Worm Breeder's Gazette 9(1): 82

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.

Freeze Fracture Studies of C. elegans (Are There Multiple Forms of Gap Junctions?)

D.H. Hall

When viewed in thin section, gap juctions are problematic in C.  
elegans neurons because of their small size.  Gap junctions in other 
cell types are often more extensive, but otherwise look pretty much 
the same using this technique.  The freeze fracture technique offers a 
different view of gap junctions and we are able to show some 
differences in their organization when comparing hypodermis, gut and 
neurons.  Similar results were obtained previously in Planaria by 
Quick and Johnson (1977).
One can speculate that the gap junction molecules in different 
tissues may derive from different genes, a question which C.  elegans 
is particularly well suited to explore.  There is molecular evidence 
that mammalian gjs derive from tissue-specific genes.  For instance, 2-
D peptide maps of liver and lens gjs show almost no overlap (Hertzberg,
et. al., 1982) and comparison of partial sequence data for these two 
molecules shows major differences (Nicholson, et. al., 1983).  Six of 
these gj protein subunits combine to form a 'hemichannel' which spans 
one plasma membrane.  Two hemichannels must link end-to-end to form a 
patent channel between two neighboring cells.  These channels 
aggregate into arrays within two apposed plasma membranes to form a 
typical gap junction.
Adult nematodes are fixed in glutaraldehyde, cryoprotected in 
glycerol and then sandwiched as a monolayer between two gold discs.  
After rapid freezing in liquid Freon, the gold disc sandwich is placed 
in a double replica holder and fractured in a Balzer s Freeze/Etch 
Device.  If etching is desired, one waits several minutes to allow 
water to sublimate from the fractured surfaces before shadowing.  The 
specimen is shadowed with Pt and then coated with carbon to make a 
Pt/C replica.  The replica is cleaned with bleach and mounted on a 
copper grid for examination by electron microscopy.  The fracture 
plane preferentially travels along membranes, splitting the unit 
membrane into two opposing halves (the P- and E- faces).
Most animals fracture lengthwise, often superficially, to reveal 
details of the cuticle and hypodermal membranes.  Deeper fractures can 
reveal many recognizable tissues: nerve ring, nerve cords, commissures,
gut, muscle, etc.  Muscle membranes have been difficult to identify; 
however, using the freeze etch technique, we have been able to view 
more deeply into cytoplasm for additional structural detail.  
Hypodermal membranes facing the cuticle are particularly easy to 
recognize because of their characteristic regions of parallel 
Gap junctions are all of the  A-type , with most of their 
intramembrane particles adhering to the P-face.  An array of pits on 
the E-face corresponds to the P-face particle array.  Each pit 
presumably represents a hemichannel pulling out of the E-face membrane.
Some gap junctions are densely packed and macular, including those 
found between gut cells.  Other gjs have more dispersed groups of 
particles, particularly those between hypodermal cells.  The fraction 
of particles adhering to the E-face varies in a similar pattern to 
that noted in Planaria:  in gut cells, 6%; in hypodermis, 11%; in 
neurons, ~25%.  Our sample of neuronal gap junctions is very limited 
so far.  No muscle gap junctions have yet been identified, although 
work with the freeze etch technique shows promise.
Gap junction antibodies derived from two mammalian tissues (liver, 
lens) are now available for immunocytochemistry.  It will be 
interesting to see whether these antibodies recognize C.  elegans gap 
junctions:  They might differentiate the gjs in various tissues.