To study dynein's different functions, we are using dominant temperature-sensitive (dom-ts) alleles of the
let-354 complementation group. The evidence that
let-354 encodes dynein heavy chain (DHC-1) includes: 1)
let-354 has been mapped genetically to the same region of LGI as
dhc-1 , 2) the mutant phenotype of
let-354 (dom-ts) embryos is similar to the phenotype observed in
dhc-1(RNAi) embryos, 3) injection of two overlapping cosmids (T21E12 and ZK973), which together contain the
dhc-1 gene, rescued the recessive larval-lethal phenotype of
let-354 .
let-354(dom-ts) /+ worms or their embryos were cultured at permissive temperature (15 o C) and shifted to non-permissive temperature (25 o C). At 15 o C, mutant embryos undergo apparently normal development. At 25 o C, mutant embryos show severe defects in meiotic chromosome separation, mitotic centrosome separation, mitotic spindle formation, spindle rotation, chromosome segregation during mitosis, cortical membrane integrity and cytokinesis. The dom-ts alleles respond rapidly to non-permissive temperature. With a thermal stage controlled by Peltier devices, embryos can be shifted to 25 o C in ~ 1 min. and immediately show defects. Embryos shifted to 25 o C after normal pronuclear migration display defects in mitotic spindle formation, cortical membrane integrity, and cytokinesis. This dominant ts effect is reversible. For example, failure to form a mitotic spindle and undergo cytokinesis at the restrictive temperature is followed, after a shift down to the permissive temperature, by spindle formation and cytokinesis during the next cell cycle. Using different temperature shift regimes, we are now attempting to define dyneins's role in various stages of pronuclear migration, spindle positioning and mitosis. (Thanks to Paul Mains and Ann Rose for
let-354 alleles.)