Figure 3. Clathrin is required for neuromuscular function. (A-B'') CHC is concentrated presynaptically but is excluded from the active zone. (A-A'') Live worms co-expressing mRFP-CHC (A, A'') and the synaptic vesicle protein GFP-SNB-1 (A', A'') in GABA neurons were examined by fluorescence microscopy. (B-B'') Transgenic animals expressing GFP-CHC (B, B'') in the GABA neurons were stained with anti-GFP and anti-SYD-2 antibodies. Note that anti-SYD-2 antibody labels active zones in all synapses of the nerve cord (B'), whereas GFP-CHC labels only synapses in GABA motor neurons (B). (Scale bars, 10 um.) (C-F) Thrashing assays. (C) Wild-type L4 hermaphrodites (n = 8) and
chc-1(
b1024) (n = 8) were incubated at 20C for 24 h and then transferred to M9 buffer. Head thrashes were counted. (D) Wild-type L4 hermaphrodites (n = 8) and
chc-1(
b1025ts) mutants (n = 8) were incubated at 15C for 24 h and examined. (E) Wild-type L4 hermaphrodites (n = 5),
chc-1(
b1025ts) (n = 5), and
chc-1(
b1025ts) mutant animals expressing GFP-CHC (dkEx6 [Prgef-1::GFP::
chc-1]) under the control of a neuron-specific promoter (n = 5) were incubated at 25C for 24 h before examination. (F) Wild-type young adult worms (n = 4) or
chc-1(
b1025ts) mutants (n = 8) were incubated at 30C for 10 min and examined. (G) Aldicarb resistance. Thirty young adult hermaphrodites of the wild type,
chc-1(
b1025ts), and
lev-1(
e211) were placed on aldicarb plates (0.5 mM) at the semi-permissive temperature of 22C and checked for their ability to move at the indicated time-points in response to prodding.
lev-1(
e211) mutants that lack a subunit of the muscle inotropic ACh receptor were resistant to both aldicarb and levamisole and used as a control. (H) Levamisole sensitivity of
chc-1(
b1025ts). Thirty young adult hermaphrodites of the wild type,
chc-1(
b1025ts), and
lev-1(
e211) were placed on levamisole plates (60 uM) and assayed for movement in response to prodding.