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.

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signaling1–4. Store operated Ca2+ entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously-unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially-restricted biotin-labelling in situ coupled with mass spectrometry5, 6 and a secondary screen based on bimolecular fluorescence complementation7, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches lead to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca2+-NFAT signaling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.

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The ER-resident regulatory protein STIM1 triggers store-operated Ca2+ entry by direct interaction with the plasma membrane Ca2+ channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb3+ luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca2+-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation, and inactivation of ORAI-family channels.

Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function, it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at stable ISs in Th cells.

Activation of T-lymphocytes requires stimulation of T-cell receptors (TCR) and co-stimulatory signals. Among different signalling cascades, TCR engagement induces Ca(2+) entry through plasma membrane Ca(2+) channels, which is an indispensable step for T-cells to expand clonally and to acquire effector functions. The Ca(2+) channels are activated by depletion of Ca(2+) stores and are called Ca(2+) release-activated Ca(2+) (CRAC) channels. Ca(2+) influx through CRAC channels is also controlled, directly or indirectly, by K(+) channels, Ca(2+)-ATPases, mitochondria, endoplasmic reticulum and Ca(2+) buffers. We review the functional implications of these transporters, organelles and buffers and develop a model of Ca(2+) signal generation that depends mainly on their relative mutual localization. This model offers the possibility of controlling amplitude and kinetics of Ca(2+) signals in T-cells. Decoding of various Ca(2+) signals allows differential activation of the transcription factor families nuclear factor of activated T-cells (NFAT), nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Variation of amplitude and kinetics of Ca(2+) signals thus is an important mechanism for modulating the specificity of T-cell responses.