Calcium Ions Trigger The Exposure Of Phosphatidylserine On The Surface Of Necrotic Cells

Author summaryNecrosis is a type of cell death that exhibits distinct morphological features such as cell and organelle swelling. Necrotic cells expose phosphatidylserine (PS)-a type of phospholipid-on their outer surfaces. Receptor molecules on phagocytes detect PS on necrotic cells and subsequently initiate the engulfment process. As necrosis is associated with stroke, cancer, neurodegenerative diseases, and heart diseases, studying necrotic cell clearance has important medical relevance. In the model organism the nematode C. elegans, we previously identified membrane proteins that promote the exposure of PS on necrotic cell surfaces by studying neurons that are induced to undergo necrosis by dominant mutations in ion channels. Here, in C. elegans, we have discovered that the necrotic insults trigger an increase of the cytoplasmic calcium ion (Ca2+), which in turn promotes PS externalization on necrotic cell surfaces. Furthermore, we have identified two different mechanisms that increase cytoplasmic Ca2+ levels, one dependent on the Ca2+ contribution from the endoplasmic reticulum (ER), the other independent of the ER. The Ca2+ signal targets ANOH-1, a worm homolog of mammalian proteins capable of externalizing PS, for promoting PS exposure on necrotic cells. Our findings reveal novel upstream regulatory mechanisms that promote necrotic cell clearance in animals.Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that 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 necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.