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First published online 17 February 2009
doi: 10.1242/jcs.036343

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The BTB-MATH protein BATH-42 interacts with RIC-3 to regulate maturation of nicotinic acetylcholine receptors

Department of Physiology, Hebrew University, Hadassah Medical School, Jerusalem, 91120, Israel

View larger version (35K): [in this window] [in a new window]   Fig. 1. X-gal assays and GST pull-down assays show an interaction between BATH-42 and RIC-3 (C-terminus). (A) X-Gal assay on yeast cells expressing the bath-42 prey alone, both the ric-3 bait and the bath-42 prey, or the ric-3 bait alone. (B) In vitro transcribed, translated and labeled bath-42 was loaded in lane 1. Labeled BATH-42 bound to GST:RIC-3, GST:ODZ or GST beads was loaded in lanes 2, 3 or 4, respectively.

View larger version (94K): [in this window] [in a new window]   Fig. 2. bath-42p:GFP expression in L4 stage or young adult C. elegans. (A) Pharyngeal muscles. Scale bar: 10 µm. (B) Seam cells (fluorescence on top right of the picture represents gut auto-fluorescence). Scale bar: 5 µm. (C) Vulval muscles. Scale bar: 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 3. bath-42(lf) leads to an increase in RIC-3 levels and to reduced levamisole sensitivity in vulva muscles. (A) Normalized staining intensity of RIC-3 in vulval muscles of wild-type (N2, black bars), or bath-42(tm2360), a bath-42(lf) mutant (grey bars). For each stained animal average staining intensity in vulval muscles was divided by the average staining intensity in the adjacent body-wall muscles and ventral cord neurons. Difference is significant P<0.01 using the Student's t-test for two samples (n=38-40 each, N=3). Representative images of RIC-3 staining in vulval muscles are shown on the right. Scale bar: 10 µm. (B) Number of eggs laid per animal by N2 or bath-42(lf) on food, M9 or M9 supplemented with 50 µM levamisole. Also shown is egg-laying in M9 supplemented with 50 µM levamisole for bath-42(lf) homozygotes that are heterozygous for ric-3(md1181), a ric-3(lf) mutant (white). Significant differences were found between number of eggs laid in the presence of levamisole for N2 compared with bath-42(lf), P<0.01 (n=72 each, N=6), and for bath-42;ric-3(md1181)/+ compared with bath-42(lf), P<0.05 (n=67-92, N=7-8) using the Student's t-test for two samples.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Increased RIC-3 expression leads to reduced nAChR activity in vulva muscles. (A) Number of eggs laid per animal by N2 (wild-type), ric-3(md1181) overexpressing ric-3 following 5 ng/µl injection, or ric-3(md181) overexpressing ric-3 following 20 ng/µl injection. Differences between the three strains are significant P<0.01 (following Bonferroni correction) using the Student's t-test for two samples (n=51-54 each, N=5). (B) Representative images of RIC-3:GFP distribution in vulval muscles of transgenics injected with 5 ng or 20 ng RIC-3:GFP. Arrows indicate representative puncta. Scale bar: 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Increased RIC-3 expression leads to defects in DEG-3 distribution and function. (A) Representative images of N2 or RIC-3:GFP-overexpessing transgenics. DEG-3 staining in green, GFP staining (for RIC-3:GFP) in red, merge in yellow. Scale bar: 2 µm. (B) Coefficient of variation of DEG-3 staining in N2 or in transgenics injected with 20 ng/µl RIC-3:GFP. Difference is significant P<0.01 using the Student's t-test for two samples (n=19, N=2). (C) Number of degenerating cells in deg-3(u662) animals expressing high copy number RIC-3:GFP (20ng/µl) compared with deg-3(u662) animals lacking this transgene. Difference is significant P<0.01 using the Student's t-test for two samples (n=41-42, N=2).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Overexpression of BATH-42 leads to reduced pharyngeal pumping in a ric-3- and cul-3-dependent manner. Effects of hsp-16-2p:BATH-42 transgene on pharyngeal pumping in wild-type (N2), ric-3(md158) mutants [ric-3(lf)], ric-3(lf) mutants also expressing a truncated RIC-3 TM protein, animals expressing the RIC-3 N protein, N2 treated with cul-3 dsRNA or N2 treated with vector dsRNA. Grey, control animals without heat shock; black, heat-shocked (HS) animals. Effects of heat-shock on pumping in N2 animals expressing hsp-16-2p:BATH-42 are significant P<0.01 using the Wilcoxon two-sample test (n=45-90 each, N=3-5). Effects of heat-shock on pumping in animals expressing RIC-3 N are also significant P<0.05 (n=75-78 each, N=5)

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