T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values <200 nm. Consequently, mitochondria close to the IS took up more Ca 2؉ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca 2؉ influx across the plasma membrane and Ca 2؉ -dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.calcium ͉ lymphocyte ͉ mitochondria

The STIM1-ORAI1 pathway of store-operated Ca2+ entry is an essential component of cellular Ca2+ signaling1. STIM1 senses depletion of intracellular Ca2+ stores in response to physiological stimuli, and relocalises within the endoplasmic reticulum (ER) to plasma membrane (PM)-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca2+ channels. Here we used a genome-wide RNAi screen to identify filamentous septin proteins as critical regulators of store-operated Ca2+ entry. Septin filaments and phosphatidylinositol 4,5-bisphosphate (PIP2) rearrange locally at ER-PM junctions prior to and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PIP2 reorganisation and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud, and the primary cilium, and to serve as membrane diffusion barriers and/or signaling hubs in cellular processes including vesicle trafficking, cell polarity, and cytokinesis2–4. Our data show that septins also organise the highly localised plasma membrane domains important in STIM1-ORAI1 signaling, and indicate that septins may organise membrane microdomains relevant to other signaling processes.

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.