Figure 1.
Expression of polyQ-expanded AT3CT in C. elegans body wall muscle cells.
A. Schematic representations of gene constructs. The polyQ-containing C-terminal domain (lacking the N-terminal 257 amino acids) of AT3 was fused to YFP and expressed in body wall muscle cells under the control of the unc-54 promoter to generate Punc-54257cAT3(Qn)::YFP, herein referred to as AT3CT(Qn) B. Fluorescent micrographs showing fixed N2 (wild type), AT3CT(Q45), and AT3CT(Q63) animals imaged for YFP fluorescence (green) or phalloidin-stained actin filaments (red). C. Western blot with an anti-GFP antibody showing relative protein levels for C. elegans expressing YFP alone or AT3CT(Q45) or AT3CT(Q63). The asterisk (*) represents protein running as a GFP monomer. In the AT3CT (Q45) and AT3CT (Q63) lanes, these bands seem to represent cleavage fragments that are likely artifacts of protein extraction.
Figure 2.
Aggregation of AT3CT in C. elegans body wall muscle cells.
A. Fluorescence Recovery after Photobleaching (FRAP) of diffuse fluorescent protein in YFP or AT3CT(Q45)-expressing animals, or fluorescent foci in AT3CT(Q45) or AT3(Q63)-expressing animals. Time of bleach is indicated with an arrow. Fluorescence was monitored for 60 s. B. Native gel showing the YFP-containing protein species that accumulate in lines expressing YFP alone, AT3CT(Q45), or AT3CT(Q63). Presumptive aggregates (a), oligomers (o), and monomers (m) are indicated. Fluorescent protein species were visualized under UV light.
Figure 3.
The toxic effects of AT3CT expression in C. elegans body wall muscle cells.
Motility was determined as a measure of thrashing rate in liquid for L4 larvae (A) or animals at day 4 of adulthood (B). All 30 individual data points are represented on a dot blot. The symbol “▪” represents means. 95 percent confidence intervals are indicated. P-values are the results of pairwise student t-test.
Figure 4.
Effect of aging on AT3CT aggregation.
A. Micrographs showing YFP fluorescence in YFP- or AT3CT(Q45 or Q63)-expressing animals. B. Native gels showing the relative distribution of presumptive monomers (m), oligomers (o), and aggregates (a) at day 1 as compared to day 4 of adulthood.
Figure 5.
Effect of Heat Shock on C. elegans expressing AT3CT in body wall muscle cells.
Animals expressing a reporter for the heat shock response (PC12C8.1mCherry) or co-expressing this reporter along with YFP, AT3CT(Q45), or AT3CT(Q63) were imaged at day 1 of adulthood before (A) or after (B) a 34°C/15 min heat shock, or at day 4 of adulthood before (C) or after (D) a 34°C/15 min heat shock. E) qRT-PCR showing the relative expression levels of the endogenous C12C8.1 mRNA before and after heat shock. F) Native gel showing in-gel fluorescence from samples taken from YFP, AT3CT(Q45), or AT3CT(Q63) animals after heat shock (+HS) or without heat shock (-HS) at day 1 of adulthood.