Integrin α PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans

Abstract

Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.

Figure 1. pat-2 and cdc-42 mutants are defective in apoptotic cell removal.

(A) pat-2 loss of function does not affect the number of cell death events. Embryonic cell deaths that occurred in the 200 min following the first cell death (up to about the 1.5-fold stage) were followed in wild-type (white rhombi) and pat-2(st567) (black squares) embryos. The y axis shows the total number of cell death events at the different time points shown on the x axis. The data shown are the average for two embryos for each genotype. The times corresponding to the comma and 1.5-fold stages are indicated. (B–C) Cell corpses in pat-2(st567) (B) and cdc-42 (gk388) (C) mutants persist longer than in the wild-type. The persistence of cell corpses that appeared 360–410 min after the first cleavage was recorded using four-dimensional Nomarski microscopy. The y axis shows the percentage of cell corpses that persisted for the time indicated on the x axis. Forty corpses were analyzed for each genotype. The data of wild-type (white bars), pat-2(st567) (gray bars); wild-type; Ex[P_pat-2pat-2Δcyto::gfp] (black bars) and pat-2(st567); Ex[P_pat-2pat-2Δcyto::gfp] (slashed bars) are shown in (B) and those of the wild-type (white bars) and cdc-42(gk388) (gray bars) in (C).

Figure 2. PAT-2 is strongly expressed in muscle cells and clusters around apoptotic cells.

(A–H) PAT-2::GFP, MOESIN::GFP, and GFP::CDC-42 are localized to pseudopods around apoptotic cells. GFP (A–D) and DIC (E–H) images of wild-type embryos expressing PAT-2::GFP (A–B), MOESIN::GFP (C), or GFP::CDC-42 (D) under the control of the indicated promoter. Apoptotic cells are indicated by arrows and shown enlarged in the insets. The arrowheads indicate hypodermal cells. (I–K) P_pat-2nls::gfp is not expressed in apoptotic MSpppaaa cells. GFP (I), membrane-bound mRFP (J), and DIC (K) images of an embryo co-expressing P_unc-54ced-1::mrfp and P_pat-2nls::gfp. MSpppaaa cell corpses are indicated by the arrows and shown enlarged in the insets. The arrowheads indicate the nucleus of the engulfing muscle cell. (L–M) The extracellular region of PAT-2 binds to the surface of apoptotic cells. DIC (L) and PAT-2(ex)::mCherry (M) images of a ced-1(e1735); ced-5(n1812) double mutant embryo expressing PAT-2(ex)::mCherry under the control of the heat-shock promoter. Apoptotic cells are indicated by arrows. (N–Q) GFP::CDC-42 and PAT-2::mCherry are co-localized to the pseudopods around apoptotic MSpppaaa cells. DIC (N), GFP::CDC-42 (O), PAT-2::mCherry (P), and merged (Q) images of a wild-type embryo co-expressing the transgenes P_unc-54 gfp:cdc-42 and P_pat-2pat-2::mcherry. The MSpppaaa cells are indicated by arrows and shown in the insets. All scale bars represent 5 µm.

Figure 3. PAT-2 is required for the internalization of apoptotic cells.

(A) The formation of MYRI::mRFP circles around cell corpses was followed and time-lapse MYRI::mRFP images of wild-type and pat-2(st567) embryos expressing P_unc-54myri::mrfp were shown. The time point immediately prior to the appearance of trace amounts of MYRI::mRFP adjacent to cell corpses was set as 0 min. Apoptotic cells are indicated by arrows and shown enlarged in the insets. (B) The DIC and MYRI::mRFP images of the apoptotic MSpppaaa cells in the wild-type and pat-2(st567) embryos expressing P_unc-54myri::mrfp. The MYRI::mRFP circle around the apoptotic MSpppaaa cell was observed in the wild-type but not pat-2(st567) embryos. MSpppaaa cell corpses are indicated by arrows and shown in enlarged insets. Both scale bars represent 5 µm.