A toolkit for DNA assembly, genome engineering and multicolor imaging for C. elegans

One way scientists can observe and quantify processes in living cells is to engineer the genomes of animals to express multiple fluorescent proteins and then quantify those signals by various imaging techniques. To allow our laboratories to confidently quantify mixed (overlapping) fluorescent signals for our studies in the basic biology of gene expression and aging in C. elegans, we developed a comprehensive toolkit for C. elegans that we describe here. The Toolkit consists of two components: 1) a series of vectors for DNA assembly by homologous recombination (HR) in the yeast, Saccharomyces cerevisiae, and 2) a set of ten worm strains that each express a single, spectrally distinct fluorescent protein, under control of either the daf-21 or eft-3 promoters. We measured the in vivo emission spectrum (3 nm resolution) for each fluorescent protein in live C. elegans and showed that we can use those pure spectra to unmix overlapping fluorescent signals in spectral images of intestine cells. Seven of ten fluorescent proteins had signals that appeared to be localized in vesicular/elliptical foci or tubules in the hypodermis. We conducted fluorescence recovery after photobleaching (FRAP) experiments and showed that these structures have recovery kinetics more consistent with freely diffusing protein than aggregates (Q35:YFP). This toolkit will allow researchers to quickly and efficiently generate mutlti-fragment DNA assemblies for genome editing in C. elegans. Additionally, the transgenic C. elegans and the measured emission spectra should serve as a resource for scientists seeking to perform, or test their ability to perform, multidimensional (multi-color) imaging experiments

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in <i>Caenorhabditis elegans</i>

<div><p>Mitochondrial dysfunction can increase oxidative stress and extend lifespan in <i>Caenorhabditis elegans</i>. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPR^mt) and extends lifespan. Here we report that transaldolase (<i>tald-1</i>) deficiency impairs mitochondrial function <i>in vivo</i>, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H₂O₂ levels. Lifespan extension from knockdown of <i>tald-1</i> is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (<i>fmo-2</i>). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.</p></div

Redox stress is downstream of transaldolase deficiency.

<p><b>(A)</b> H₂O₂ levels increase from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i> (N = 7 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(B)</b> NADPH levels decrease from RNAi knockdown of <i>tald-1</i> (N = 5+ biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> RNAi knockdown of <i>tald-1</i> causes sensitivity to paraquat (PQ). Percent survival of N2 worms grown on RNAi bacteria and 10 mM PQ was measured over seven days. Survival analyses were performed at 25°C (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Model of transaldolase deficiency mediated longevity.

<p>Reduced activity of the pentose phosphate pathway enzyme transaldolase has several consequences, including inhibition of mitochondrial respiration, induction of a mitochondrial stress response, alterations in redox homeostasis, and activation of a starvation-like metabolic response. Lifespan extension in response to transaldolase deficiency appears to be mediated by both MAPK signaling and HLH-30 mediated induction of autophagy and activation of FMO-2.</p

Alkaline gradient (Run time 11 min).

<p>Alkaline gradient (Run time 11 min).</p

The flavin-containing monooxygenase FMO-2 is upregulated in a HLH-30 and PMK-1 dependent fashion and regulates the lifespan extension from <i>tald-1(RNAi)</i>.

<p><b>(A)</b><i>fmo-2p</i>::<i>mCherry</i> reporter expression is increased by <i>tald-1(RNAi)</i> or BD in a HLH-30 and PMK-1 dependent fashion. BD animals were starved for 24 hours on FUDR plates prior to imaging. Scale bar, 200 μm. <b>(B)</b> Mean relative fluorescence of <i>fmo-2p</i>::<i>mCherry</i> reporter animals in the context of the <i>hlh-30(tm1978)</i> mutation. Fluorescence is calculated relative to N2 <i>EV(RNAi)</i> controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(C)</b> Mean relative fluorescence of <i>fmo-2p</i>::<i>mCherry</i> reporter animals in the context of the <i>pmk-1(km25)</i> mutation. Fluorescence is calculated relative to N2 <i>EV(RNAi)</i> controls (N = 5 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(D)</b> Gene expression of <i>fmo-2</i> is upregulated by <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> (N = 11 biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(E)</b> Gene expression of <i>fmo-2</i> is upregulated by <i>tald-1(RNAi)</i> in a HLH-30 and PMK-1 dependent fashion (N = 3–6 biological replicates, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(F)</b> Percent of animals displaying HLH-30 nuclear localization. BD animals were starved for 8 hours on FUDR plates prior to imaging (N = 5 independent experiments, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(G)</b> FMO-2 is required for the lifespan extension from <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 15.3±0.1 days, n = 341), N2 fed <i>tald-1(RNAi)</i> (mean 17.8±0.1 days, n = 353), <i>fmo-2(ok2147)</i> fed <i>EV(RNAi)</i> (mean 18±0.2 days, n = 314), <i>fmo-2(ok2147)</i> fed <i>tald-1(RNAi)</i> (mean 17.4±0.2 days, n = 382). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(H)</b> FMO-2 is partially required for the lifespan extension from <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 15.7±0.1 days, n = 562), N2 fed <i>cco-1(RNAi)</i> (mean 23.3±0.2 days, n = 616), <i>fmo-2(ok2147)</i> fed <i>EV(RNAi)</i> (mean 18.3±0.1 days, n = 474), <i>fmo-2(ok2147)</i> fed <i>cco-1(RNAi)</i> (mean 20.5±0.2 days, n = 473). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. <b>(I)</b> Lifespan extension from <i>fmo-2</i> overexpression is not additive with <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16.5±0.1 days, n = 453), N2 fed <i>tald-1(RNAi)</i> (mean 20.6±0.1 days, n = 421), <i>eft-3p</i>::<i>fmo-2</i> fed <i>EV(RNAi)</i> (mean 18.2±0.1 days, n = 439), <i>eft-3p</i>::<i>fmo-2</i> fed <i>tald-1(RNAi)</i> (mean 19.1±0.1 days, n = 435). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(J)</b> Lifespan extension from <i>fmo-2</i> overexpression is additive with <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16.5±0.1 days, n = 453), N2 fed <i>cco-1(RNAi)</i> (mean 23.3±0.2 days, n = 259), <i>eft-3p</i>::<i>fmo-2</i> fed <i>EV(RNAi)</i> (mean 18.2±0.1 days, n = 439), <i>eft-3p</i>::<i>fmo-2</i> fed <i>cco-1(RNAi)</i> (mean 25.5±0.2 days, n = 352). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Inhibition of the pentose phosphate pathway activates the UPR^mt and extends lifespan.

<p><b>(A)</b> Diagram of both the oxidative and non-oxidative branches of the PPP. The oxidative branch produces NADPH, while the non-oxidative branch produces ribose-5-P and interconverts sugar carbon backbones. The white boxes contain enzyme names with the human gene listed above the <i>C</i>. <i>elegans</i> homolog. <b>(B)</b> PPP gene knockdown increases <i>hsp-6p</i>::<i>gfp</i> reporter expression. <b>(C)</b> Mean relative fluorescence of <i>hsp-6p</i>::<i>gfp</i> animals grown on PPP RNAi. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(D)</b> RNAi knockdown of PPP genes extends <i>C</i>. <i>elegans</i> lifespan. N2 fed <i>EV(RNAi)</i> (mean 17.4±0.1 days, n = 455), N2 fed <i>tald-1(RNAi)</i> (mean 19.9±0.2 days, n = 391), N2 fed <i>tkt-1(RNAi)</i> (mean 18.4±0.1 days, n = 461), N2 fed T25B9.9<i>(RNAi)</i> (mean 18.8±0.2 days, n = 311). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(E)</b> RNAi knockdown of <i>tald-1</i> extends lifespan independently of the UPR^mt. N2 fed <i>EV(RNAi)</i> (mean 19.3±0.2 days, n = 192), N2 fed <i>tald-1(RNAi)</i> (mean 22.1±0.2 days, n = 251), <i>atfs-1(tm4525)</i> fed <i>EV(RNAi)</i> (mean 19.6±0.2 days, n = 230), <i>atfs-1(tm4525)</i> fed <i>tald-1(RNAi)</i> (mean 24.5±0.3 days, n = 228), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>EV(RNAi)</i> (mean 18.9±0.2 days, n = 205), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>tald-1(RNAi)</i> (mean 23.1±0.3 days, n = 220). Lifespans were performed at 20°C, with pooled data from two independent experiments shown. <b>(F)</b> RNAi knockdown of <i>cco-1</i> extends lifespan independently of the UPR^mt. N2 fed <i>EV(RNAi)</i> (mean 19.3±0.2 days, n = 192), N2 fed <i>cco-1(RNAi)</i> (mean 32.3±0.5 days, n = 187), <i>atfs-1(tm4525)</i> fed <i>EV(RNAi)</i> (mean 19.6±0.2 days, n = 230), <i>atfs-1(tm4525)</i> fed <i>cco-1(RNAi)</i> (mean 29±0.6 days, n = 194), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>EV(RNAi)</i> (mean 18.9±0.2 days, n = 205), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>cco-1(RNAi)</i> (mean 32.6±0.5 days, n = 228). Lifespans were performed at 20°C, with pooled data from two independent experiments shown. <b>(G)</b> RNAi knockdown of <i>tald-1</i> extends lifespan only when knockdown occurs during development. N2 fed <i>EV(RNAi)</i> (mean 14.2±0.1 days, n = 361), N2 fed <i>tald-1(RNAi)</i> from hatching (mean 16.4±0.2 days, n = 468), N2 fed <i>tald-1(RNAi)</i> from L4 (mean 14.4±0.1 days, n = 330). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p