The earliest intracellular signals determined in T cell activation are local, sub-second Ca2+ microdomains (1). Here we identify a Ca2+ entry component involved in Ca2+ microdomain formation in both non-stimulated and stimulated cells. In non-stimulated cells, spontaneous small Ca2+ microdomains depend on expression of ORAI1, STIM1, and STIM2. Using T cells stably transfected with ORAI1 fused to a genetically encoded Ca2+ indicator for optical imaging spontaneous Ca2+ microdomains depending on ORAI1 were also detected. Super resolution microscopy of non-stimulated T cells resulted in identification of a circular subplasmalemmal region with a diameter of approx. 300 nm with preformed patches of co-localized ORAI1, ryanodine receptors (RYR), and STIM1. Preformed complexes of STIM1 and ORAI1 in non-stimulated cells were confirmed by co-immunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second of T cell receptor (TCR) stimulation, Ca2+ microdomain numbers increase in the subplasmalemmal space, an effect not observed upon genetic deletion of Orai1, Stim2 or Ryr1 or upon antagonism of the Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Taken together, while preformed clusters of STIM and ORAI1 allow for local Ca2+ entry events in non-stimulated cells, upon TCR activation, NAADP-evoked Ca2+ release via RYR1, in tight interplay with Ca2+ entry via ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

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Car drivers can employ a number of strategies to negotiate curves. The tangent point strategy proposes the use of the angle between the tangent point of the inner lane markings and the car's current heading direction, which is proportional to the required steering angle. The gaze-sampling strategy proposes to fixate points on the future path and measure the curvature of optic flow vectors which can inform the drivers whether they over- or under-steer. Nine subjects drove repeatedly on the four loops of a motorway junction for which street parameters were available, while eye-movements, steering parameters and relations of the car to the lane were recorded. In the first half of the trials, we observed which strategy drivers normally use, whereas in the second half, we instructed subjects to use exclusively either the tangent-point or the gaze-sampling strategy and observed their steering behavior. Our results confirm that subjects normally look at the tangent point whereas they do not use gaze sampling of their own accord. Further, subjects drive more smoothly in terms of position on the lane and steering stability in the tangent-point condition.