Fig 1.
ANC-1 functions with SLT-1 to polarize ALM axon growth.
(A) Example of a wild-type ALM neuron, with a single process growing from the anterior side of the cell body. (B) Example of a multipolar defect in an anc-1 mutant ALM neuron, with a second process growing from the posterior side of the cell body (arrow). (C) Quantification of the ALM axon polarity defects. Analysis of double mutants indicates that anc-1 and slt-1 function together in a genetic pathway to polarize ALM axon growth. In addition, anc-1 functions in parallel to unc-6 to polarize ALM axon growth. ALM neurons were analyzed in L4 hermaphrodites using the jsIs973 transgene that encodes Pmec-7::rfp. N>198 for all genotypes. Asterisks above individual bars indicate statistically significant difference relative to wildtype control, “N-1” Chi-squared test for proportions (*p = .041, **p < .01, ****p < .0001). Asterisks above brackets indicate statistically significant difference relative to wildtype control, “N-1” Chi-squared test for proportions (**p < .01Asterisks). Error bars represent the standard error of the proportion. Anterior is to the left. Scale bars are 5 μm.
Fig 2.
The ANC-1A and ANC-1C isoforms are required for the axon growth-polarizing activity of the anc-1 gene.
(A) Diagram of the ANC-1A, ANC-1B and ANC-1C proteins. The W621* mutation removes function of ANC-1A and ANC-1C and the oxTi902 and Q1363* mutations remove function of all three isoforms. (B) Quantification of ALM axon polarity defects. The ANC-1A and ANC-1C isoforms are required for the polarization of ALM axon growth. ALM neurons were analyzed in L4 hermaphrodites using the muIs32 transgene that encodes Pmec-7::gfp. N>194 for all genotypes. Asterisks indicate statistically significant difference (***p = .007, ****p < .0001) relative to wild type, “N-1” Chi-squared test for proportions. Error bars represent the standard error of the proportion. anc-1(W621*) is anc-1(gk109018[W621*]); anc-1(ΔCH-GFP) is anc-1(yc41[Δch::gfp3xFLAG::anc-1]); anc-1(ΔF0) is anc-1(syb5659).
Fig 3.
ANC-1A and ANC-1C are enriched in touch receptor neurons and are localized to the base of the proximal axon.
(A) Expression of ANC-1ΔCH-GFP, a marker for ANC-1A and ANC-1C, in the ALM neuron. (B) Expression of ANC-1ΔCH-GFP in the PLM neuron. Also see S1 Fig for colocalization with the Touch Receptor Neuron marker. (C) Expression of Scarlet::ANC-1A in the ALM (red circle) and AVM (yellow circle). Expression also seen in the seam cells (see arrow). (D) Expression of Scarlet::ANC-1A in the PLM (blue circle). (E) Expression of Scarlet::ANC-1C in the ALM (red circle) and the AVM (yellow circle). (F) Expression of Scarlet::ANC-1C in the PLM (blue circle). (G) ANC-1 visualized by split GFP. The ANC-1 protein was endogenously tagged with 2XGFP11 and GFP1-10 was expressed by a transgene (Pmec-4::gfp1-10). (H) Schematic of mutant forms of ANC-1. Anterior is to the left. Scales bars are 5 μm.
Fig 4.
ANC-1A and ANC-1C promote clustering of mitochondria at the base of the proximal axon.
(A-B) Example of ALM neurons with wildtype shown in (A) and the anc-1(oxTi902) null mutant shown in (B). Mitochondria are shown in red and are labeled with the mitochondrial targeting sequence of COX8 fused to TagRFP (MITO::TagRFP), which was expressed from the jsIs1073 transgene (Pmec-7::mito::tagRFP). The green label is cytosolic GFP, which was used to visualize the entire neuron and was expressed from the muIs32 transgene (Pmec-7::gfp). Scale bars are 5 μm. (C) Quantification of mitochondrial clustering defect at the base of the proximal ALM axon. N>35 for all genotypes. Asterisks indicate statistically significant difference, “N-1” Chi-squared test for proportions (*p = .016, ***p < .001). Error bars represent the standard error of the proportion. Data displayed as mean ± SEP. (D) Quantification of the length of the mitochondria cluster at the base of the proximal axon. N>35 for all genotypes. Data was compared using Welch’s ANOVA and Dunnet’s T3 multiple comparisons tests. Asterisks indicate statistically significant difference, (*p = .011, **p < .01, ***p < .001, ****p < .0001). Data displayed as mean ± SEM (E) Correlation between absence of mitochondria at the base of the proximal axon and the ALM axon polarity phenotype. Asterisk indicate statistically significant difference, “N-1” Chi-squared test for proportions (*p = .017). Data displayed as mean ± SEP.
Fig 5.
ANC-1 functions with mitochondria to polarize ALM axon growth.
(A) Mutations that disrupt the electron transport chain cause ALM axon polarity defects. (B) Dosage-dependent disruption of ALM axon polarity by Antimycin and Rotenone. (C) Antimycin disrupts ALM axon polarity but fails to enhance ALM axon polarity defects caused by the anc-1(oxTi902) null mutant. (D) Rotenone disrupts ALM axon polarity but fails to enhance ALM axon polarity defects caused by null mutants in anc-1, slt-1, or unc-6. N>196 for each concentration. Asterisks indicate statistically significant difference, “N-1” Chi-squared test for proportions (*p < .05, **p < .01). Error bars represent the standard error of the proportion. Scale bars are 5 μm. Alleles are: anc-1(oxTi902), mcu-1(ju1154), spg-7(ad2249), isp-1(qm150), gas-1(fc21), unc-6(ev400), and slt-1(eh15).