Figure 1.
copa-1 missense mutants experience heightened ER stress phenotypes after exposure to 10 µg/mL tunicamycin:a. COPI heptameric coatomer. Illustration showing the alpha, beta-prime, and epsilon subcomplex, as well as the delta, zeta, beta, and gamma subunits of the COPI coat protein assemble onto a budding vesicle from cis-Golgi bound for the ER. COPI coats vesicles moving in retrograde from the Golgi to the ER. Panel created in BioRender. b. Protein alignment between human and C. elegans COPA and COPA-1, respectively, at indicated residues. Nine amino acids implicated in COPA Syndrome are conserved (highlighted in aqua) and one differs (highlighted in magenta) between the two species. c.
copa-1 mutants generated with CRISPR/Cas9 gene editing. The genomic sequence and protein sequence are listed for each line at the indicated position. K322N mutation is indicated in green and R235H mutation is indicated in pink. Silent mutations used for genotyping with restriction enzyme digest are noted in orange. d.
copa-1 mutants have higher
hsp-4::GFP expression when stressed by TM compared to WT: WT,
copa-1[K232N], and
copa-1[R235H] animals were synchronized via egg-lay on MeOH (vehicle) or TM plates, and representative images show hatched larval animals at 24, 48, and 72 hours since they were laid on plates as embryos. Left channel is maximum projection of the GFP signal and right channel is the maximum projection overlaid with the top slice of a DIC stack. Images taken at 10x magnification with scale bar for 100 µm. Thirteen 0.5 µm z-stacks/image. e.
copa-1 mutants have higher quantified
hsp-4::GFP expression when stressed by TM compared to WT throughout larval development. While on control conditions
copa-1 mutants have comparable levels of
hsp-4::GFP expression compared to WT, they have a higher increase when stressed by TM, indicating greater activation of the UPRER. Intensity calculated by the mean fluorescent value of the whole area of the animal corrected for background intensity. Values normalized to average
hsp-4::GFP intensity of WT animals on control conditions. n = 20-30 animals/strain/condition. f.
copa-1 mutants have decreased length when stressed by TM compared to WT starting at 48 hours post-egg lay. n = 20-30 animals/strain/condition. g.
copa-1 mutants experience a developmental delay on TM during larval stages: Synchronized animals grown from an egg-lay on control or 10 µg/mL conditions reached fertile adulthood about a day later with
copa-1 mutations compared to control conditions or WT animals on either condition. This suggests an organismal phenotype brought on by higher ER stress sensitivity in
copa-1 mutants. N = 33-65 animals/strain/condition. Differences analyzed by Two-Way ANOVA with Tukey Kramer post hoc tests. * = p<0.05, **** = p<0.0001