Worm Breeder's Gazette 14(4): 33 (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.

ImmunoEM Study of MH27 Reveals Sticky Junctions

David H. Hall

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.