Fig 1.
AT variants and transgenic constructs.
A ribbon diagram of α1-antitrypsin (PBD, 3NE4) highlighting the positions of the amino acid residues associated with deficient and null alleles (A). A schematic depicting the C. elegans expression construct (B). Pnhx-2, intestine-specific promoter; sGFP, green fluorescent protein with an N-terminal signal peptide; AT, α1-antitrypsin.
Fig 2.
Confocal images of AT variant lines.
Representative images of adult animals expressing various AT transgenes. Each panel shows a widefield DIC image of the entire worm (upper panel), corresponding fluorescence image (middle panel), and 2.5 x magnification of the region outline by a white box (lower panel, inset). Animals expressing the sGFP control protein efficiently secrete GFP out of the intestine (int) into the pseudocelomic space (A, asterisks). However, animals expressing sGFP::KDEL (ER-retention signal) control retain GFP in the ER (B).sGFP::ATM is efficiently secreted into the pseudocoelomic space (C, asterisks) similar to that observed in sGFP expressing controls. Animals expressing the deficient alleles, sGFP::ATZ (D), sGFP::Siiyama (E) and sGFP::Mmalton (F) accumulate protein in the ER as large globules (arrows) and show no evidence of secretion. Animals expressing sGFP::ATS accumulate very low steady-state levels of fusion protein with an occasional small globule detected near the tail (G, arrows). Animals expressing the null alleles, sGFP::Saar (H) and sGFP::NHK (I) accumulate barely detectable levels of protein in the ER.
Fig 3.
Quantification of aggregate size.
Aggregate sizes accumulating in animals expressing deficient alleles. GFP-positive globules were imaged using confocal microscopy and rendered in 3D. Aggregate volumes were calculated using Volocity software. Representative images of globules from sGFP::ATZ, sGFP::Mmalton and sGFP::Siiyama are shown. Statistical significance of average size compared against ATZ was determined using two-tailed student's t-test, with the probabilities of results reported as *P<0.05. ns, not significant.
Fig 4.
Immunoblots of AT variant transgenic lines.
Western blot analysis of lysates from animals expressing different AT variant transgenes. The steady-state levels of WT and mutant sGFP::AT fusion proteins under denaturing conditions (A, upper panel). Actin serves as a loading control (A, lower panel). Relative AT protein levels as determined by densitometry (B). Statistical significance was determined using two-tailed student's t-test, comparing each AT variant line to ATM. Relative mRNA levels as determined by qPCR (C). Statistical significance was determined using two-tailed student's t-test, comparing each AT variant line to ATM. Lysates from null mutants sGFP::Saar and sGFP::NHK, exposed to vector(RNAi) or sel-1(RNAi) probed with GFP (D, upper panel) or α-tubulin (D, lower panel).
Fig 5.
Proteostasis pathways used to clear AT variant proteins.
Effect of ERAD (hrd-1 or sel-1) RNAis on clearance of deficient (A) and null variants (B). Effect of autophagy (bec-1 or unc-51) RNAis on clearance of deficient (C) and null variants (D). The data is representative of 6 experiments. Statistical significance was determined by comparing treatments to their respective vec(RNAi) controls using an unpaired, two-tailed students t-test, *P<0.05, **P<0.01, and ***P<0.001.
Fig 6.
Effect of AT variant protein expression on longevity.
Representative Kaplan-Meier curves of deficient mutants (A). N2 (black), ATM (green), Siiyama (red), Mmalton (blue) and ATZ (lavender). Median survival times (in parenthesis). Representative Kaplan-Meier curves of ATS and null alleles (B). N2 (black), ATS (green), NHK (red) and Saar (blue). Statistical significance determined using the Log-Rank (Mantel-Cox) test, with probabilities of results reported as **P<0.001, or ***P<0.0001.
Fig 7.
Effect of AT variant protein expression on larval growth and brood sizes.
Assessment of slow growth (Gro) phenotype during post-embryonic development (A). Statistical significance was determined comparing Gro phenotypes of AT variants to wild-type ATM-expressing lines using an unpaired, two-tailed students t-test. Reversal of Gro phenotype by GFP(RNAi) (B). Statistical significance was determined by comparing treatments to their respective vec(RNAi) controls using an unpaired, two-tailed students t-test. Brood size assessment of AT variant lines (C). Statistical significance was determined comparing brood size of AT variants compared to wild-type ATM-expressing lines using an unpaired, two-tailed students t-test. Brood size comparison between integrated and non-integrated Siiyama expressing lines (D). Statistical significance was determined comparing brood size of AT variants was compared to wild-type ATM-expressing lines using an unpaired, two-tailed students t-test. In all experiments, the probabilities of results were reported as *P<0.05, **P<0.01, or ***P<0.001.