JCS034496 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Comparison on the kinetics of STIM1 oligomerization and clustering in the superficial (ROI 1) and deeper ER (ROI 2) domains upon store depletion with histamine/BHQ (100 and 15 µM). (A) Images illustrate the location of the two regions of interest (ROI) measured. (B) Representative tracings of the effect of 100 µM histamine and 15 µM BHQ on STIM1 oligomerization (lines, left graph), STIM clustering (dotted lines, middle graph) and overlay in ROI 1 (red) and ROI 2 (blue). (C) Schematic illustration of the kinetics of STIM1 oligomerization and redistribution upon ER depletion in both ROIs.

• Supplemental Figure S2 -

  Fig. S2. Determination of the effective correlation concentrations (ECC₅₀) of STIM1. Oligomerization upon ER Ca²⁺ depletion (ECC₅₀/_{oligomerization}) and of the disassembly of STIM1 oligomers upon ER Ca²⁺ refilling (ECC₅₀/_disassambly). (A) Representative tracings of the effect of 100 µM histamine and 15 µM BHQ on Ca²⁺_ER (upper panel) and STIM1 oligomerization (lower panel), in the presence of 2 mM extracellular Ca²⁺ followed by the removal of extracellular Ca²⁺ (i.e. EGTA containing solution), and a subsequent re-addition of extracellular Ca²⁺ in the absence of histamine and BHQ. (B) The correlation between ER Ca²⁺ depletion and STIM1 oligomerization (continuous line, black circles, n=9 for STIM1 oligomerization; n=10 for Ca²⁺_ER) is compared with the correlation between ER Ca²⁺ refilling and the disassembly of STIM1 oligomers (dotted line open squares, n=9 for STIM1 oligomerization; n=10 for Ca²⁺_ER). Ca²⁺_ER was recorded in single endothelial cells that transiently expressed D1_ER and STIM1-YFP, respectively. STIM1 oligomerization was measured by following FRET between STIM1-CFP and STIM1-YFP. For STIM1 oligomerization, the ratios (F₅₃₅/F₄₈₀)/F₀ were normalized (Δ_max=100%). Curves were fitted using Prism 4.0 for Mac (GraphPad Software, San Diego, CA).

• Supplemental Figure S3 -

  Fig. S3. Simultaneous recordings of the disassembly/reformation of STIM1 clusters (red traces) with Ca²⁺_cyto (black traces). Ca²⁺_cyto was recorded simultaneously with STIM1 dynamics in cells that transiently expressed YFP-STIM1 and loaded with fura-2-am. Images for subplasmalemmal STIM1 clustering were captured using an array confocal laser scanning microsope and for fura-2 measurements a conventional high-resolution imaging at the same device was used.

• Movie 1 -

  Movie 1. Histamine-nduced degradation of basal STIM1 clusters. The cellular dynamics of STIM1-YFP is shown upon cell stimulation with the cytosolic-Ca²⁺-elevating agonist histamine (100 µM) in the presence of 2 mM extracellular Ca²⁺.

• Movie 2 -

  Movie 2. STIM1 clustering upon moderate and strong ER Ca²⁺ depletion in the presence and absence of extracellular Ca²⁺. The cellular dynamics of STIM1-YFP is shown upon moderate ER Ca²⁺ depletion using 100 µM histamine in the presence of 2mM extracellular Ca²⁺ followed by strong ER Ca²⁺ depletion using the combination of 100 µM histamine and 15 µM BHQ first in the presence of 2mM extracellular Ca²⁺ and subsequently in the absence of extracellular Ca²⁺ (i.e. EGTA-containing solution).