Pressure sensing of lysosomes enables control of TFEB responses in macrophages.

Polymers are endocytosed and hydrolysed by lysosomal enzymes to generate transportable solutes. While the transport of diverse organic solutes across the plasma membrane is well studied, their necessary ongoing efflux from the endocytic fluid into the cytosol is poorly appreciated by comparison. Myeloid cells that employ specialized types of endocytosis, that is, phagocytosis and macropinocytosis, are highly dependent on such transport pathways to prevent the build-up of hydrostatic pressure that otherwise offsets lysosomal dynamics including vesiculation, tubulation and fission. Without undergoing rupture, we found that lysosomes incurring this pressure owing to defects in solute efflux, are unable to retain luminal Na+, which collapses its gradient with the cytosol. This cation 'leak' is mediated by pressure-sensitive channels resident to lysosomes and leads to the inhibition of mTORC1, which is normally activated by Na+-coupled amino acid transporters driven by the Na+ gradient. As a consequence, the transcription factors TFEB/TFE3 are made active in macrophages with distended lysosomes. In addition to their role in lysosomal biogenesis, TFEB/TFE3 activation causes the release of MCP-1/CCL2. In catabolically stressed tissues, defects in efflux of solutes from the endocytic pathway leads to increased monocyte recruitment. Here we propose that macrophages respond to a pressure-sensing pathway on lysosomes to orchestrate lysosomal biogenesis as well as myeloid cell recruitment.

Dynamic glucose uptake, storage, and release by human microvascular endothelial cells.

Endothelia determine blood-to-tissue solute delivery, yet glucose transit is poorly understood. To illuminate mechanisms, we tracked [3H]-2-deoxyglucose (2-DG) in human adipose-tissue microvascular endothelial cells. 2-DG uptake was largely facilitated by the glucose transporters GLUT1 and GLUT3. Once in the cytosol, >80% of 2-DG became phosphorylated and ∼20% incorporated into glycogen, suggesting that transported glucose is readily accessible to cytosolic enzymes. Interestingly, a fraction of intracellular 2-DG was released over time (15-20% over 30 min) with slower kinetics than for uptake, involving GLUT3. In contrast to intracellular 2-DG, the released 2-DG was largely unphosphorylated. Glucose release involved endoplasmic reticulum-resident translocases/phosphatases and was stimulated by adrenaline, consistent with participation of glycogenolysis and glucose dephosphorylation. Surprisingly, the fluorescent glucose derivative 2-NBD-glucose (2-NBDG) entered cells largely via fluid phase endocytosis and exited by recycling. 2-NBDG uptake was insensitive to GLUT1/GLUT3 inhibition, suggesting poor influx across membranes. 2-NBDG recycling, but not 2-DG efflux, was sensitive to N-ethyl maleimide. In sum, by utilizing radioactive and fluorescent glucose derivatives, we identified two parallel routes of entry: uptake into the cytosol through dedicated glucose transporters and endocytosis. This reveals the complex glucose handling by endothelial cells that may contribute to glucose delivery to tissues.