A Deficiency in the Autophagy Gene Atg16L1 Enhances Resistance to Enteric Bacterial Infection

SummaryPolymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohn’s disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes, such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyperimmune phenotype and its protective effects are lost in Atg16L1/Nod2 double-mutant mice. These results reveal an immunosuppressive function of Atg16L1 and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections

Roles for UNC-6/Netrin Signaling During Cell Invasion in C. Elegans

<p>Basement membranes are dense, sheet-like forms of extracellular matrix that</p><p>surround the epithelial tissues of metazoan organisms. While these structures are</p><p>critical for epithelial support and tissue organization, basement membranes also pose</p><p>formidable barriers to most cells. However, certain specialized cells are able to breach</p><p>these barriers and move between tissues. Acquisition of cell invasive behavior by some</p><p>tumor cells is thought be an important step in cancer progression. Due to the clear basic</p><p>and clinical importance of understanding the mechanisms underlying cell invasion</p><p>through basement membranes, cell invasive behaviors has been an area of intense study.</p><p>In this work I examine a developmentally regulated model of cell invasive behavior in</p><p>the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels</p><p>basement membrane to connect two epithelial tissues, the uterus and vulva. Using this</p><p>model I identify a novel role for UNC-6/Netrin signaling during this process through basement membranes. I show that Netrin signaling is a third regulatory input for AC invasion that functions partially in parallel to fos-1a and the vulval signal. Further I link netrin signaling to the formation of invasive protrusions that penetrate basement membrane.</p>Dissertatio

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue

F-actin polarization is dependent on the vulval precursor cells and CDC-42.

<p>(A-E) 3D projections (left) and isosurface renderings of a fluorescent F-actin probe (<i>cdh3>mCherry</i>::<i>moeABD</i>) in wild type animals (A), animals treated with RNAi against <i>cdc-42</i> (B), RNAi agianst <i>gdi-1</i> (C), double RNAi targeting <i>cdc-42</i> and <i>gdi-1</i> (D, <i>cdc-42; gdi-1</i> RNAi), and animals lacking the vulval precursor cells (E, <i>lin-3</i> RNAi). F-actin was mispolarized after targeting <i>cdc-42</i> (^a p < 0.05), <i>cdc-42</i> and <i>gdi-1</i> in combination (^b, p = 0.004), and the vulval precursor cells (<i>lin-3</i> RNAi; ^c p = 0.001). Comparisons were made using Fisher’s exact tests. (F) Scatter plot showing the percentage of F-actin polarized to the invasive membrane (line shows mean). The most severe of F-actin mispolarization defect was observed after loss of the vulval precursor cells (<i>lin-3</i> RNAi; n <u>></u> 8 animals per group; **^d p < 0.01 vs. wild type, *^e p < 0.05 vs. <i>cdc-42</i> RNAi, **^f p < 0.01 vs. <i>gdi-1</i> RNAi). Comparisons were made using a Tukey’s multiple comparisons test. Scale bars = 5 μm.</p

GDI-1 regulates invadopodia formation.

<p>(A) A transcriptional reporter (<i>gdi-1>GFP</i>) revealed <i>gdi-1</i> expression in the AC (arrow). (B) Uterine-specific RNAi against <i>gdi-1</i> blocked AC invasion (arrowhead marks intact BM). (C) A ventral time series shows decreased invadopodia (marked with F-actin) formation in animals with reduced <i>gdi-1</i> (RNAi; bottom) relative to wild type (top; overlaid text reports average number of invadopodia per timepoint from 6 wild type animals and 5 <i>gdi-1</i> RNAi treated animals; p < 0.0001, Tukey’s multiple comparisons test). (D) BM breaching was delayed in <i>gdi-1</i> RNAi treated animals (n ≥ 25 animals per stage, per genotype; *** p < 0.0001, * p < 0.05, Fisher’s exact test; bars represent 95% confidence intervals). (E) Animals treated with <i>gdi-1</i> RNAi have longer-lived invadopodia (n = 6 wild type and 7 <i>gdi-1</i> RNAi treated animals; p < 0.01, Chi-square test). (F) Ventral views showing that the wild type, punctate pattern of GFP::CDC-42 (top) was not affected by loss of <i>gdi-1</i> (bottom; overlaid text reports average number of puncta from 8 wild type animals and 9 <i>gdi-1</i> RNAi treated animals; p = 0.05, Student’s t-test). (G) CDC-42 was still activated normally (as visualized with the GTP-CDC-42 sensor) after loss <i>gdi-1</i> (overlaid text reports average number of puncta from 18 wild type animals and 6 <i>gdi-1</i> RNAi treated animals; p = 0.53, Student’s t-test). Scale bars = 5 μm.</p

Loss of <i>gdi-1</i> does not affect other AC trafficking and polarization processes.

<p>(A) In wild type animals (left) the primary vulval cells (expressing <i>egl-17>GFP</i>) are specified in response to the EGF-like ligand LIN-3 secreted by the AC. Primary vulval cell specification was normal after loss of <i>gdi-1</i> (RNAi; n <u>></u> 10 in each group) indicating proper AC trafficking and secretion of LIN-3. (B) The extracellular matrix component HIM-4 (<i>cdh3>him-4</i>::<i>GFP</i>) is secreted by the AC (visualized with <i>cdh3>mCherry</i>) and forms puncta within the BM of wild type animals (top; arrowheads). HIM-4::GFP deposition into the BM was unaffected by loss of <i>gdi-1</i> (bottom; arrowheads). In ventrally viewed images, the number of puncta was normal (overlaid text reports average number of puncta from 8 wild type and 9 <i>gdi-1</i> RNAi treated animals; p = 0.80; Student’s t-test). (C) The β-integrin subunit PAT-3::GFP (left; <i>pat-3>pat-3</i>::<i>GFP</i>) overlaid on a DIC image (right). PAT-3::GFP is polarized along the AC invasive membrane (top panels; brackets) and is also found between the vulval precursor cells (arrowheads). Loss of <i>gdi-1</i> (RNAi; bottom panels) did not alter PAT-3::GFP enrichment at the invasive membrane (brackets). Scatter plot shows the ratio of PAT-3::GFP at the AC invasive membrane relative to the apicolateral membrane (line shows the mean; n = 8 wild type and 9 <i>gdi-1</i> RNAi treated animals; n.s. = not significant, p = 0.11, Student’s t-test). Scale bars = 5 μm.</p

A Model of CDC-42 and GDI-1 function in promoting invadopodia formation.

<p>(A) A secreted cue or cues from the vulval precursor cells coordinates the activities of CDC-42 and F-actin formation (red puncta) at the invasive membrane by activating CDC-42 and through an unknown mechanism also regulates a GDI-1-dependent pathway that directs invadopodial membrane trafficking (blue) to invadopodia. Loss of <i>cdc-42</i> leads to a decrease in the number of invadopodia and some mispolarized F-actin, while loss of GDI-1 results in mistargeting of the invadopodial membrane to apical and lateral plasma membranes and also a reduction in the number of invadopodia. Absence of the vulval cells leads to severe disruptions in invadopodia formation, invadopodial membrane trafficking, and F-actin formation and polarization.</p

GDI-1 and the vulval precursor cells regulate invadopodial membrane trafficking.

<p>(A-D) Laterally viewed time series showing dynamic trafficking of the invadopodial membrane (visualized with marker for PI(4,5)P_2, <i>cdh3>mCherry</i>::<i>PLC</i>δ^<i>PH</i>). Graphs show the average percentage of total PI(4,5)P₂ fluorescence present at or near the basal invasive cell membrane of the AC plotted over time (n = 5 animals per group). (A) In wild type animals PI(4,5)P₂ was trafficked at the ACs invasive cell membrane and remained strongly polarized over time. (B, C) In animals with reduced <i>gdi-1</i> function (B) and animals lacking vulval precursor cells (C; <i>lin-3</i> RNAi), PI(4,5)P₂ trafficking was not restricted to the basal invasive membrane and PI(4,5)P₂ was found in apical and lateral membranes. (D) Animals with reduced <i>cdc-42</i> function (RNAi) trafficked PI(4,5)P₂ normally. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test. Scale bars = 5 μm.</p