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.

Lean and Mean--Starvation Rescue of Unc-45(ts) mutants

Lee Venolia1,2, Amy Matthews1

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