Worm Breeder's Gazette 14(1): 42 (October 1, 1995)

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.

Dye-coupling of wild-type and eat-5 pharynxes.

Cristin Panzarella, Leon Avery (leon@eatworms.swmed.edu)

Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038.

Pharyngeal action potentials are tightly synchronized: the corpus (the front half of the pharynx) and the terminal bulb depolarize within a few milliseconds of each other. Because the nervous system is not required for synchronization, Avery and Horvitz (Neuron 3: 473-485) proposed that it was accomplished by electrical coupling of pharyngeal muscles through gap junctions.

To test whether pharyngeal muscle cells are coupled, we used the intracellular recording techniques developed by Wayne Davis (1995 Worm Meeting abstract 179) to inject a fluorescent dye into terminal bulb muscles of four adult wild-type pharynxes. In every case the dye spread from a single terminal bulb muscle cell into all 15 large muscle cells of the terminal bulb (6 cells), isthmus (3 cells), and corpus (6 cells) (Figure part a).

The only thing known to cause the corpus and terminal bulb to become unsynchronized is a mutation in the gene eat-5 (Avery, Genetics 133: 897). eat-5 encodes a new member of a large protein family that also includes UNC-7 and the products of the Drosophila genes ogre and Passover (T Starich et al, 1995 Worm Meeting abstract 487; TM Barnes, TIG 10: 303; WBG 13(2): 68). Even before the addition of eat-5 this family had been proposed to encode a new type of gap junction protein (TM Barnes, TIG 10: 303-305; T Starich, personal communication).

Dye injected into the terminal bulbs of four eat-5 mutant pharynxes spread through the terminal bulb and isthmus, but failed to spread into the corpus (Figure part b). (This experiment was reported once before, with different results: eat-5 pharynxes were dye-coupled (Avery, WBG 12(2): 87). The injection technique used previously was much cruder: the impaled pharynxes died almost instantly, and the amount of dye injected was much larger.)

Terminal bulb muscles of dissected pharynxes were impaled with sharp electrodes containing a solution of fluorescent dye, and the dye was injected by iontophoresis. a. A wild-type pharynx, anterior to the left. Dye spread throughout the terminal bulb (TB), isthmus (Is), and corpus (Co). b. An eat-5 mutant pharynx, anterior to the left. Dye spread through the terminal bulb and isthmus, but did not cross into the corpus. This pharynx was still impaled at the time the picture was taken--the electrode filled with fluorescent dye can be seen at the upper right.

A much prettier version of this figure can be found on the WWW page http://eatworms.swmed.edu/Worm_labs/Avery/Pictures.html.

These results are the best evidence we know of that the family of proteins of which EAT-5 is a member is indeed involved in the function of gap junctions. In addition, they strongly support the hypothesis that the pharynx is synchronized by muscle electrical coupling.