Figure Legends for Supplemental Figures
Figure S1. Localization of soluble GFP, YFP-GPI, SNB-1 and SNG-1.
(A)Ventral view of RIA interneurons in wild type animals expressing soluble GFP. The neurites of RIA neurons are composed of a distal presynaptic region (Pre), proximal postsynaptic region (Post) and the narrow isthmus-like area connecting them (I). Dorsal is up. Scale bar, 5 µm.
(B)Localization indexes calculated for distribution of soluble GFP (left) and for YFP-GPI (right) specifically expressed in RIA neurons. (Left) Indexes for soluble GFP under the following conditions: untreated wild type animals, cultivated with LiCl overnight at adult stages (LiCl at adult), cultivated with LiCl from birth (LiCl from birth) and untreated ttx-7(nj50) mutants, cultivated with inositol from birth (Ins from birth), with transgene RIAp::ttx-7a (RIAp::ttx-7a), with transgene hsp::ttx-7a to which a 4-hr heat shock was applied at the adult stage (hsp::ttx-7a + hs at adult). No significant difference was detected in any combination by the Steel-Dwass multiple comparison test (n=10 animals for each condition). (Right) Indexes for YFP-GPI of wild type and ttx-7(nj50) animals (p>0.05, Mann-Whitney U test; n=11 and 12 for wild type animals and ttx-7 mutants, respectively). The index of YFP-GPI in wild type animals is significantly higher than the index of soluble GFP in wild type animals (p<0.01, Mann-Whitney U test).
(C)YFP-GPI localization in the cell body of wild type and ttx-7 animals. Scale bar, 2 µm.
(D)Localization of the synaptic vesicle protein SNG-1 fused to GFP in RIA neurons of wild type and ttx-7 animals. Scale bar, 5 µm.
(E)Localization of SNB-1::VENUS in RIA neurons of wild type and ttx-7 animals. SNB-1::VENUS was expressed from the integrated transgenic array njIs9. Similar to the transgenes expressed from extrachromosomal arrays shown in Fig. 3C, SNB-1::VENUS from an integrated array mislocalized over the whole neurite in ttx-7 mutants.
Figure S2. Localization of SNB-1 in other mutants and mutant SNB-1 in wild type animals.
(A)Localization of SNB-1::VENUS in RIA neurons of gsk-3, syd-1, sad-1, unc-11 and unc-11 ttx-7 double mutants. In unc-11 mutants, SNB-1::VENUS appeared diffuse on the plasma membrane but was largely restricted in the presynaptic region. As unc-11 ttx-7 double mutants showed a different distribution of SNB-1 from that in ttx-7 mutants, and exhibited severer diffusion than unc-11 mutants, unc-11 and ttx-7 mutants have defects in at least partly non-overlapping molecular processes.
(B)Localization of SNB-1(M38A)::VENUS in RIA neurons of wild type animals. The M38A substitution corresponds to the M46A substitution of human synaptobrevin, which is known to prevent endocytosis of synaptobrevin(Grote and Kelly, 1996). Fluorescence appeared diffuse on the plasma membrane.
Figure S3. Thermotaxis behavior of ttx-7 mutants and RIA-killed wild type animals
(A)Results of thermotaxis (TTX) assays of wild type and ttx7 animals cultivated at various temperatures. Each track of an individual animal on the assay plate was classified according to Mori and Ohshima (Mori and Ohshima 1995), whose classification method is also illustrated above the graphs here: The graphs show the fractions of tracks belonging to each class, namely 17 for tracks mainly in the coldest region, 20 for tracks mainly in the 20C region, 25 for tracks mainly in the hottest region and 17/25 for tracks spread all over the plate. ttx-7 mutants showed severe athermotactic (17/25) defects independent of the cultivation temperature, although some effects of cultivation temperature could be seen especially when cultivated at 25C. This may indicate that the ttx-7 mutation allows thermophilic signaling to emerge as thermophilic behavior to some extent, which is consistent with the result of ttx-7; tax-6 double mutants in Fig. 2E (n=60 to 160 animals for each strain in each condition.)
(B)Results of TTX assays of RIA-killed wild type and naïve ttx7(nj50) animals cultivated at 20C. The classification of tracks was done as shown in (A).Data for RIA-killed wild type animals were from a previous study (Mori and Ohshima 1995). The difference between the results of the two strains did not reach statistical significance (Chi-square test; n=46 and 155 animals for RIA-killed wild type and ttx-7 animals, respectively.).
Figure S4. Extent of rescue with varying periods of heat shock at adult stages.
(A)Different periods of heat shock–induced expression of ttx-7 cDNA in ttx-7 mutants rescued thermotaxis defects proportional to the duration of heat shock, except for the 6-hr heat shock. The reason for less rescue with the 6-hr heat shock may be due to toxicity from prolonged exposure to heat (n=3 assays).
(B)Different periods of heat shock–induced expression of ttx-7 cDNA in ttx-7 mutants rescued the defect in SNB-1 localization indexes (n=10 animals).
(C)Different periods of heat shock–induced expression of ttx-7 cDNA in ttx-7 mutants rescued the defect in SYD-2 localization indexes (n=10 animals).
(A-C) Pearson’s correlation coefficient was calculated for each combination and is shown below the graphs. Correlation was significant between two of the three combinations as indicated by p values.
Figure S5. Detailed analyses of the effects of various reagents on thermotaxis and synaptic component localization.
(A)Exogenously applied inositol did not affect thermotaxis behavior of wild type animals (Student’s t test, n=3 assays).
(B)Exogenously applied inositol did not affect the localization index of SNB-1 in wild type animals (Mann-Whitney’s U test, n=10 animals).
(C)Exogenously applied inositol did not affect the localization index of SYD-2 in wild type animals (Mann-Whitney’s U test, n=10 animals).
(D)LiCl applied from birth abolished thermotaxis behavior of wild type animals as with the ttx-7 mutation. Dunnett’s multiple comparison test was performed. n=3 or more assays.
(E)LiCl applied from birth abolished SNB-1 localization in wild type animals as with the ttx-7 mutation. Steel’s multiple comparison test was performed. n=10 animals.
(F)LiCl applied from birth abolished SYD-2 localization in wild type animals as with the ttx-7 mutation. Steel’s multiple comparison test was performed, n=10 or more animals.
(G)Overnight cultivation with 15 mM or more concentration of LiCl abolished thermotaxis of adult wild type animals; n≥3 assays). Dunnett’s multiple comparison test was performed.
(H)Thermotaxis tracks of wild type animals treated with LiCl overnight during adulthood. Animals overexpressing ttx-7 or grown with inositol showed normal thermotaxis behavior against the effect of LiCl.
(I)Animals overexpressing ttx-7 in RIA neurons were resistant to overnight cultivation with 15 mM LiCl but not to 30 mM LiCl in thermotaxis; n=3 assays). Student’s t test was performed only on 30 mM results and did not detect a significant difference.
Figure S6. Localization of SNB-1 in neurons other than RIA neurons.
(A)SNB-1::GFP expression driven by the snb-1 own promoter from integrated array jsIs1. Expression is induced in almost all neurons. Upper panels show head region, and lower panels show nervecords of wild type animals (left), ttx-7 mutants (middle), and wild type animals cultivated overnight with 15 mM LiCl (right). Anterior is to the left, dorsal is up; scale bar, 5 m for all panels in this figure.
(B)SNB-1::GFP expression driven by the unc-25 promoter from integrated array juIs1. Expression is induced in GABAergic motor neurons. Upper panels show nervecords in adult animals, and lower panels shows nervecords in L1 larvae. In L1 larvae, the unc-25 promoter drives gene expression in DD motor neurons, which innervate ventral muscles (Hallam, Goncharov et al. 2002).
(C)SNB-1::VENUS expression driven by the acr-2 promoter from an extrachromosomal array. Expression is induced in cholinergic motor neurons. Photographs show nervecords.
(D)SNB-1::VENUS expression driven by the gcy-5 and gcy-7 promoters from an extrachromosomal array. Expression is induced in ASE gustatory neurons. Photographs show the head region.
(E)SNB-1::VENUS expression driven by the str-2 promoter from an extrachromosomal array. Expression is induced in an AWC olfactory neuron. Photographs show the head region.
(F)SNB-1::VENUS expression driven by the gcy-8 promoter from an extrachromosomal array. Expression is induced in AFD thermosensory neurons. Photographs show the head region.