Necrotic cells actively expose PS on their surfaces.

<p>(A) The position and name of the six touch neurons in <i>C</i>. <i>elegans</i>. (B) Necrotic touch neurons are engulfed by hypodermal cells. DIC (a) and GFP (b) images of 2 necrotic touch neurons ALML and ALMR (marked by white lines) engulfed inside larval hypodermis labeled with CED-1C::GFP expressed and distributed in the cytoplasm in hypodermal cells under the control of P_<i>ced-1</i> in the <i>mec-4(e1611dm)</i> genetic background. Scale bars are 10μm. (C) PS is exposed on the surface of necrotic cells. (a) DIC and (b) corresponding epifluorescence images detecting MFG-E8::GFP on the surface of a necrotic touch neuron (white arrowheads) in the tail of a L1 larva. P_<i>dyn-1</i><i>mfg-e8</i>::<i>gfp</i> is expressed in the <i>mec-4(e1611dm)</i> background. (c) The number of necrotic touch neurons labeled with MFG-E8::GFP on their surfaces and the total number of necrotic touch neurons analyzed in 30 animals. Dorsal is to the top. Scale bars are 10μm. (D) PS is not detected on the outer surface of live touch neurons. (a) DIC, (b) mCherry/DIC merged, and (c) GFP images of a live touch neuron (arrows) in the tail region of a wild-type L3 larva co-expressing P_<i>mec-7</i> mCherry (touch neuron marker) and P_<i>dyn-1</i> MFG-E8::GFP. (d) MFG-E8::GFP is not observed in any of the 20 live touch neurons characterized. Dorsal is to the top. Scale bars are 5μm. (E) Time-lapse images monitoring the appearance of PS on the outer surface of necrotic PLML and PLMR during embryogenesis. The touch neuron-specific reporter construct is P_<i>mec-7</i><i>gfp</i>. The PS reporter construct is P_<i>dyn-1</i><i>mfg-e8</i>::<i>mCherry</i>. Time points are marked as min post 1^st embryonic division. Recording started at the 2-fold stage (460 min) (a) and ended when the embryo hatched (i), in 5-min interval. White arrowheads in (j) indicate living PLML and PLMR. White arrowheads in all other panels indicate the same cells, which are PLMR and/or PLML, that undergo necrosis. The white open arrowhead in (m, o, q) mark intestinal lumen, within which the secreted MGF-E8::mCherry is visible. The scale bar is 5μm. (F) Summary of the quantitative analysis of the time-lapse recording monitoring the dynamics of three features of necrotic PLML and PLMR: (1) the specification of touch neurons (the expression of P_<i>mec-7</i> GFP), (2) the distinct swelling morphology of necrotic cells observed under DIC microscope, and (3) the appearance of PS on the surface of necrotic cells (MFG-E8::mCherry enrichment on necrotic cells). The time points of event initiation are represented as “min-post the 1st cleavage”.</p

Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms

<div><p>Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode <i>Caenorhabditis elegans</i>, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS <i>in vitro</i>. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the <i>C</i>. <i>elegans</i> homolog of the mammalian Ca²⁺-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the <i>C</i>. <i>elegans</i> homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me” signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.</p></div

CED-7 promotes necrotic cell removal by acting in two different cell types.

<p>(A) CED-7 domain diagram. TM: transmembrane region (each has 6 transmembrane domains). HH: highly hydrophobic motif. NBD: nucleotide-binding domain. (B) CED-7::GFP expressed in necrotic cells is localized to cell surface. DIC (a) and corresponding epifluorescence (b) images of the tail of a newly hatched <i>mec-4(e1611dm)</i> L1 larva (hatched within 1hr) expressing P_<i>mec-7</i><i>ced-7</i>::<i>gfp</i>. Arrowheads mark two CED-7::GFP-positive necrotic corpses. Dorsal is to the top. Scale bars are 5μm. (C) Expression of <i>ced-7</i> cDNA either in touch neurons or engulfing cells each partially rescues the necrotic cell removal defects. <i>ced-7(n1996)</i>; <i>mec-4(e1611dm)</i> larvae expressing either P_<i>ced-1</i><i>ced-7</i> or P_<i>mec-7</i><i>ced-7</i> were scored for the number of necrotic corpses in indicated larvae stages. Each data point represents the mean of 3 groups of 20 animals per group. Error bars represent sd. (D) CED-7 is required for PS exposure. (a) DIC and (b) the corresponding epifluorescence images of a necrotic cell (arrowheads) lacking MFG-E8::GFP signal on its surface in the tail of a L1 larva hatched within 1hr. Dorsal is to the top. Scale bar: 5μm. (c) Bar graph showing the percentage of necrotic cells on which the PS is detected by MFG-E8::GFP in newly hatched L1 larvae in different engulfment mutant backgrounds. Alleles used here: <i>mec-4(e1611dm)</i>, <i>ced-1(e1735)</i>, <i>ced-6(n2095)</i>, <i>ced-7(n1996)</i>, and <i>ced-10(n1993)</i>. Each data point represents the mean percentage calculated from 6 groups of necrotic cells (10 in each group). Error bars represent sd. ns: no significant difference (p>0.05); ***, p<0.001, Student <i>t</i>-test. (E) The function of CED-7 in necrotic cells specifically rescues the PS-exposure defect of <i>ced-7</i> mutants. The percent of necrotic cell corpses labeled by MFG-E8::GFP were scored in <i>ced-7; mec-4</i> mutants expressing each transgene of P_<i>mec-7</i><i>ced-7</i> or P_<i>ced-1</i><i>ced-7</i> individually.</p

<i>anoh-1</i> is required for the efficient removal of necrotic touch cells but not apoptotic cells.

<p>(A) Gene structure of two isoforms of <i>anoh-1</i>. The blue open boxes indicate the region deleted in the <i>anoh-1(tm4762)</i> allele. (B) Domain structure of ANOH-1b protein. Dark grey bars indicate 8 predicted transmembrane domains. Red color labels amino acids 1–18, which exists in ANOH-1b but missing from ANOH-1a. (C) The <i>anoh-1(tm4762)</i> mutant embryos are normal in the removal of somatic apoptotic cells. (D) The <i>anoh-1(tm4762)</i> mutant adults are normal in the removal of apoptotic germ cells. (E) The engulfment and degradation processes of embryonic apoptotic cells C1, C2, and C3 are normal in <i>anoh-1(tm4762)</i> mutant embryos, measured by time-lapse microscopy. (F) The <i>anoh-1(tm4762)</i> mutation perturbs the removal of necrotic cells, and the <i>anoh-1b</i> but not the <i>anoh-1a</i> form rescues the necrotic cell-removal defect when expressed in touch cells under the control of P_<i>mec-7</i>. For each sample, 6 groups of 10 worms at indicated stages were scored and displayed as mean. Error bars indicate standard deviations. *, 0.01t-test). ***, p<0.001 (student <i>t</i>-test). ns, no significant difference, p>0.05 (student <i>t</i>-test). (G) The expression of <i>anoh-1b</i> in the necrotic cells, not engulfing cells, rescues <i>anoh-1(tm4762)</i> mutant phenotype. In <i>anoh-1(tm4762); mec-4(e1611dm)</i> mutant animals expressing indicated transgenes, the number of necrotic cells in the tail of each of the 6 groups of 10 newly hatched L1 larvae were scored. Data are presented as mean ± sd. (H) The GFP fusion forms of ANOH-1b but not ANOH-1a are localized to the plasma membrane. GFP (a, c, e, g) and DIC (b, d, f, h) images of the tails of <i>mec-4(e1611dm)</i> L1-stage larvae. Arrowheads in (a, b, e, f) label necrotic cells on which ANOH-1b is observed on the cell surface. Arrows in (c, d, g, h) label necrotic cells in which ANOH-1a is observed on the nuclear surface. All GFP reporters are expressed under the P_<i>mec-7</i> promoter control. (I) The <i>anoh-1b</i> promoter is expressed in touch neurons. Shown here are epifluorescence (b-c) and the corresponding DIC (a) images of the tail region of a wild-type L2 larva co-expressing P_<i>mec-7</i> mCherry (b), which is a touch neuron-specific reporter, and P_{<i>anoh-1b</i>} NLS::GFP (c). White arrowheads mark a touch neuron. Dorsal is up. Scale bars are 5μm.</p

Effects of NEMO amount on NF-κB activation.

<p>(A) Cell extracts from equal numbers of 1.3E2 clones expressing different amounts of NEMO protein were generated using either 2x SDS Laemmli sample buffer or Totex buffer. The cell extracts were separated on SDS-PAGE gel and immunoblotted with NEMO, IKKα/β, and actin antibodies. (B, C) The 1.3E2 clones as in A were left untreated or treated with etoposide (VP16, 5 μM, 1 hr) in B or LPS (10 μg/ml, 30 min) in C. P represents pool of the 1.3E2 clones before isolation of individual clones. Protein extracts were examined by EMSA and Western blotting using NEMO, IKKα/β, and tubulin antibodies.</p

Inactivating both <i>ced-7</i> and <i>anoh-1</i> results in enhanced phenotypes.

<p>Alleles used: <i>anoh-1(tm4762)</i>, <i>ced-7(n1996)</i>, <i>mec-4(e1611dm)</i>. (A) Double mutants display persistent necrotic cells more frequently than each of the single mutants. Mutants of indicated genotypes were scored at four larval stages for the persistence of necrotic corpses in their tails. Sixty animals of each genotype were scored in six groups of 10 worms. Error bars indicate standard deviations of each data point. “***”, “*”, and “ns”, p<0.001, 0.010.05, respectively, student <i>t-</i>test. (B) ANOH-1 and CED-7 are both needed for presenting PS to the necrotic cell surfaces. DIC (a-d) and corresponding epifluorescence (e-h) images of the tails of newly (within 1hr) hatched L1 larvae expressing P_<i>dyn-1</i><i>mfg-E8</i>::<i>gfp</i> showing different GFP signal on necrotic cell surfaces. White arrowheads label necrotic corpses. Dorsal is up. Scale bars are 10μm. (C) Relative signal intensity of MFG-E8::GFP was calculated as the ratio between GFP signal intensity on surfaces of necrotic cells in the tail and that in a nearby region inside the same worm. Signal intensity measurement was performed using L1 larvae aged within 1-hr of hatching. Each grey circle represents one necrotic cell analyzed. “n” indicates the number of necrotic cells analyzed for each genotype. Red lines represent the median value of each group of sample. “***” and “**”, p<0.001 and 0.001t-test.</p

The extracellular domain of CED-1 directly associates with PS.

<p>The entire extracellular region of CED-1 fused to GST (CED-1-GST) was tested for the binding to phospholipids. Data were representative of three independent experiments that yielded similar results. (A) Detecting the purified GST or CED-1-GST using SDS-PAGE. Molecular weight markers are marked on the side. Arrowheads mark the purified proteins. (B) The binding affinities of identical amount of CED-1-GST and GST to PS and PC were measured in ELISA-like reactions. Data shown here are results after subtracting the OD value corresponding to GST from that corresponds to CED-1-GST at each data point. (C) The binding affinity of CED-1-GST (6.3 p mole in 0.1ml volume) to PS was analyzed in ELISA-like reactions in the presence of PC-only liposomes (0.5 mM) or PS-containing liposomes (0.5 mM) as competitors. (D) The binding of GST and CED-1-GST (both in 6.3 pmol in 0.1ml volume) to various phospholipids, including PC, PS, phosphatidylethanolamine (PE) and phosphatidylinositol (PI), was examined in ELISA-like reactions. *, 0.002<<i>p</i><0.005; **, 0.001<<i>p</i><0.002; ***, <i>p</i><0.001; ns, no significant difference; Student <i>t-</i>test.</p

Determination of the average number of NEMO molecules in multiple human cell lines.

<p>Representative NEMO Western blots of protein extracts from the indicated number of Jurkat, HEK293, HeLa, and RPE cells compared to protein extracts from the indicated number of C5 cells are shown. * represents non-specific band. Each blot was performed three biological replicates except for HEK293 which was two biological replicates.</p

Effects of NEMO amount on NF-κB activation in response to etoposide.

<p>(A) Time course analysis. The indicated 1.3E2 clones were left untreated or treated with 5 μM VP16 for indicated times and analyzed by EMSA. Three independent experiments were performed as above on different days. The NF-kB bands were quantified and fold inductions of NF-κB DNA binding activity were graphed below. Error bars represent S.E.M. (B) Dose response analysis. The indicated 1.3E2 clones were left untreated or treated with the indicated concentration of etoposide (VP16) for 1 hr. The quantification and graph were done as in A.</p

Purification of full length recombinant human NEMO protein.

<p>(A) SDS–PAGE gel stained with Coomassie Brilliant Blue R-250 for the protein purification profile. (B) 10 μl of purified recombinant NEMO proteins were loaded with a serial dilution of BSA at known concentrations (C) GST or GST-NEMO protein was incubated with <i>in vitro</i> translated mock or IKKβ for GST pull-down assay (lane 1–4), or GST or GST-cleaved NEMO protein was incubated with <i>in vitro</i> translated mock or flag-IKKβ for co-immunoprecipitation with anti-NEMO antibody (lane 7–10). Flag-IKKβ in the complexes or inputs (lanes 5 and 6) were separated on SDS-PAGE gel and immunoblotted with flag antibodies.</p