Black widow spider venom (BWSV) contains high contents of molecular weight. protein called latrotoxins (LTX) that induce catastrophic neurotransmitter. release from nerve terminals, and is known to bind with high affinity to. three neural proteins in mammals including latrophilin. We have established. C.elegans as a model organism to study the function of the binding protein,. latrophilin (a member of the class B family of G-protein coupled. receptors), and its role in regulating neurotransmitter release by. latrotoxins, by showing that specific proteins in BWSV kill C. elegans (Mee. et al. 2004). However, a latrophilin null worm is required for determining. the function of the latrophilin gene, and we have investigated the lat-. 1
(ok1465) allele.. The
lat-1(
ok1465) allele has a deletion of the
lat-1 gene, and ~97% of lat-. 1
(ok1465) homozygous worms arrest or die before adulthood, with only 3. adult offspring per animal. However, it is unclear if this lethality is due. to the deletion in the
lat-1 gene, or to adventitious mutations introduced. during the process of mutagenesis to create this allele.. The B0457 cosmid contains the full sequence of the
lat-1 gene, and. injection of this cosmid into
lat-1(
ok1465) worms rescued the lethality of. the
lat-1(
ok1465) allele. Because it is not clear whether the
lat-1 gene or. other genes on the B0457 cosmid rescued
lat-1(
ok1465) worms, we injected. the full-length
lat-1a cDNA fused to a gfp reporter construct and driven by. ~1.5kb, 5''end of
lat-1 promoter into
lat-1(
ok465) worms, and obtained. rescue of the lethality. Deletion of various C-terminal or N-terminal. regions of
lat-1a cDNA yielded full or partial rescue of
lat-1(
ok1465). worms, defining essential functional regions of the
lat-1a cDNA.. It has been demonstrated by Mee et al that BWSV was toxic to C.elegans, and. we studied the effect of BWSV on the transgenic animals generated from. micro-injection of the B0457 cosmid, the full-length
lat-1a cDNA, or C-. terminal and N-terminal truncated variants of
lat-1a cDNA. The results. showed that BWSV was highly toxic to wild-type C.elegans (EC50 ~4ng/ml),. whereas the
lat-1(
ok1465) worms were highly (>105-fold) resistant to BWSV.. The
lat-1(
ok1465) worms that were transgenic for B0457cosmid, or
lat-1a. cDNA, were as sensitive to BWSV as wild-type worms. Truncation of the C-. terminus of
lat-1a cDNA to the transmembrane domain yielded worms that had. 105-fold resistance to BWSV, compared to wild-type; thus the intracellular. domain of
lat-1 is required for mediating BWSV toxicity.. Therefore, these data showed that the
lat-1 gene was responsible for the. lethality of animals carrying the
ok1465 deletion allele of the
lat-1 gene,. and the absence of this gene causes embryonic or larval lethality in ~97%. of offspring. The absence of the
lat-1 gene in
lat-1(
ok1465) worms was. responsible for the resistance of these worms to BWSV, and the requirement. for the C-terminus of
lat-1 for lethality of the BWSV shows that the. ability of the
lat-1 gene to signal through its C-terminal domain is. required for toxicity of BWSV. This finding contradicts models that suggest. latrophilin merely acts as a tether to allow BWSV toxins to form pores in. the membrane (Volynski et al, 2000).. References. Mee, C.J., Tomlinson, S.R, Perestenko, P.V, dePomerai, D., Duce, I.R,. Usherwood , P.N.R, Bell, D.R, 2004. Latrophilin is required for toxicity. of black widow spider venom in Caenorhabditis elegans. J.Biochem. 378 (Pt. 1), 185-191. Volynski, K.E., Meunier, F.A, Lelianova, V.G, Dudina, E.E, Volkova, T.M,. Rahman, M.A, Manser, C., Grishin, E.V, Dolly, J.O, Ashley, R.H, Ushkaryov,. Y.A, 2000. Latrophilin, neurexin, and their signaling-deficient mutants. facilitate alpha -latrotoxin insertion into membranes but are not involved. in pore formation. J.Biol.Chem. 275 (52), 41175-41183. Do not add objects such as pictures, boxes, headers, footers, footnotes,. etc.