Interaction of ARF-1.1 and neuronal calcium sensor-1 in the control of the temperature-dependency of locomotion in Caenorhabditis elegans

Abstract

Neuronal calcium sensor-1 (NCS-1) mediates changes in cellular function by regulating various target proteins. Many potential targets have been identified but the physiological significance of only a few has been established. Upon temperature elevation, Caenorhabditis elegans exhibits reversible paralysis. In the absence of NCS-1, worms show delayed onset and a shorter duration of paralysis. This phenotype can be rescued by re-expression of ncs-1 in AIY neurons. Mutants with defects in four potential NCS-1 targets (arf-1.1, pifk-1, trp-1 and trp-2) showed qualitatively similar phenotypes to ncs-1 null worms, although the effect of pifk-1 mutation on time to paralysis was considerably delayed. Inhibition of pifk-1 also resulted in a locomotion phenotype. Analysis of double mutants showed no additive effects between mutations in ncs-1 and trp-1 or trp-2. In contrast, double mutants of arf-1.1 and ncs-1 had an intermediate phenotype, consistent with NCS-1 and ARF-1.1 acting in the same pathway. Over-expression of arf-1.1 in the AIY neurons was sufficient to rescue partially the phenotype of both the arf-1.1 and the ncs-1 null worms. These findings suggest that ARF-1.1 interacts with NCS-1 in AIY neurons and potentially pifk-1 in the Ca(2+) signaling pathway that leads to inhibited locomotion at an elevated temperature.

Figure 1. Changes in locomotion over time following temperature elevation.

N2 worms were examined following elevation of temperature from 20 °C ± 0.5 to a final temperature of 28.0 °C ± 0.5. The rate of thrashing of individual worms was determined and the data shown as the mean rate of thrashing for the population ± SEM (A). The inset shows the rate of thrashing of those worms that were not paralysed, showing that they thrashed at the same rate before and during the time when other worms became paralysed and after the initial recovery period. The percentage of worm that were paralysed at each time point was also determined (B).

Figure 2. Analysis of the temperature-dependent locomotion of null ncs-1 and NCS-1 rescue strains.

Various C. elegans strains, as indicated, were analysed using the TDL assay as described in the Methods section. Locomotion of worms was initially assessed at 20 °C and then following the temperature shift to 28.5 °C. Multiple animals were tested for each strain and mean values for time to paralysis (A) and the duration of paralysis (B) were determined and expressed as mean ± SEM. The numbers of animals used for each strain were N ≥ 30. All data sets were compared with N2 control worms and statistical differences for the onset and duration of paralysis for each strain was determined using one-way ANOVA and Dunnett’s correction for multiple comparisons (*p < 0.001 versus N2; **p < 0.001 versus qa401).

Figure 3. Assay of the temperature dependent locomotion of various mutant C. elegans strains.

Temperature-dependent locomotion assays were carried out on various strains of C. elegans, as indicated, to provide mean values for the time to the start of paralysis after the shift to 28.5 °C and the duration of paralysis. Multiple animals were tested for each strain (N = ≥20) and mean values for time to paralysis (A) and the duration of paralysis (B) were determined. All data are expressed as mean ± S.E.M. Statistical differences were identified by comparing averaged data to that of N2 wild-type worms, using one-way ANOVA with Dunnett’s correction for multiple comparisons (*p < 0.05 versus N2).

Figure 4. Assay of the temperature-dependent locomotion of the ncs-1 mutation in the presence or absence of trp-1 and trp-2.

Temperature-dependent locomotion assays were carried out on various indicated strains of C. elegans to determine the onset and duration of paralysis after the shift to 28.5 °C. The double mutants were derived from genetic crosses of qa401 with either sy691 or ok323. Multiple animals (N ≥ 40) were tested for each strain and mean values for time to paralysis (A) and the duration of paralysis (B) were determined. All data are expressed as mean ± S.E.M. Statistical differences were identified by comparing averaged data to that of N2 wild-type worms, using one-way ANOVA with Dunnett’s correction for multiple comparisons (*p < 0.001 versus N2).

Figure 5. Assay of the temperature dependent locomotion of arf-1.1; ncs-1 mutants.

Temperature-dependent locomotion assays were carried out on various indicated strains of C. elegans to determine the onset and duration of paralysis after the shift to 28.5 °C. Multiple animals (N ≥ 60) were tested for each strain and mean values for time to paralysis (A) and the duration of paralysis (B) determined. All data are expressed as mean ± S.E.M. Statistical differences were identified by comparing averaged data to that of N2 wild-type worms, using one-way ANOVA with Dunnett’s correction for multiple comparisons (*p < 0.001).

Figure 6. Effect of PIK-93 on temperature-dependent locomotion in N2, pifk-1 and ncs-1 null strains.

Various worm stains as indicated were pre-treated for one hour with vehicle (1% DMSO) or 19 μM PIK-93. Temperature-dependent locomotion assays were carried out to determine the onset and duration of paralysis after the shift to 28.5 °C. Multiple animals (N = 30) were tested for each strain and mean values for time to paralysis (A) and the duration of paralysis (B) were determined. All data are expressed as mean ± S.E.M. Statistical differences were identified by comparing averaged data to that of N2 wild-type worms, using one-way ANOVA with Dunnett’s correction for multiple comparisons (*p < 0.001 versus vehicle control).

Figure 7. Assay of the temperature-dependent locomotion after ARF-1.1 overexpression or rescue.

GFP expression is driven by pAIY specifically in the two AIY neurons indicated by arrows. No expression was observed elsewhere (A). The asterisk shows the position of the posterior tip of the imaged worm. Transgenic worms were generated to express ARF-1.1 within AIY neurons in a wild-type background (N ≥ 56) (B,C), in arf-1.1 mutant worms (N ≥ 40) (D,E) or in ncs-1 null mutant worms (N ≥ 56) (F,G). Temperature-dependent locomotion assays were carried out on various indicated strains of C. elegans to determine the time to the start of paralysis after the shift to 28.5 °C and the duration of paralysis. Multiple animals were tested for each strain and mean values for time to paralysis (B,D,F) and the duration of paralysis (C,E,G) were determined. Each transgenic data set presented used a conglomerate of at least 3 separate transgenic lines, which were pooled together. All data are expressed as mean ± S.E.M. Statistical differences were identified by comparing averaged data to those of N2 wild-type worms using one-way ANOVA with Dunnett’s correction for multiple comparisons (*p < 0.001).