Worm Breeder's Gazette 14(4): 70 (October 1, 1996)
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||Dept. of Molecular Biology and Biochemistry, Rutgers University, New Brunswick, NJ 08855|
|2||Dept. of Biochemistry, New York University, Medical School, New York, NY 10016|
unc-8 encodes a member of the C. elegans degenerin family (as was predicted by Shreffler and co-workers), that is extensively expressed in the nervous system including motor neurons, interneurons and sensory neurons. Degenerins are thought to assemble into mechanically-gated ion channels. Gain-of-function mutations in unc-8 result in transient swelling of neurons, reminiscent of the vacuolation phenotype induced by specific mutations in other family members. As a consequence, movement is severely uncoordinated and animals are unable to back up. These phenotypes are suppressed by lesions in the mec-6 gene that also suppress the effects of analogous mutations in other degenerins. We have sequenced the semi-dominant e15, e49 and n491 unc-8 alleles. The two strongest mutations, e15 and n491, alter the same residue in the extracellular region before the cysteine-rich domain II (G387E). Interestingly, these mutations map in the center of a domain previously implicated by Garcia-Anoveros and co-workers in regulation of channel closing; mutations affecting this domain in two other degenerins, deg-1 and mec-4, result in vacuolation and degeneration of cells expressing the mutant alleles. The mutation in e49 affects a conserved residue within cysteine-rich domain III (A586T). Shreffler and Wolinsky had generated additional second-site mutations in unc-8 that either suppressed (allele e15lb129) or enhanced (allele n491lb82) the phenotype of the e15 and n491 unc-8 alleles when in trans to the wild type gene. lb129 results in a substitution of tyrosine for an absolutely conserved histidine at position 114, in the intracellular part of the protein before the membrane-spanning domain I. lb82 encodes a threonine to isoleucine change at position 664 in the extracellular region between cysteine-rich domain III and membrane-spanning domain II. The unusual genetic properties of these two alleles suggest that at least two UNC-8 molecules participate in the formation of a channel and that these molecules interact both through intracellular and extracellular domains. These interactions might serve to ensure a rigid channel structure that could respond to rather than absorb mechanical forces. C. elegans strains carrying unc-8 loss-of-function mutations do not exhibit readily noticeable defects in locomotion. However, close examination of the sinusoidal path inscribed by unc-8(lf) worms on bacterial lawns revealed that the amplitude as well as the wave-length of the track is substantially reduced. This striking phenotype suggests that UNC-8 functions to regulate sinusoidal locomotion. unc-8 together with an additional member of the degenerin family, del-1 (for degenerin-like) that resides on cosmid clone E02H4 derived from the X chromosome, is expressed at high levels in command motor neurons of the ventral nerve cord (unc-8 in the D and V class and del-1 in the V class of motor neurons). In early electron-microscopy studies of the C. elegans nervous system White and co-workers had noticed an unusual feature of these neurons. The endings of their long longitudinally running processes were undifferentiated and devoid of any synaptic input. This morphology (which resembles the morphology of another group of sensory neurons-the six touch receptors) prompted investigators to hypothesize that motor neuron endings functioned as stretch receptor areas that could modify the overall activity of the neurons, thus modulating locomotion. This attractive scenario called for the existence of mechanically-gated channels in motor neurons. Given the expression of two degenerins, unc-8 and del-1, in these neurons and the striking phenotype of unc-8(lf) mutations, we propose that UNC-8 and DEL-1 assemble into stretch-activated channels in motor neurons. By receiving input from parts of the body stretched by muscle contraction, such channels would provide the feedback needed to accordingly modulate muscle activity and sustain a normal sinusoidal pattern of movement.