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”.