A Directed RNAi Screen Based on Larval Growth Arrest Reveals New Modifiers of C. elegans Insulin Signaling

Genes regulating Caenorhabditis elegans insulin/IGF signaling (IIS) have largely been identified on the basis of their involvement in dauer development or longevity. A third IIS phenotype is the first larval stage (L1) diapause, which is also influenced by asna-1, a regulator of DAF-28/insulin secretion. We reasoned that new regulators of IIS strength might be identified in screens based on the L1 diapause and the asna-1 phenotype. Eighty- six genes were selected for analysis by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest. ykt-6, mrps-2, mrps-10 and mrpl-43 were identified as genes which, when inactivated, caused larval arrest without any associated feeding defects. Several tests indicated that IIS strength was weaker and that insulin secretion was defective in these animals. This study highlights the role of the Golgi network and the mitochondria in insulin secretion and provides a new list of genes that modulate IIS in C. elegans

Analysis of DAF-28::GFP and ss::GFP secretion.

<p>A) Quantification of DAF-28::GFP secretion defects in control and experimental RNAi worms. Error bars are +/− SE for three individual experiments. B) Paired DIC (top) and fluorescence (bottom) images of <i>daf-28::gfp</i> expressing worms exposed to control RNAi (top) or <i>ykt-6(RNAi)</i> (bottom). Arrows indicate coelomocytes. No DAF-28::GFP accumulation is seen in the coelomocytes of the <i>ykt-6(RNAi)</i> animal shown here. C) Paired DIC (left) and fluorescence (right) images of two <i>ykt-6</i>(RNAi) worms that did not secrete <i>daf-28::gfp</i>. DAF-28::GFP protein now accumulates around the gonad. n = intestinal nucleus. The anterior/posterior axis is indicated. D) Overlay of DIC and fluorescence images of worms with control RNAi and RNAi against indicated genes to test for the secretion and uptake of ss::GFP. In all cases GFP accumulates only in the coelomocytes (green cells).</p

Effect of depletion of gene function of identified genes on insulin/IGF signaling.

<p>A) The growth defect by RNAi against <i>ykt-6</i>, <i>mrps-2</i>, <i>mrps-10</i>, <i>mrpl-43</i> and <i>tomm-40</i> is suppressed in <i>daf-16</i> mutants. Bar plot showing mean growth rate coefficients between wild-type and <i>daf-16(mgDf50)</i> animals after RNAi. 0 corresponds to no difference in growth above stage L3 between <i>daf-16(mgDf50)</i> mutants and wild-type animals. Positive values indicate better growth in the <i>daf-16</i> background, where 1 corresponds to a twice as large fraction growing past stage L3 in RNAi treated <i>daf-16</i> worms vs. RNAi treated wild-type worms. 2 corresponds to a three times as large fraction growing past stage L3 etc. Error bars are +/− SE for two individual experiments. NS = not significant, *** P<0.001. Significance levels were calculated using <i>z</i>-tests. B) Fluorescence micrographs showing nuclear/cytoplasmic localization of DAF-16::GFP after RNAi against indicated genes. Arrows indicate nuclei that accumulate DAF-16::GFP. C) DIC micrographs showing dauer-specific alae in dauer larvae with the indicated RNAi treatments. D) DIC micrographs showing radially constricted pharynges in dauer larva with the indicated RNAi treatments compared to a control larva in stage L3. Arrows indicate outline the region between the two pharyngeal bulbs. E) Quantification of dauer formation in animals with RNAi against the indicated genes in wild-type, <i>daf-2</i>, <i>rrf-3</i>, and <i>daf-7</i> mutants in two experiments. <b>Control (RNAi):</b> N2(n = 600), <i>daf-2</i>(n = 220), <i>rrf-3</i>(n = 660), <i>daf-7</i>(n = 655). <b><i>mrps-2(RNAi)</i></b><b>:</b> N2(n = 600), <i>daf-2</i>(n = 197), <i>rrf-3</i>(n = 640), <i>daf-7</i>(n = 630). <b><i>mrps-10(RNAi)</i></b><b>:</b> N2(n = 600), <i>daf-2</i>(n = 202), <i>rrf-3</i>(n = 920), <i>daf-7</i>(n = 600). <b><i>mrpl-43(RNAi)</i></b><b>:</b> N2(n = 600), <i>daf-2</i>(n = 189), <i>rrf-3</i>(n = 730), <i>daf-7</i>(n = 615). <b><i>mrrf-1(RNAi)</i></b><b>:</b>N2(n = 600), <i>daf-2</i>(n = 189), <i>rrf-3</i>(n = 980), <i>daf-7</i>(n = 753). The N numbers are the total of two experiments. Error bars are +/− SE for two individual experiments.</p

Analysis of mitochondrial function after RNAi against mitochondrial ribosomal protein encoding genes.

<p>A) Fluorescence micrographs showing induction of phsp-6::GFP after RNAi against the indicated genes. Five animals are displayed in each panel. B) Fluorescence micrographs depicting TMRE staining in animals after RNAi against the indicated genes. Five animals are displayed in each panel. C) Mean TMRE fluorescence intensities relative to the control in RNAi-treated animals. Control animals were fed with empty vector L4440. Error bars represent a 95% confidence interval of the mean fluorescence intensity. Significance levels were calculated by student's t-tests vs. control. *** P<0.0001 and * P = 0.017.</p

Analysis of pharyngeal pumping, feeding and endocytosis in RNAi treated animals.

<p>A) Quantification of pharyngeal pumping rates after feeding RNAi. Each circle represents a single animal. Error bars represent a 95% confidence interval of the mean pumping rate. B) Overlay of differential interference contrast microscopy (DIC) and fluorescence images of worms exposed to RNAi against indicated genes and control RNAi to assay for their ability to ingest fluorescent beads. Arrows indicate accumulation of beads in the intestinal lumen. C) Fluorescence images of worms tested for uptake of FM4-64 dye by intestinal cells after exposure to RNAi against indicated genes and control RNAi.</p

Feeding RNAi analysis of the genes affecting <i>C. elegans</i> larval development.

<p>A) Feeding RNAi against several genes, but not <i>mrrf-1</i>, caused larval growth phenotypes. Gro: Growth arrest; Lva: larval arrest; Clr: Clear body; WT: wild-type. Empty vector (L4440) feeding RNAi was used as a control in all experiments. B) Fraction of animals above stage L3 after RNAi treatments in one representative experiment.</p

FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase

Aberrant activation of anaplastic lymphoma kinase (ALK) has been described in a range of human cancers, including non-small cell lung cancer and neuroblastoma (Hallberg and Palmer, 2013). Vertebrate ALK has been considered to be an orphan receptor and the identity of the ALK ligand(s) is a critical issue. Here we show that FAM150A and FAM150B are potent ligands for human ALK that bind to the extracellular domain of ALK and in addition to activation of wild-type ALK are able to drive 'superactivation' of activated ALK mutants from neuroblastoma. In conclusion, our data show that ALK is robustly activated by the FAM150A/B ligands and provide an opportunity to develop ALK-targeted therapies in situations where ALK is overexpressed/activated or mutated in the context of the full length receptor