Calcium ions trigger the exposure of phosphatidylserine on the surface of necrotic cells

Intracellular Ca 2+ level is under strict regulation through calcium channels and intracellular Ca 2+ storage pools such as the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which result in the mis-regulation of Ca 2+ influx, cause the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans , six mechanosensory (touch) neurons are induced to undergo excitotoxic necrosis by dominant mutations in the DEG/ENaC sodium channel subunits. These necrotic neurons are subsequently engulfed and degraded by neighboring hypodermal cells. We previously reported that the necrotic touch neurons actively expose phosphatidylserine (PS), an “eat-me” signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca 2+ level occurs prior to the exposure of PS on the surfaces of necrotic neurons. We further found that inhibiting the release of Ca 2+ from the ER, either pharmacologically or genetically through mutations in the gene encoding calreticulin, the ER Ca 2+ chaperon, impairs PS exposure on necrotic neurons. On the contrary, inhibiting the re-uptake of cytoplasmic Ca 2+ into the ER induces ectopic necrosis and PS exposure. These findings indicate that high levels of cytoplasmic Ca 2+ is necessary and sufficient for PS exposure. Remarkably, we found that PS exposure occurred independently of other necrosis events. On the other hand, apoptotic cells, unlike necrotic cells, do not depend on the ER Ca 2+ pool for PS exposure. Our findings reveal a necrotic neuron-specific, “two-step Ca 2+ -influx” pathway that promotes PS exposure on cell surfaces. This pathway is initiated by the modest influx of Ca 2+ from the extracellular space and further boosted by the release of Ca 2+ from the ER into the cytoplasm. Author Summary Necrosis is a type of cell death that exhibits distinct morphological features such as cell swelling. Many environmental insults induce cells to undergo necrosis. Necrotic cells expose phosphatidylserine (PS) – a type of phospholipid – on their outer surfaces. Receptor molecules on phagocytes detect phosphatidylserine on necrotic cells and subsequently initiate the engulfment process. As necrosis is associated with stroke, cancer, neurodegenerative diseases, heart diseases, and inflammatory diseases, studying necrotic cell clearance has important medical relevance. In the model organism the nematode C. elegans , by utilizing dominant mutations in ion channels that induce neurons to undergo necrosis, we previously identified membrane proteins that promote the exposure of phosphatidylserine on necrotic cell surfaces. Here, using the same experimental system, we further discover that the necrosis insults trigger an increase of the cytoplasmic Ca 2+ level, which in turn promotes PS externalization in necrotic cells. Furthermore, we find that the Ca 2+ pool in the endoplasmic reticulum is necessary for the rapid increase of cytoplasmic Ca 2+ that helps initiate necrosis. This Ca 2+ -regulated event is not observed when cells undergoing apoptosis (a form of cell suicide) expose PS. Our findings reveal a novel upstream regulatory mechanism that promotes necrotic cell clearance in animals.