The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.

No abstract

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.

The yeast high osmolarity glycerol (HOG) pathway signals via the Pbs2 MEK and the Hog1 MAPK, whose activity requires phosphorylation of Thr and Tyr in the activation loop. The Ptc1-type 2C Ser/Thr phosphatase (PP2C) inactivates Hog1 by dephosphorylating phospho-Thr, while the Ptp2 and Ptp3 protein tyrosine phosphatases dephosphorylate phospho-Tyr. In this work, we show that the SH3 domain-containing protein Nbp2 negatively regulates Hog1 by recruiting Ptc1 to the Pbs2-Hog1 complex. Consistent with this role, NBP2 acted as a negative regulator similar to PTC1 in phenotypic assays. Biochemical analysis showed that Nbp2, like Ptc1, was required to inactivate Hog1 during adaptation. As predicted for an adapter, deletion of NBP2 disrupted Ptc1-Pbs2 complex formation. Furthermore, Nbp2 contained separate binding sites for Ptc1 and Pbs2: the novel N-terminal domain bound Ptc1, while the SH3 domain bound Pbs2. In addition, the Pbs2 scaffold bound the Nbp2 SH3 via a Pro-rich motif distinct from that which binds the SH3 domain of the positive regulator Sho1. Thus, Nbp2 recruits Ptc1 to Pbs2, a scaffold for both negative and positive regulators.

During apoptosis, phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and serves as an “eat-me” signal to trigger phagocytosis. It is poorly understood how PS exposure is activated in apoptotic cells. Here we report that CED-8, a C. elegans protein implicated in controlling the kinetics of apoptosis and a homolog of the XK family proteins, is a substrate of the CED-3 caspase. Cleavage of CED-8 by CED-3 activates its proapoptotic function and generates a carboxyl terminal cleavage product, acCED-8, that promotes PS externalization in apoptotic cells and can induce ectopic PS exposure in living cells. Consistent with its role in promoting PS externalization in apoptotic cells, ced-8 is important for cell corpse engulfment in C. elegans. Our finding identifies a crucial link between caspase activation and PS externalization, which triggers phagocytosis of apoptotic cells.