Figure S3.
Regulation of klp-7 by the PCP components of Wnt signaling in the PLM neuron, related to Fig. 6. (A) Confocal images of PLM neurons expressing a constitutive marker Pmec-4::mScarlet (shrEx143; magenta) and Pmec-4-GFP::KLP-7B (shrEx127; green) in WT and lin-17(0) at the L4 stage. (B) Histogram showing the normalized GFP::KLP-7 measured from the 30-µm ROIs placed on the anterior (A) and posterior (P) processes of the PLM as shown in A from L1 to L4 stages. N = 3–4 independent replicates; n (number of neurons) = 13–25. (C) Sequence alignment of the consensus amino acid stretch known to undergo phosphorylation in the neuron in the motor domain of Kinesin-13 homologues in various species. Sites of A-type phosphorylation catalyzed by ROCK (amino acid 549) and B-type phosphorylation catalyzed either by PAK-1 (aa 546 and 555) are highlighted. (D) Localization of LET-502 in the PLM (white arrows) using a rat anti–LET-502 antibody (magenta). TRN expressing GFP is stained using an anti-GFP mAb (green). (E) Representative image of worms expressing Pnmy-2:NMY-2::GFP. Touch neurons are labeled by tbIs222 (Pmec-4:mCherry), and enrichment of NMY-2::GFP in the PLM posterior process is indicated by white arrowheads. (F) The normalized length of the anterior process (length of anterior process/distance between PLM cell body and vulva) due to RNAi of daam-1 or rho-1.(G) The change in the normalized length of the anterior process due to the overexpression of Pmec-4-RHO-1WT (shrEx265) or a constitutively activated form of RHO-1, Pmec-4-RHO-1G14V (shrEx259). N = 3–8 independent replicates; n (number of neurons) = 20–49. (H) Overexpression of the A-type phosphorylated version of KLP-7 using Punc-86-KLP-7T549E (shrEx181) makes a longer anterior process (orange arrow) and shorter posterior process (yellow arrow). (I and J) Normalized posterior (I) and anterior (J) lengths of a PLM expressing various mutant forms of KLP-7 in the WT background. Punc-86-KLP-7T549E (shrEx181) and Punc-86-4-KLP-7T549A (shrEx216) transgenes were used to express the constitutively phosphorylated and phosphodead versions of KLP-7, respectively. N = 3 independent replicates; n (number of neurons) = 22–55. (K) Bar chart showing the percentage of PLM neurons with polarity reversal phenotype when various mutant forms of KLP-7 and RHO-1 are overexpressed in lin-17(0) or lin-17(0);klp-7(0) background. N = 3–4 independent replicates; n (number of neurons) = 28–63. Error bars represent SEM. Statistical comparisons were done using ANOVA with Tukey’s multiple comparison test (B, D, E, G, and H) or Fisher’s exact test (I). *, P < 0.05; ***, P < 0.001.

Regulation of klp-7 by the PCP components of Wnt signaling in the PLM neuron, related to Fig. 6. (A) Confocal images of PLM neurons expressing a constitutive marker Pmec-4::mScarlet (shrEx143; magenta) and Pmec-4-GFP::KLP-7B (shrEx127; green) in WT and lin-17(0) at the L4 stage. (B) Histogram showing the normalized GFP::KLP-7 measured from the 30-µm ROIs placed on the anterior (A) and posterior (P) processes of the PLM as shown in A from L1 to L4 stages. N = 3–4 independent replicates; n (number of neurons) = 13–25. (C) Sequence alignment of the consensus amino acid stretch known to undergo phosphorylation in the neuron in the motor domain of Kinesin-13 homologues in various species. Sites of A-type phosphorylation catalyzed by ROCK (amino acid 549) and B-type phosphorylation catalyzed either by PAK-1 (aa 546 and 555) are highlighted. (D) Localization of LET-502 in the PLM (white arrows) using a rat anti–LET-502 antibody (magenta). TRN expressing GFP is stained using an anti-GFP mAb (green). (E) Representative image of worms expressing Pnmy-2:NMY-2::GFP. Touch neurons are labeled by tbIs222 (Pmec-4:mCherry), and enrichment of NMY-2::GFP in the PLM posterior process is indicated by white arrowheads. (F) The normalized length of the anterior process (length of anterior process/distance between PLM cell body and vulva) due to RNAi of daam-1 or rho-1.(G) The change in the normalized length of the anterior process due to the overexpression of Pmec-4-RHO-1WT (shrEx265) or a constitutively activated form of RHO-1, Pmec-4-RHO-1G14V (shrEx259). N = 3–8 independent replicates; n (number of neurons) = 20–49. (H) Overexpression of the A-type phosphorylated version of KLP-7 using Punc-86-KLP-7T549E (shrEx181) makes a longer anterior process (orange arrow) and shorter posterior process (yellow arrow). (I and J) Normalized posterior (I) and anterior (J) lengths of a PLM expressing various mutant forms of KLP-7 in the WT background. Punc-86-KLP-7T549E (shrEx181) and Punc-86-4-KLP-7T549A (shrEx216) transgenes were used to express the constitutively phosphorylated and phosphodead versions of KLP-7, respectively. N = 3 independent replicates; n (number of neurons) = 22–55. (K) Bar chart showing the percentage of PLM neurons with polarity reversal phenotype when various mutant forms of KLP-7 and RHO-1 are overexpressed in lin-17(0) or lin-17(0);klp-7(0) background. N = 3–4 independent replicates; n (number of neurons) = 28–63. Error bars represent SEM. Statistical comparisons were done using ANOVA with Tukey’s multiple comparison test (B, D, E, G, and H) or Fisher’s exact test (I). *, P < 0.05; ***, P < 0.001.

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