Summary Autophagy is a lysosomal degradation pathway that is important in cellular homeostasis. Prior work showed a key role for the autophagy related 5 (Atg5) in resistance to Toxoplasma gondii. Here we show that the cassette of autophagy proteins involved in the conjugation of microtubule associated protein 1 light chain 3 (LC3) to phosphatidylethanolamine, including Atg7, Atg3, and the Atg12-Atg5-Atg16L1 complex play crucial roles in the control of T. gondii in vitro and in vivo. In contrast, pharmacologic modulation of the degradative autophagy pathway or genetic deletion of other essential autophagy genes had no substantial effects. Rather the conjugation system was required for targeting of LC3 and interferon-γ effectors onto the vacuolar membrane of T. gondii and its consequent disruption. These data suggest that the ubiquitin-like conjugation systems that reorganize intracellular membranes during canonical autophagy are necessary for proper targeting of immune effectors to the intracellular vacuole membranes utilized by pathogens.

Mitochondria play a central role in the regulation of programmed cell death signaling. Here, we report the finding of a mitochondrial matrix-targeted protein phosphatase 2C family member (PP2Cm) that regulates mitochondrial membrane permeability transition pore (MPTP) opening and is essential for cell survival, embryonic development, and cardiac function. PP2Cm is highly conserved among vertebrates, with the highest expression levels detected in the heart and brain. Small hairpin RNA (shRNA)-mediated knockdown of PP2Cm resulted in cell death associated with loss of mitochondrial membrane potential in cultured cardiac mycoytes and an induction of hepatocyte apoptosis in vivo. PP2Cm-deficient mitochondria showed elevated susceptibility to calcium-induced MPTP opening, whereas mitochondrial oxidative phosphorylation activities were not affected. Finally, inactivation of PP2Cm in developing zebrafish embryos caused abnormal cardiac and neural development as well as heart failure associated with induced apoptosis. These data suggest that PP2Cm is a novel mitochondrial protein phosphatase that has a critical function in cell death and survival, and may play a role in regulating the MPTP opening.[Keywords: Mitochondrial permeability transition pore; protein phosphatase; cell death; heart failure; developmental defects; zebrafish] Supplemental material is available at http://www.genesdev.org.

Flk1 is the major receptor for VEGF on endothelial cells. During embryogenesis, flk1 is required for both vasculogenesis and angiogenesis and abnormally elevated flk1 expression is often associated with pathological conditions in adults. While the biological function of flk1 has been studied extensively, very little is known about how the flk1 gene is regulated at the transcriptional level. Our transgenic study led to the identification of a flk1 endothelial enhancer positioned approximately 5 kb upstream of the flk1 translation initiation site. Binding sites for FoxH1, scl, ets and gata factors are found in the zebrafish flk1 endothelial enhancer, as well as in upstream sequences of mouse flk1 and human kdr genes, suggesting that the regulatory machinery for flk1/kdr is conserved from fish to mammals. The roles of scl, ets and gata factors in hemangioblasts have been well defined, but the significance of FoxH1 in vessel formation has not been explored previously. Here we show that FoxH1 binds to the flk1 endothelial enhancer in vitro and functions as a repressor for flk1 transcription in cultured cells. Consistent with these findings, the expression level of flk1 is elevated in embryos lacking both maternal and zygotic FoxH1. We further show that overexpression of FoxH1 has a negative effect on vascular formation that can be counteracted by the down-regulation of smad2 activity in zebrafish embryos. Taken together, our data provide the first evidence that flk1 is a direct target of FoxH1 and that FoxH1 is involved in vessel formation in zebrafish.

SUMMARY All viruses with positive-sense RNA genomes replicate on membranous structures in the cytoplasm, called replication complexes (RCs). RCs provide an advantageous microenvironment for viral replication, but it is unknown how the host immune system counteracts these structures. Here we show that interferon-gamma (IFNG) disrupts the RC of murine norovirus (MNV) via evolutionarily conserved autophagy proteins and the induction of IFN-inducible GTPases, which are known to destroy the membrane of vacuoles containing bacteria, protists, or fungi. The MNV RC was marked by the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system of autophagy and then targeted by immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) upon their induction by IFNG. Further, the LC3 conjugation system and the IFN-inducible GTPases were necessary to inhibit MNV replication in mice and human cells. These data suggest that viral RCs can be marked and antagonized by a universal immune defense mechanism targeting diverse pathogens replicating in cytosolic membrane structures.

Cardiac fibrillation, a form of cardiac arrhythmia, is the most common cause of embolic stroke and death associated with heart failure. The molecular mechanisms underlying cardiac fibrillation are largely unknown. Here we report a zebrafish model for cardiac fibrillation. The hearts of zebrafish tremblor (tre) mutants exhibit chaotic movements and fail to develop synchronized contractions. Calcium imaging showed that normal calcium transients are absent in tre cardiomyocytes, and molecular cloning of the tre mutation revealed that the tre locus encodes the zebrafish cardiac-specific sodium-calcium exchanger (NCX) 1, NCX1h. Forced expression of NCX1h or other calcium-handling molecules restored synchronized heartbeats in tre mutant embryos in a dosage-dependent manner, demonstrating the critical role of calcium homeostasis in maintaining embryonic cardiac function. By creating mosaic zebrafish embryos, we showed that sporadic NCX1h-null cells were not sufficient to disrupt normal cardiac function, but clustered wild-type cardiomyocytes contract in unison in tre mutant hearts. These data signify the essential role of calcium homeostasis and NCX1h in establishing rhythmic contraction in the embryonic zebrafish heart. calcium homeostasis ͉ cardiac arrhythmia ͉ heart T he heart is a muscular pump that drives circulation throughout the body. It is of the utmost importance to establish rhythmic and synchronized cardiac contraction early in development to ensure proper growth and survival of the embryo.Calcium plays an essential role in regulating cardiac cycles. As a wave of depolarization passes through the heart, a small amount of calcium is permitted to enter the cardiomyocytes through voltage-dependent L-type calcium channels. This small calcium influx then triggers the release of a larger amount of calcium from the sarcoplasmic reticulum via ryanodine receptors, resulting in an abrupt increase in cytosolic calcium levels and cardiac contraction. Relaxation is accomplished by resequestering of calcium to the sarcoplasmic reticulum by sarcoendoplasmic reticular Ca 2ϩ -ATPase2 (SERCA2) and extrusion of calcium from the cell by NCX1 and plasma membrane Ca 2ϩ -ATPase (PMCA). Abnormal calcium handling caused by altered expression levels or protein activities of NCX1 and SERCA2, or by mutations in ryanodine receptors, have been associated with cardiac diseases, such as heart failure and arrhythmia, and with sudden death in humans and animal models (1-6). In addition, genetic studies in the zebrafish demonstrate that L-type calcium channels and the sodium pump (a modulator of NCX activity) are indispensable for embryonic cardiac function (7,8). These findings underscore the critical roles of calcium in embryonic and adult cardiac physiology.Three NCX genes have been identified in mammals. NCX2 and NCX3 are expressed predominantly in the brain and skeletal muscle, respectively, whereas NCX1 is virtually ubiquitous (9). NCX1 is highly expressed in the heart and is considered to be the primary mechanism for calcium extrusion...