Figure 4

Effect of single and double mutations on the ability of expressed NCS-1 to rescue the temperature-dependent changes in locomotion. (A) In each assay locomotion rates of wild-type Bristol N2 and ncs-1 null (qa406) worms were determined alongside wild-type or mutant expressing transgenic worms at room temperature (20°C; open bars) and then after elevation of temperature to 28°C for 10 min (filled bars). For each condition locomotion at 20°C and 28°C of three separate lines of transgenic worms expressing NCS-1 or its mutants on the ncs-1 null background was assayed, the data from separate worm lines were pooled and the data from experiments on different days normalised to the locomotion rate of N2 worms at 20°C in that particular assay. The rate of movement at 20°C and 28°C was compared for each condition (*; P < 0.01, n = 28-84 worms). The inset shows a Western blot probed with anti-human-NCS-1of ncs-1 null worms and transgenic worms with ncs-1 bearing the indicated mutations. (B) Within the same assay the locomotion of N2, ncs-1 null and transgenic worms expressing W103A, V125A or W103A.V125A was directly compared at room temperature (20°C; open bars) and then after elevation of temperature to 28°C for 10 min (filled bars). The rate of movement at 20°C and 28°C was compared for each condition (*; P < 0.01, n = 15 worms). Statistical significance was determined using the Mann–Whitney U test with use of the Bonferonni correction for multiple comparisons. The inset shows a Western blot probed with anti-human-NCS-1of ncs-1 null worms and transgenic worms with ncs-1 bearing the indicated mutations.