Worm Breeder's Gazette 13(5): 76 (February 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.

BODIPY-ceramide staining of plasma membranes in living worms

Mike Roth and Leon Avery (leon@eatworms.swmed.edu)

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

Fluorescent ceramide analogs that can be used as vital stains of the Golgi
complex have been developed in Pagano's lab (Lipsky and Pagano, 1983, PNAS
80: 2608; Pagano et al, 1991, JCB 113: 1267).  These dyes traverse
membranes passively, but once in the Golgi get trapped by glycosylation. 
Unable to cross the lipid bilayer, they label Golgi membranes and mark
membrane traveling through the secretory pathway. In particular, ceramide
coupled to the fluorophore boron dipyromethane difluoride (BODIPY, or C5-
DMB-ceramide) is bright, resistant to photobleaching, and stains the Golgi
complex more efficiently than other membranes.  BODIPY shifts from red to
green fluorescence as it becomes concentrated -- this effect can be used
to increase the prominence of Golgi labeling over the background of
labeling in the endoplasmic reticulum and other membranes.  At least, it
does these things in cultured mammalian cells.  We decided to test whether
it works the same way in worms, in the hope of doing some membrane
trafficking genetics.

BODIPY-ceramide is taken up into worms, and it stains membranes.  The
brightest fluorescence, not surprisingly, was in amphid and phasmid
neurons, which are known to take up hydrophobic dyes from outside, and in
gut granules.  Gut granules are of course fluorescent even in unstained
worms, but they appeared brighter after staining, so there may have been
some preferential uptake.  Unfortunately, we were not able to see
convincing Golgi staining.  The intestinal cells, being large and probably
active in secretion, were our Great Hope for seeing labeled Golgi, but the
gut granules defeated us.

After gut granules and amphid/phasmid neurons, the brightest staining was
in the nerve ring and the lining of the intestinal lumen.  What
distinguishes these two places from the rest of the worm is that both have
a lot of plasma membrane (covering neuronal processes and intestinal
microvilli) in a small space.  In fact, the fluorescence was consistent
with staining of all plasma membranes.  Our prettiest pictures came from
starved L1s.  After overnight staining in M9 containing 10 ug/ml BODIPY-
ceramide and 1 mM defatted BSA, we could see not only the ring and
intestinal lumen, but also, more faintly, the outlines of the packed
neurons in the lumbar and ring ganglia, the outlines of cells in the
terminal bulb of the pharynx, and the pharyngeal nerve ring at the border
of the metacorpus and isthmus. Plasma membranes in the gonad of starved
spermless females (fem-1(ts) raised at 25C) were also clearly stained.  In
the distal arm the meshwork of membrane invaginations surrounding the
individual germline nuclei were visible.  Around the bend the plasma
membranes of immature oocytes were stained.  In addition there was some
perinuclear staining in these cells: the only hint of possible Golgi
staining we saw.

The stained worms were alive and healthy, and in fact annoyingly active,
even in the presence of 20 mM NaN3.  We don't know if we will ever be able
to recognize Golgi, but as a vital stain for plasma membrane BODIPY-
ceramide may be useful.

We will put some confocal pictures on the C elegans WWW server.  (Use the
"Open URL" or "Open Location" function of your browser to get