Volume 22, Issue 14, Pages 1276-1284 (July 2012) C. elegans Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on Phagocytes for Cell Corpse Engulfment  Yan Zhang, Haibin Wang, Eriko Kage-Nakadai, Shohei Mitani, Xiaochen Wang  Current Biology  Volume 22, Issue 14, Pages 1276-1284 (July 2012) DOI: 10.1016/j.cub.2012.06.004 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 nrf-5 Is Required for Cell Corpse Clearance (A) Time-course analysis of cell corpses during embryonic development was performed in wild-type (N2, open bar), nrf-5(qx16) (black bar), or nrf-5(sa513) (gray bar). At least 15 embryos were scored at every stage. Data are shown as mean ± SEM. Data derived from WT and nrf-5(qx16) or WT and nrf-5(sa513) were compared by unpaired t test. ∗∗p < 0.0001; all other points had p > 0.05. (B) Embryonic cell deaths were followed in WT (N2, n = 3) and nrf-5(qx16) (n = 3) embryos. The average number of total cell deaths (±SEM) is shown in parenthesis. (C) The duration of 33 corpses from WT (N2, open bar, n = 3) and 61 corpses from nrf-5(qx16) (black bar, n = 4) was monitored. The average duration (±SEM) is in parenthesis. (D) nrf-5(sa513) does not enhance the engulfment defect in ttr-52 and ced-7 mutants. Cell corpses were scored in the head region of 4-fold embryos. At least 15 animals were scored in each strain. Data are shown as mean ± SEM. The number of cell corpses in WT was compared with that in ttr-52(tm2078) or nrf-5(sa513). For other points, single ced mutants were compared with ced;nrf-5 double mutants, and ttr-52;nrf-5 double mutants were compared with nrf-5 single mutants. ∗∗p < 0.0001; all other points had p > 0.05. (E) Schematic diagram of the cell corpse engulfment pathway. See also Figure S1. Current Biology 2012 22, 1276-1284DOI: (10.1016/j.cub.2012.06.004) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 NRF-5 Is a Secretory Protein from Body Wall Muscle Cells and Clusters around Apoptotic Cells (A–F′) Differential interference contrast (DIC) and fluorescent images of WT (A–B′, D–E′) or ced-3(n717) embryos (C, C′, F, F′) that carry PhspNRF-5::mCHERRY (A, A′, C, C′), PhspNRF-5(21-551)::mCHERRY (B, B′), Pced-1ssGFP::mNRF-5 (D, D′, F, F′), or Pced-1GFP::mNRF-5 (E, E′). Arrows indicate apoptotic cells, and arrowheads point to accumulated fluorescent proteins in the extraembryonic cavity. Cell corpses indicated by yellow arrows are shown at ×3 magnification in the insets. (G–H″′) DIC and fluorescent images of WT animals transgenic for both Pnrf-5GFP and Pmyo-3mCHERRY. Scale bars in all panels represent 5 μm. See also Figure S2. Current Biology 2012 22, 1276-1284DOI: (10.1016/j.cub.2012.06.004) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 ced-7 Is Required for the Clustering of NRF-5 around Cell Corpses (A–J′) DIC and fluorescent images of WT (A, A′, F, F′), ced-1(e1735) (B, B′, G, G′), ced-7(n2094) (C, C′, H, H′), ttr-52(tm2078) (D, D′), nrf-5(qx16) (I, I′), or tat-1(qx30) (E, E′, J, J′) embryos expressing ssGFP::mNRF-5 (A–E′) or TTR-52::mCHERRY (F–J′). Arrows indicate apoptotic cells. Cell corpses indicated by yellow arrows are shown at ×3 magnification in the insets. Scale bars represent 5 μm. (K–N) The percentage of cell corpses labeled by ssGFP::mNRF-5 (K) or TTR-52::mCHERRY (M) was quantified at the 1.5-fold (K) or comma (M) stage in the strains indicated. The ratio of fluorescence intensity on apoptotic cells versus their living neighbors (X-fold) was determined at the 1.5-fold (L) or comma (N) stage in indicated strains expressing ssGFP::mNRF-5 (L) or TTR-52::mCHERRY (N). At least 15 embryos (K, M) or 30 cell corpses (L, N) were quantified in each strain, and data are shown as mean ± SEM. Data derived from different mutant backgrounds were compared to WT. ∗∗p < 0.0001, ∗p < 0.05; all other points had p > 0.05. Current Biology 2012 22, 1276-1284DOI: (10.1016/j.cub.2012.06.004) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Loss of nrf-5, ced-7, or ttr-52 Blocks PS Appearance on Engulfing Cells (A–H′) DIC and fluorescent images of WT (A, A′), nrf-5(qx16) (B, B′, G, G′), ced-7(n2094) (C, C′, F, F′), ced-1(e1735) (E, E′), or ttr-52(tm2078) (D, D′, H, H′) embryos at the early (A–D′) or 4-fold (E–H′) stage expressing Annexin V::GFP. Arrows indicate Annexin V::GFP-labeling of living cells that surround cell corpses (A′) or apoptotic cells (E–H′). Yellow boxed regions are shown at ×3 magnification in the insets. Scale bars represent 5 μm. (I–K) The percentage of cell corpses surrounded by Annexin V::GFP-positive cells (I) or labeled by Annexin V::GFP (J) was quantified at the comma stage (I) or the 4-fold embryonic stage (J). The ratio of fluorescence intensity on apoptotic cells versus their living neighbors (X-fold) was determined at the 4-fold embryonic stage in indicated strains expressing Annexin V::GFP (K). At least 15 animals (I, J) or 30 cell corpses (K) were scored in each strain and data are shown as mean ± SEM. Unpaired t tests were performed to compare all other data sets with that of WT (I) or ced-1 (J, K). ∗∗p < 0.0001, ∗p < 0.05; all other points had p > 0.05. See also Figures S3 and S4. Current Biology 2012 22, 1276-1284DOI: (10.1016/j.cub.2012.06.004) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Loss of nrf-5, ced-7, or ttr-52 Causes PS Accumulation on Apoptotic Cells (A–G′) DIC and fluorescent images of WT (A, A′), tat-1(qx30) (B, B′, F, F′), ced-1(e1735);tat-1(qx30) (C, C′), ced-7(n2094)tat-1(qx30) (D, D′), tat-1(qx30);nrf-5(sa513) (F, F′), or tat-1(qx30)ttr-52(tm2078) (G, G′) embryos expressing TTR-52::mCHERRY (A–E′) or GFP::Lact-C2 (F–G′). Arrows indicate apoptotic cells. Scale bars represent 5 μm. (H) The ratio of fluorescence intensity on apoptotic cells versus their living neighbors (X-fold) was determined in indicated strains expressing TTR-52::mCHERRY (left) or GFP::Lact-C2 (right). At least 30 cell corpses were quantified in each strain. Data are shown as mean ± SEM. Unpaired t tests were performed to compare all other data sets with that of tat-1(qx30). ∗∗p < 0.0001; all other points had p > 0.05. See also Figure S3. Current Biology 2012 22, 1276-1284DOI: (10.1016/j.cub.2012.06.004) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 6 NRF-5 Binds PS and Possesses Lipid Transfer Activity In Vitro (A) NRF-5 binds PS in vitro. Affinity-purified EGFP-NRF-5-Flag but not EGFP-Flag bound PS spotted on a membrane strip. (B) Transfer of [3H]cholesterol from donor proteins to acceptor liposomes (left) and from donor liposomes to acceptor proteins (right) was examined as described in Experimental Procedures. At least two independent experiments were performed. (C) Cell corpses are internalized by the PS-positive neighbor. The clearance of apoptotic cells is followed in WT (a, b) and nrf-5(qx16) embryos (c, d) carrying Pttr-52TTR-52::mCHERRY. The apoptotic cells (white arrowheads) are quickly internalized and cleared by the PS-positive neighbor (yellow arrowheads) in WT but persist much longer in nrf-5(qx16) embryos, which lack PS labeling on the neighboring cell. Scale bar represents 1 μm. See also Figure S5 and Movies S1, S2, S3, and S4. Current Biology 2012 22, 1276-1284DOI: (10.1016/j.cub.2012.06.004) Copyright © 2012 Elsevier Ltd Terms and Conditions