Dendritic cells (DCs) capture Ags or viruses in peripheral tissue to transport them to lymphoid organs to induce cellular T cell responses. Recently, a DC-specific C-type lectin was identified, DC-specific ICAM-grabbing non-integrin (DC-SIGN), that functions as cell adhesion receptor mediating both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R that captures HIVgp120 and facilitates DC-induced HIV transmission of T cells. Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function of DC-SIGN as endocytic receptor. In this study we demonstrate that on DCs DC-SIGN is rapidly internalized upon binding of soluble ligand. Mutating a putative internalization motif in the cytoplasmic tail reduces ligand-induced internalization. Detailed analysis using ratio fluorescence imaging and electron microscopy showed that DC-SIGN-ligand complexes are targeted to late endosomes/lysosomes. Moreover, ligands internalized by DC-SIGN are efficiently processed and presented to CD4⁺ T cells. The distinct pattern of expression of C-type lectins on DCs in situ and their nonoverlapping Ag recognition profile hint to selective functions of these receptors to allow a DC to recognize a wide variety of Ags and to process these to induce T cell activation. These data point to a novel function of the adhesion receptor DC-SIGN as an efficient DC-specific Ag receptor that can be used as a target to induce viral and antitumor immunity.

Superoxide and its derivatives are increasingly implicated in the regulation of physiological functions from oxygen sensing and blood pressure regulation to lymphocyte activation and sperm-oocyte fusion. Here we describe a novel superoxide-generating NADPH oxidase referred to as NADPH oxidase 5 (NOX5). NOX5 is distantly related to the gp91^phox subunit of the phagocyte NADPH oxidase with conserved regions crucial for the electron transport (NADPH, FAD and heme binding sites). However, NOX5 has a unique N-terminal extension that contains three EF hand motifs. The mRNA of NOX5 is expressed in pachytene spermatocytes of testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes. When heterologously expressed, NOX5 was quiescent in unstimulated cells. However, in response to elevations of the cytosolic Ca²⁺concentration it generated large amounts of superoxide. Upon Ca²⁺ activation, NOX5 also displayed a second function: it became a proton channel, presumably to compensate charge and pH alterations due to electron export. In summary, we have identified a novel NADPH oxidase that generates superoxide and functions as a H⁺ channel in a Ca²⁺-dependent manner. NOX5 is likely to be involved in Ca²⁺-activated, redox-dependent processes of spermatozoa and lymphocytes such as sperm-oocyte fusion, cell proliferation, and cytokine secretion.

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP ₃ receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca ²⁺ storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca ²⁺ that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca ²⁺ store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca ²⁺ uptake. Direct measurements with ER-targeted Ca ²⁺ -sensitive fluorescent “cameleon” proteins revealed that Bcl-2 decreased the free Ca ²⁺ concentration within the lumen of the ER, [Ca ²⁺ ] _ER . Analysis of the kinetics of Ca ²⁺ store depletion in response to the Ca ²⁺ -ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca ²⁺ concentration within the ER lumen by increasing the Ca ²⁺ permeability of the ER membrane. The increased ER Ca ²⁺ permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ_m) and pH gradient (ΔpH_m) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH_m, whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH_m in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpH_m (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH_m decreased during cytosolic Ca²⁺ elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca²⁺-ATPases and transmitted to mitochondria by P_i/H⁺ symport and K⁺/H⁺ exchange, whereas the decrease in ΔpH_m reflected the low H⁺-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca²⁺ and pH, mitochondria alkalinized and ΔpH_m increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca²⁺ caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH_m are regulated by opposing Ca²⁺-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.