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First published online 2 September 2008
doi: 10.1242/jcs.034496

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Cytosolic Ca²⁺ prevents the subplasmalemmal clustering of STIM1: an intrinsic mechanism to avoid Ca²⁺ overload

¹ Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Graz, Austria
² Institute of Biophysics, University of Linz, Linz, Austria

View larger version (38K): [in this window] [in a new window]   Fig. 1. STIM1 oligomerization upon maximal ER Ca2+ depletion correlated with ER Ca2+ depletion, while subplasmalemmal STIM1 clustering was delayed and followed the decay of cytosolic Ca2+ levels. (A-D) Representative tracings of the effect of 100 µM histamine on [Ca2+]ER (A), STIM1 oligomerization (B), subplasmalemmal STIM1 clustering (C) and [Ca2+]cyto (D) in EGTA-containing solution followed by re-addition of 2 mM Ca2+ in the absence of histamine. (E) Respective statistical evaluation of the experiments displayed in A (n=11), B (n=7), C (n=12) and D (n=12). (F) Schematic illustration of STIM1 activation by ER Ca2+ depletion. (G) Images of the subcellular distribution of YFP-STIM1 under conditions of high ER Ca2+ levels in resting cells (left images) and low ER Ca2+ levels upon cell stimulation with histamine in EGTA (right images) at the middle z-plane (upper images) and at a TIRF-like bottom plane (lower images). (H) Normalized tracings of [Ca2+]ER and [Ca2+]cyto were inverted and plotted with the respective normalized traces for STIM1 oligomerization and STIM1 clustering (max=100%).*P<0.05 versus the respective compared data set.

View larger version (55K): [in this window] [in a new window]   Fig. 2. The dynamics of STIM1 oligomerization and subplasmalemmal clustering upon moderate and strong ER Ca2+ depletion was limited, while subplasmalemmal STIM1 clustered repetitively at focal points. (A-D) Representative tracings of the effect of 100 µM histamine on [Ca2+]ER (A), STIM1 oligomerization (B), subplasmalemmal STIM1 clustering (C) and [Ca2+]cyto (D) in Ca2+-containing buffer followed by removal of Ca2+ in the presence of histamine. (E) Respective statistical evaluation of the experiments displayed in A (n=12), B (n=8), C (n=6) and D (n=6). (F) Spatial reversibility of subplasmalemmal STIM1 clusters and the histamine-induced degradation of pre-existing STIM1 clusters: i, pre-existing STIM1 clusters under resting conditions; ii, histamine induced disassembly of STIM1 cluster; iii, reassembly of STIM1 clusters upon removal of extracellular Ca2+.

View larger version (86K): [in this window] [in a new window]   Fig. 3. BAPTA-am loading reversed the disassembly of STIM1 clusters by histamine. (A) Representative images of the sensitivity of pre-existing subplasmalemmal STIM1 clusters to histamine and their recovery upon reduction of [Ca2+]cyto (n=6). (B) BAPTA reversed the decomposition of pre-existing subplasmalemmal STIM1 clusters upon histamine stimulation independently from extracellular Ca2+ (n=6). (C,D) Cytosolic Ca2+ signaling (C) and kinetics of subplasmalemmal STIM1 clustering (D) in response to 100 µM histamine in the presence of 2 mM extracellular Ca2+ in YFP-STIM1-expressing cells that were loaded with either fura-2-am (n=6) or with fura-2-am and BAPTA-am (n=6). *P<0.05 versus the respective compared data set.

View larger version (34K): [in this window] [in a new window]   Fig. 4. STIM1 oligomerization and STIM1 clustering upon strong ER Ca2+ depletion, impeded ER Ca2+ replenishment at high and low [Ca2+]cyto levels. (A-C) Representative tracings (left graphs) and statistical summary (right graphs) of [Ca2+]cyto (A, n=12), [Ca2+]ER (B, n=9) and STIM1 oligomerization (C, n=28) in response to stimulation with 100 µM histamine and the SERCA inhibitor BHQ (15 µM). (D) Subcellular distribution of STIM1 at a TIRF-like plane (left images) and at a middle z-plane (right images) under resting conditions (`prior stimulation', upper panel), under cell stimulation with 100 µM histamine and 15 µM BHQ in 2 mM Ca2+ (`His/BHQ in Ca2+', upper middle panel) and cell stimulation in EGTA (`His/BHQ in EGTA', lower middle panel). Along the lines indicated, fluorescence intensity of YFP-STIM1 is presented at the corresponding z-plane and activation stages in the right graphs. The lower images and graphs show overlays of the upper illustrations. (E) Average data and respective statistical evaluation of the experiments displayed in D (n=11 for each condition). (F) The time course of STIM1 clustering (n=3 for each condition). *P<0.05 versus the respective compared data set.

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