3 research outputs found
A genome scale screen for isolating mutants with extended mitotic lifespan in the yeast <i>S</i>. <i>cerevisiae</i>.
<p><b>A.</b> The screen work flow. The design rationale is detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s001" target="_blank">S1 Fig</a>. <b>B.</b> Cy5:Cy3 signal ratios of mutants maintained in a dividing state for 16 days. Ranked values were log<sub>2</sub> normalized and projected. Of the starting collection of 3762, 52 mutants that maintained negative log<sub>2</sub> Cy5:Cy3 signal ratios at both day 6 and day 16 and displayed signal ratios <-2.3 at day 16 were classified as potentially long-lived (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s003" target="_blank">S3 Fig</a>). <b>C.</b> Broad functional clustering of the putative longevity genes isolated in this screen using GO Ontology. Genes that function in protein modification and trafficking across the ER-Golgi network are outlined.</p
Enhanced clearance of protein aggregates in yeast and worm <i>rer1</i> mutants.
<p><b>A.</b> Live cell imaging of α-syn-GFP after a 4 hr induction in 0.2% galactose. TM (0.02 μg/ml) was added to the cultures after an initial induction in galactose and cells were imaged 2 hours later. In a marked contrast to untreated cells, the GFP foci were largely vacuolar in <i>rer1Δ</i> or wild type cells treated with TM (arrows). <b>B.</b> GFP immunoblot in whole cell lysates prepared from cells in (<b>A)</b>. The intravacuolar proteolysis of α-syn-GFP generates the transiently stable GFP moiety detected as a discrete fragment in SDS-PAGE. <b>C.</b> Fluorescent images of transgenic hermaphrodites stably expressing α-syn-GFP from a body wall muscle promoter (<i>Punc-</i><sub><i>54</i></sub>::<i>α-syn</i>::<i>GFP</i>). Arrows denote a subset of the α-syn-GFP foci. Worms co-expressing heat shock protein torsinA (<i>tor-2</i>) in the same cells (<i>P</i><sub><i>unc-54</i></sub>::<i>tor-2</i>) served as a positive experimental control. The average number of α-syn-GFP foci per worm scored from digitized Z-stacked images ± s.e.m are plotted in <b>D</b> (n >20).</p
ER stress extends reproductive lifespan in yeast and worms.
<p><b>A.</b> Mitotic lifespan of wild type yeast grown on YPD supplemented with the indicated concentrations of TM. Mean mitotic lifespans for WT (18.5) and cells treated with TM at 0.005 (24.5) and 0.02 (27.5) μg/ml were statistically significant (p-values<0.01). <b>B.</b> Reproductive lifespan in worms with or without dietary supplementation with TM. <b>C.</b> Live cell imaging of GFP-Rer1 in wild type yeast and <i>ire1Δ</i> mutants treated with DMSO or after a 2 hr exposure to 0.02 μg/ml TM. (Box) In tandem with inducing canonical UPR, ER stress increases trafficking from ER to Golgi, reflected in the Golgi redistribution of GFP-Rer1 in cells treated with TM. Conversely, deleting <i>RER1</i> increases ER load denoted by the high basal UPR in <i>rer1Δ</i> mutants. <b>D</b>. Mitotic lifespan of WT (16.5 days) and isogenic <i>rer1∆</i> (26.5), <i>ire1∆</i> (16.5) and <i>rer1∆ ire1∆</i> (24.5) mutants at 30°C. Differences between WT mean mitotic lifespan were significant for <i>rer1∆</i> and <i>rer1∆ ire1∆</i> mutants (p-values<0.001).</p