Apoptotic timing in <i>unc-38</i> and duration of cell corpse clearance in <i>unc-38</i> or <i>unc-29</i> mutants.

<p>(A) The time of onset of the first 13 apoptotic cell deaths of the AB lineage were followed by 4D microscopy. Data shown are average ± standard deviation. (B) Duration of cell corpses clearance was determined. Phenotype analysis was performed in three different animals of the indicated genotypes (n = 3 x 13 cells). Cells indicated as unengulfed floated off from the tissue into the egg-shell cavity or their lineage could no longer be followed due to the beginning of muscle contraction at the 1.5-fold stage (120 minutes post-onset of cell death). Each circle represents a single cell. Red lines mark the median. Alleles: <i>unc-38(x20)</i>, <i>unc-29(e1072)</i> and <i>ced-6(n1813)</i>. Statistical significance was assessed using the Wilcoxon signed-rank test.</p

Loss of <i>unc-38</i> reduces cell corpse numbers in all embryonic developmental stages of engulfment mutants.

<p>4D microscopy was performed on developing embryos of the indicated genotypes. The number of cell corpses was scored at discrete time points during embryonic development. Data shown are average ± standard deviation, n = 10. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.005, determined by <i>t</i>-test, between <i>ced-6</i> and <i>unc-38; ced-6</i>, no statistical significance was observed between wild type and <i>unc-38</i> mutants.</p

UNC-38 acts in germ cells (dying cells) to modulate engulfment efficiency.

<p>(A) Tissue-specific RNAi: cell corpses were scored in the germ line of animals of the indicated genotypes 12 hours post L4/adult molt. The positive controls <i>ced-3</i> and <i>gla-1</i> (reduced or increased corpse number) are known to act in dying germ cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149274#pone.0149274.ref004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149274#pone.0149274.ref013" target="_blank">13</a>]. AHR–Ahringer RNAi library clone; ORF–Orfeome RNAi library clone. Error bars show standard deviation, * p<0.005, n = 20. (B) Left panel—DIC picture of an embryo at the “bean” stage; Right panel–AnxV::GFP reporter expression. White arrowheads show cell corpses positive for AnxV::GFP; yellow arrowhead shows a cell corpse negative for AnxV::GFP; scale bar: 10 μm. (C) Quantification of the fraction of germline corpses visible by DIC that are also positive for the AnxV::GFP reporter. Data shown are average ± standard deviation of 3 experiments (n≥10 worms, ≥200 corpses per experiment). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.005, determined by <i>t</i>-test.</p

Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in <i>Caenorhabditis elegans</i>

<div><p>The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in <i>Caenorhabditis elegans</i> have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the “eat-me” signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.</p></div

Loss of subunits of the levamisole receptor but not of other nAChRs reduces cell corpse numbers.

<p>Cell corpses were scored in the head region of freshly hatched L1 larvae of the indicated genotypes. <i>ced-x</i> stands for the genotypes on the x-axis. Data shown are average ± standard deviation, n = 20.*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.005, determined by <i>t</i>-test.</p

Loss of <i>unc-38</i> reduces cell corpse numbers in soma and germ lines of engulfment mutants.

<p>(A) Cell corpses were scored in the head region of freshly hatched L1 larvae of the indicated genotype. (B) Germ cell corpses were scored in adult animals of the indicated genotypes 12 hours post-L4/adult molt. <i>ced-x</i> stands for the genotypes on the x-axis. Data shown are average ± standard deviation, n = 20. All tests had a significance of <i>p</i><0.005 with the exception of the case noted, *<i>p</i><0.05, determined by <i>t</i>-test.</p

CCL2 alters the structure of the intestinal brush border and terminal web in living animals.

<p>(A-E) <i>C</i>. <i>elegans</i> L4 larvae of the indicated genotype were fed for 24 h on control (empty vector) or CCL2-expressing <i>E</i>. <i>coli</i>. Various fluorescent transgenic reporters were used to detect changes in intestinal cell architecture by confocal microscopy. (A) PGP-1::GFP is expressed at the intestinal apical plasma membrane. (B) RAB-8::GFP labels the intestinal cytoplasm. (C) ERM-1::GFP links the intestinal apical plasma membrane to the actin filaments within the microvilli. (D) ACT-5::GFP labels actin filaments within the microvilli and the terminal web underlying the intestinal apical plasma membrane. (E) IFB-2::CFP labels intermediate filaments in the terminal web. Scale bar: 10 μm; inset: 2x magnification of the lumenal section of the intestine.</p

CCL2 leads to microvilli loss, intestinal plasma membrane invaginations, and terminal web gaps.

<p>Wild-type <i>C</i>. <i>elegans</i> L4 larvae were fed on control (empty vector) (A,C,E) or CCL2-expressing <i>E</i>. <i>coli</i> (B,D,F-H) for the indicated hours and then observed under a transmission (A-D, G,H) or focused ion beam scanning electron microscope (E,F). (A,C) The intestinal apical surface of control animals showed the typical dense brush border made of microvilli (asterisk) and glycocalyx (arrow), and an intact terminal web (arrowhead) underlying the intestinal apical plasma membrane. (B,D) Exposure to CCL2 prompts missing microvilli (asterisks); invaginations of intestinal apical plasma membrane filled with lumenal content (arrows); and disruption of the terminal web (arrowheads). (E,F) 3D reconstruction of selected microvilli in control (E) and CCL2-treated (F) animals showing the disorganized brush border following CCL2 exposure; fused microvilli (yellow; filled arrow); cell border (open arrow); adherens junction (arrowhead). (G,H) Morphological changes are already visible at the ultrastructural level after 3 h (G) and 6 h (H) of exposure to CCL2; invaginations of plasma membrane (filled arrows); terminal web (filled arrowheads); dark cap at the tip of microvilli (open arrows); intact actin filament bundles (open arrowheads); depolymerized actin filament bundles (asterisks). Scale bar: 500 nm.</p

Mutations in <i>zfp-1</i> or <i>lin-35</i> enhance axon guidance defects in an <i>oxIs12</i>-dependent manner.

<p>A: The <i>zfp-1</i>(op481) point mutation, the <i>zfp-1</i>(ok554) deletion and <i>lin-35</i>(n745) lead to a very similar enhancement of defects when combined with <i>sdn-1</i>(zh20) or <i>hse-5</i>(tm472). However <i>zfp-1</i>(ok554) and <i>lin-35</i>(n745) are not enhancing each other. The defects of <i>zfp-1</i>(ok554)<i>; sdn-1</i>(zh20) and <i>lin-35</i>(n745)<i>; sdn-1</i>(zh20) disappear if <i>oxIs12</i> is replaced by either <i>oxIs268</i> or <i>juIs76</i>. B: Attempts to reconstitute the defects. (i) <i>pkIs296</i> enlarges the X-chromosome of <i>oxIs268; zfp-1</i>(ok554)<i>; sdn-1</i>(zh20) animals leading to a slight increase in defects, however not to the level of <i>zfp-1</i>(ok554)<i>; sdn-1</i>(zh20) <i>oxIs12</i> animals (ii). The <i>dpy-21</i>(e428) mutation behaves the same as <i>zfp-1</i> or <i>lin-35</i> mutations. The <i>dpy-21</i>(e428)<i>; sdn-1</i>(zh20) double mutant has severe D-type axon guidance defects if <i>oxIs12</i> is present but not if <i>oxIs268</i> is used to stain the D-type motor neurons. The phenotypes of <i>zfp-1</i>(ok554)<i>; sdn-1</i>(zh20) and <i>lin-35</i>(n745)<i>; sdn-1</i>(zh20) with either <i>oxIs12</i> or <i>juIs76</i> are shown as comparison. Grey bars represent the number of commissural axons growing away from the VNC; white bars indicate the number of commissural axons reaching the DNC. Dashed lines indicate limits according to Figure 1A. Numbers are from 50 L1 animals +/- SEM. Statistical test results are indicated as follows: ns = not significant, * = p<0.05, ** = p<0.005, *** = p<0.0005. Superscripts (also shown in the bars of the corresponding strains) indicate to which strain the comparison was made: 1: <i>hse-5</i>(tm472), 2: <i>sdn-1</i>(zh20), 3: <i>zfp-1</i>(ok554), 4: <i>zfp-1</i>(ok554)<i>; sdn-1</i>(zh20)<i>; oxIs268</i>, 5: <i>dpy-21</i>(e428)<i>; sdn-1</i>(zh20) <i>oxIs12</i>, 6: <i>zfp-1</i>(ok554)<i>; sdn-1</i>(zh20) <i>oxIs12</i>, 7: <i>lin-35</i>(n745)<i>; sdn-1</i>(zh20) <i>oxIs12</i>. The table summarizes the different transgenes used, indicates their composition and the chromosome in which they are integrated.</p

CCL2 does not induce pore formation in the intestinal apical plasma membrane.

<p>(A-C) <i>C</i>. <i>elegans</i> L4 larvae expressing PGP-1::GFP were fed on control (empty vector pQE30) (A), Cry21A- (B) or CCL2- (C) expressing <i>E</i>. <i>coli</i> for 24 h, transferred into wells containing propidium iodide (PI; red) for 2 h and observed using confocal microscopy. Cry21A is a pore-forming toxin of <i>B</i>. <i>thuringiensis</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129381#pone.0129381.ref031" target="_blank">31</a>]. PI entered the intestinal cytoplasm of Cry21A-fed (B), but not of control- (A) or CCL2- (C) fed animals. Scale bar: 10 μm.</p