Worm Breeder's Gazette 14(4): 33 (October 1, 1996)
Dept. Neuroscience, Albert Einstein College of Medicine Bronx, NY 10461
Immunofluorescence studies using MH27 antibody have shown a wide variety of tissues to be labelled in patterns which define apical cell borders in C. elegans. Disappearance of MH27 binding during development often marks cell fusions in hypodermis and in the male tail (Podbilewicz and White, 1994; Fitch and Emmons, 1995). We have used an immunoEM method to learn the nature of membrane structures marked by MH27. A post-embedding technique was used to apply MH27 Ab to thin sectioned worms, followed by a gold-linked secondary Ab (for methods, see Selkirk et al., 1991; Hall, 1995). Electron microscopy reveals that MH27 binds only to a few types of cell junctions. By immunoEM, MH27 can be seen to bind to zonula adherens junctions in intestine, at hypodermal/seam cell borders, and at pharyngeal muscle/support cell borders. In adult pharyngeal muscle, small longitudinal stripes of adherens "junction" are also retained on the apical surface where pairs of muscle cells had fused to become syncytial. Thus MH27 binding can sometimes mark the vestiges of an old cell border, long after cell fusion. Spermathecal cells are held closely together by two types of "septate" junction, which together cover almost all of their lateral borders. Extensive, dark-staining apical junctions, which look vaguely adherens-like, are not labelled by MH27. In more basal regions, MH27 binds heavily to another class of extensive sinuous junctions, which lack any dramatic staining characteristics. Neither class of junction has osmiophilic septa visible in ordinary sections. The apical junctions are rather closely apposed and show periodic striping of the thick cytoplasmic density bordering the junctions. The basal, sinuous junctions show an even, widened extracellular space between adjacent plasma membranes, with faint periodic striations crossing the extracellular space between the cells. There is no cytoplasmic density associated with the sinuous junctions. Lanthanum infiltration has been used to negatively stain extracellular septa in the spermathecal junctions. The apical junctions have long, wavy septa; they may comprise a novel class of septate junction, characterized by the thick densely-staining coat on the cytoplasmic face of each cell. The sinuous junctions have not been well infiltrated with lanthanum yet. In one instance we did observe short septa at regular intervals, spanning the extracellular space, similar to those expected in "smooth septate junctions" as described in other invertebrate species. These two classes of septate junction may resist the stresses which would otherwise tear apart the spermatheca during the passage of an oocyte. Our current technique does not permit resolution of the exact locus of MH27 antigen within the adherens junction or the smooth septate junction. It could be cytoplasmic, extracellular, or within the plasma membrane. In glancing sections of both types of junction, it is clear the antigen is found evenly along the entire face of the cell-cell appositions, possibly at a fixed distance in relation to the plasma membrane. High resolution studies would require a more direct labelling method, such as attaching a small gold tag directly to a Fab fragment of the MH27 antibody. This would bring the gold tag into closer proximity to the true antigenic site within a thin section. We thank Jim Waddle and Ross Francis (Washington U.) for generously supplying MH27 antibody. Fitch and Emmons (1995) Dev. Biol. 170: 564-582. Hall (1995) In C. elegans: Modern Biological Analysis of an Organism. H.F. Epstein and D.C. Shakes (eds.). Academic Press, New York, pp. 395-436. Podbilewicz and White (1994) Dev. Biol. 161: 408-424. Selkirk et al. (1991) J. Biol. Chem. 266: 11002-11008.