Figure 1. Amino acid substitutions in the UNC-104 stalk suppress PH domain-disrupting
unc-104(
e1265). Protein schematic showing domain organization with amino acid changes and alignment of part of the Stalk region. Grey highlighted amino acids in alignment are predicted beta strands in Alpha Fold structure. Green highlighted amino acids in alignment compose conserved loop containing the suppressor positions D948 and A950 (red highlight). SPG30 variant position I1026 denoted with cyan highlight. (B-F) Bright field images showing movement tracks of wild type (juIs1) (B), unc- 104
(e1265) (encoding D1497N) (C), and suppressors
unc-104(
ju250 e1265) (encoding M1540I D1497N) (D),
unc-104(
ju254 e1265) (encoding D948N D1497N) (E), and
unc-104(
ju249 e1265) (encoding A950V D1497N) (F). (B'-F') Expression of the Punc-25-SNB-1::GFP(juIs1) synaptic vesicle marker along the dorsal and ventral nerve cords in wild type (B'), unc- 104
(e1265) (encoding D1497N) (C"), and suppressors (D'-F'). (G-I) Bright field images showing movement tracks of wild type (juIs137) (G),
unc-104(
ju1894) (encoding D948N) (H), and
unc-104(
ju1895) (encoding A950V) (I). (G'-I') Expression of the Pflp-13-SNB-1::GFP(juIs137) synaptic vesicle marker along the dorsal and ventral processes of DD motor neurons. (J) Chart showing sequencing results of
ju249 CRISPR rescue of
unc-104(
e1265). Ratio is number of F1 animals with indicated codon change out of the 44 total suppressed animals that were sequenced.