Cytoskeleton rotation relocates mitochondria to the immunological synapse and increases calcium signals.

Ca2+ microdomains and spatially resolved Ca2+ signals are highly relevant for cell function. In T cells, local Ca2+ signaling at the immunological synapse (IS) is required for downstream effector functions. We present experimental evidence that the relocation of the MTOC towards the IS during polarization drags mitochondria along with the microtubule network. From time-lapse fluorescence microscopy we conclude that mitochondria rotate together with the cytoskeleton towards the IS. We hypothesize that this movement of mitochondria towards the IS together with their functionality of absorption and spatial redistribution of Ca2+ is sufficient to significantly increase the cytosolic Ca2+ concentration. To test this hypothesis we developed a whole cell model for Ca2+ homoeostasis involving specific geometries for mitochondria and use the model to calculate the spatial distribution of Ca2+ concentrations within the cell body as a function of the rotation angle and the distance from the IS. We find that an inhomogeneous distribution of PMCA pumps on the cell membrane, in particular an accumulation of PMCA at the IS, increases the global Ca2+ concentration and decreases the local Ca2+ concentration at the IS with decreasing distance of the MTOC from the IS. Unexpectedly, a change of CRAC/Orai activity is not required to explain the observed Ca2+ changes. We conclude that rotation-driven relocation of the MTOC towards the IS together with an accumulation of PMCA pumps at the IS are sufficient to control the observed Ca2+ dynamics in T-cells during polarization.

Mutations of the Ca2+-sensing stromal interaction molecule STIM1 regulate Ca2+ influx by altered oligomerization of STIM1 and by destabilization of the Ca2+ channel Orai1.

A drop of endoplasmic reticulum Ca(2+) concentration triggers its Ca(2+) ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca(2+) entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca(2+) homeostasis.