Gunther-Cummins, L. | bioRxiv Science

bioRxiv2022

mTORC2 couples fasting to mitochondrial fission

Fasting triggers diverse cellular and metabolic adaptations to facilitate organismal survival1,2. During nutrient deprivation, increases in circulating fatty acids support mitochondrial respiration2. The mechanisms driving mitochondrial adaptations and respiratory sufficiency during nutrient deprivation remain incompletely understood. Here we show that extended periods of fasting, or lipid availability stimulates mTORC2 activity. Activation of mTORC2 and phosphorylation of its target NDRG13 at S336 sustains mitochondrial fission and respiratory sufficiency. Timelapse imaging reveals that wildtype NDRG1, but not phosphorylation-deficient NDRG1S336A mutant, engages with mitochondria to facilitate its scission. Using proteomics, and an siRNA screen, we show that mTORC2-phosphorylated NDRG1 cooperates with the small GTPase Cdc424 and Cdc42-specific effectors and regulators to orchestrate fission. Accordingly, RictorKO, NDRG1S336A mutants, and Cdc42-deficient cells each display mitochondrial phenotypes reminiscent of fission failure. During nutrient surplus, mTOR complexes perform anabolic functions5; however, paradoxical reactivation of mTORC2 during fasting plays an unexpected role in driving mitochondrial fission and respiration.

cell biology↗

bioRxiv2021

Dense granule protein, GRA64 interacts with host cell ESCRT proteins during Toxoplasma gondii infection

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In attempts to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a trans-membrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrate that GRA64 appears to interact with certain components of the host Endosomal Sorting Complexes Required for Transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence in mice nor encystation as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of {Delta}gra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection. IMPORTANCEToxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immune compromised individuals. Part of this parasites success lies in its ability to infect diverse organisms and host cells, as well as to persist as a latent infection within parasite constructed structures called tissue cysts. In this study, we characterized a protein secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuole, this protein is exposed to the host cell and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.

microbiology↗