletal muscle myosin had a velocity of 6 m/sec (Sheetz et al., J. of Cell Biology 99:1867 1984). Both S95 and e190 were tested in the same conditions. S95 produced a maximum velocity similar to N2, while e190 dispIayed a reduced maximum velocity. The addition of calcium to the assay buffer raised the maximum observed velocity of N2 and S95 to approximately 2.5 m/sec. In several cases, myosin coated beads that had been firmly attached to actin cables increased in velocity or released from the actin cables with the addition of calcium. We hope to utilize the Nitella assay system to analyze the movement of the four myosin isozymes present in C. elegans and to investigate various unc-54 head mutants.