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.

In Vitro Measurements of C. elegans Myosin Motility on Nitella Actin Cables

L. Schriefer, M.P. Sheetz, and R. Waterston

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.