Worm Breeder's Gazette 13(2): 72 (February 1, 1994)
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.
Mec-4 encodes a subunit of an ion channel required for the function of the six touch receptor neurons. Dominant alleles of mec-4 induce degeneration of these neurons. As part of a detailed study of how the MEC-4 (d)protein kills cells we wanted to determine when mec-4 is first expressed in the postembryonic touch receptor neurons AVM and PVM. We have done this by synchronizing worm populations and monitoring expression of an integrated mec-4 /lacZarray.
To generate a synchronized population for staining, we picked about 100 gravid worms from plates that contained lots of bacteria (20°C) onto a prewarmed plate. Worms were allowed to lay eggs for one hour and then adults were removed. In theory, all remaining eggs would have been fertilized within an hour of each other. (In practice, a wider window of ages probably occurs as eggs are not all laid at precisely the same moment in development). Larvae were stained for ß-galactosidase activity at hour intervals.
We find that AVM expresses ß-galactosidase in 50% of animals by 22 hours after the egg is laid (see figure below). PVM expresses ß-galactosidase in 50% of animals a bit later, by 25 hours. AVM and PVM are generated at about 9 hours after hatching at 20°C, about 19 hours after egg deposition. Thus, mec-4 appears to be expressed very soon after the birth of the touch cell. The fact that we do not see a sharp transition point for the activation of mec-4 expression may reflect the degree of asynchrony in the culture or may result from stochastic activation of mec-4 expression over a three hour period.
Interestingly, we have observed that at 27-28 hours the intensity of staining drops dramatically or disappears altogether. According to our calculations, the loss of staining coincides with the time of the L1 -L2molt. We are considering two explanations. First, standard-staining conditions may not be appropriate for molting animals. Alternatively, we wonder if proteins that project into the extracellular space and contact the cuticle are degraded at the time of the molt. The processes of the touch receptor neurons run along the cuticle and are surrounded by an extracellular material called the mantle. MEC-4 is predicted to have an extracellular domain that could be involved in contacting the mantle. Perhaps during molting proteins that associate with the cuticle are temporarily destroyed.
Use of mec-4 /lacZto probe the relationship of mec-4 and mec-6 . mec-6 is required for mec-4 (d)-induceddegenerations to occur. We have now looked in detail at staining of mec-4 /1ac-Zin the mec-6 ( u450 )background. We find that in the wild-type background, mec-4 /lacZstains all six touch receptors intensely. However, in the mec-6 ( u450 )background, mec-4 /lacZactivity is consistently and significantly lower than in the wild-type background. After one day of
incubation with X-gal, staining can barely be observed and after two days of incubation, the touch cells still stain faintly. Lessened staining is clearly not due to a change in the configuration of our array in the mec-6 ( u450 )strains because when the mec-4 /lac-Zarray is crossed out of the mec-6 ( u450 )background, intense staining is restored.
Our observations indicate that mec-6 mutations may block mec-4 (d)-induceddegeneration by significantly lowering levels of MEC-4 protein. Why this occurs, however, is unclear. Data are consistent with the idea that MEC-6 could be required for high levels of transcription of mec-4 .Data are also consistent with the idea that MEC-6 could be required for insuring the translational stability of MEC-4 .If, for example, MEC-6 was a subunit of the channel complex, its absence could result in turnover of other channel subunits that could not be properly assembled.