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. Author manuscript; available in PMC: 2010 Nov 1.
Published in final edited form as: Nat Cell Biol. 2010 Apr 25;12(5):436–446. doi: 10.1038/ncb2045

Figure 5.

Figure 5

An EF-hand mutant of CRACR2A causes spontaneous clustering of STIM1. (a) CRACR2A binds Ca2+ via its EF-hands. 45Ca2+ overlay experiments were performed with purified full-length CRACR2A, CRACR2A truncated in its N terminus (ΔN, amino acids 119–395), C terminus (ΔC, amino acids 1–154), or mutated in its EF-hand2 (EF2MUT). 2 or 10 µg of each protein were used to examine Ca2+ binding. Similar amount of purified calmodulin (CaM) was run (left panel) to compare Ca2+ binding affinity. Compare Ca2+ binding of CaM with ΔC mutant of CRACR2A for similar molar concentrations of each protein. Panels on the right show Ponceau S staining of the same blots to compare protein amounts. (b) Live cell epifluorescence and TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP and CRACR2AEF2MUT-mCherry in the absence or presence of store depletion. Left panels show epifluorescence images from the middle of the cell and right panels show TIRF images from the footprint of the cell. Arrowheads in the top panel show clustering of STIM1-YFP in cells co-expressing CRACR2AEF2MUT. An arrow in the same panel depicts minimal clustering in a cell expressing only STIM1-YFP. Arrowheads in the bottom TIRF panels show co-accumulation of CRACR2AEF2MUT-mCherry with STIM1-YFP at sites of newly formed clusters after store depletion (+TG). Bar, 10 µm. (c) TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP in the absence (top) or presence (bottom) of CRACR2AEF2MUT. Cells co-expressing mCherry (top) or CRACR2AEF2MUT-mCherry (bottom) were selected. CRACR2AEF2MUT induced clustering of STIM1-YFP without ER Ca2+ depletion (compare images at T = 0 s in the top and bottom panels). Thapsigargin was added at T = 0 s and images of STIM1 clustering are shown at 200 and 500 s. Arrowheads represent preformed clusters of STIM1 that expand upon store depletion. Bar graph depicts averaged number of HEK293 cells (± s.d.) showing STIM1 clustering in the presence (n = 112 cells) or absence (n = 106) of CRACR2AEF2MUT. Cells moderately expressing STIM1-YFP were examined. Bar, 10 µm.