The Journal of Neuroscience, June 7, 2006, ():
The Caenorhabditis elegans Choline Transporter CHO-1 Sustains Acetylcholine Synthesis and Motor Function in an Activity-Dependent Manner
J. Neurosci. Matthies et al.
26: 6200
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1: Expression of a CHO-1:GFP fusion protein rescues the 3Hcholine uptake deficit present in cho-1 mutant primary cultures. Data are from primary cultures prepared from WT, CHO-1 KO, and transgenic animals expressing a CHO-1:GFP fusion protein in the cholinergic nervous system in the cho-1 knockout background (BY506). 3Hcholine uptake was measured in the presence and absence of 1 µM HC-3, and expressed as percent WT control (mean + S.E.M.). The CHO-1:GFP fusion protein restored choline uptake to 73.1 + 10.6% of WT control when expressed in the cho-1 mutant background. Lack of complete rescue may indicate that choline is transported less efficiently with the inclusion of the GFP tag.
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Supplementary Figure 2: Pre- and post-synaptic pharmacological profiles of cho-1 mutant animals. A) cho-1 mutant animals had normal sensitivity to the acetylcholinesterase inhibitor aldicarb over a 0 – 1.0 mM concentration range. Shown is a representative experiment, plotted as percent animals paralyzed at each aldicarb concentration. B) cho-1 mutants showed wildtype responses to the nicotinic acetylcholine receptor agonist Levamisole. Shown is a time course of treatment using 0.1 mM Levamisole, plotted as percent animals paralyzed.
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Supplementary Figure 3: cho-1(tm373) mutant animals have overtly normal cholinergic neuroanatomy and synapses. Shown are representative confocal projections of cholinergic neuron processes making normal contact with the dorsal nerve cord in WT (BY505) and CHO-1 KO (BY507) animals. Soluble GFP was used as a reporter, and expressed under the control of the cho-1 promoter. Inset: the density and morphology of cholinergic synapses in WT (BY509) and CHO-1 KO (BY510) animals were similar as observed by VAMP:mRFP1 expression. Scale bar = 20 µm.
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Supplementary Figure 4: Locomotor behavior of WT, cho-1 mutant, BY506 (pcho-1:CHO-1:GFP expressed in the cho-1 mutant background) and characterized synaptic mutants. Individual thrashing behavior was monitored using an automated tracking system, and the time each animal spent immobile during each 30 minute window plotted (mean + S.E.M.). Results are from 2 hour assays performed in choline-free liquid media. Data was analyzed using ANOVA, comparing mutants to WT for all time segments. Non significant data: p>0.05.
