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

Abstract

Intracellular Ca2+ level is under strict regulation through calcium channels and intracellular Ca2+ storage pools such as the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which result in the mis-regulation of Ca2+ 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 Ca2+ level occurs prior to the exposure of PS on the surfaces of necrotic neurons. We further found that inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically through mutations in the gene encoding calreticulin, the ER Ca2+ chaperon, impairs PS exposure on necrotic neurons. On the contrary, inhibiting the re-uptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. These findings indicate that high levels of cytoplasmic Ca2+ 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 Ca2+ pool for PS exposure. Our findings reveal a necrotic neuron-specific, "two-step Ca2+-influx" pathway that promotes PS exposure on cell surfaces. This pathway is initiated by the modest influx of Ca2+ from the extracellular space and further boosted by the release of Ca2+ from the ER into the cytoplasm. Author SummaryNecrosis 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 Ca2+ level, which in turn promotes PS externalization in necrotic cells. Furthermore, we find that the Ca2+ pool in the endoplasmic reticulum is necessary for the rapid increase of cytoplasmic Ca2+ that helps initiate necrosis. This Ca2+-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.