Volume 23, Issue 2, Pages 522-534 (April 2018) STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions  Krishna Prasad Subedi, Hwei Ling Ong, Ga-Yeon Son, Xibao Liu, Indu Suresh Ambudkar  Cell Reports  Volume 23, Issue 2, Pages 522-534 (April 2018) DOI: 10.1016/j.celrep.2018.03.065 Copyright © 2018 Terms and Conditions

Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions

Figure 1 C-Terminal Domain Interactions and Conformation Determine STIM2 Activation of Orai1 (A) Domain structure of STIM2 showing different mutations made in the CC1, CC2, and CC3 regions of STIM2. (B and C) Ca2+ entry was measured in HEK293 cells expressing: (B) CFP-tagged STIM2-WT (S2-WT), STIM2-CCmt (S2-CCmt), or STIM2-R517L (S2-R517L); and (C) siControl (siCtrl)+S2-WT, siOrai1 (siO1)+S2-WT, siControl+S2-CCmt, or siOrai1+S2-CCmt. Representative traces and quantitative data are shown. (D–F) TIRFM images of unstimulated cells expressing YFP-Orai1 (Orai1), CFP-STIM2 (STIM2), or mCherry-ER3 (ER) either alone or in combinations as indicated: (D) ER, Orai1, and STIM2 individually expressed; (E) ER+STIM2; and (F) ER+STIM2+Orai1. Fluorescence images of each protein and line scans are shown. (G) CFP, YFP, and FRET images of cells expressing YFP-OASF2-CFP (Y-OASF2-C), YFP-OASF-CFP, and YFP-OASF-L251S-CFP. (H) Bar graphs showing FRET of YFP-OASF-CFP (OASF), YFP-OASF-L251S-CFP (OASF-L251S), and YFP-OASF2-CFP (OASF2). (I) Bar graphs showing FRET of YFP-OASF2-CFP (OASF2), YFP-OASF2-R517L-CFP (OASF2-R517L) and YFP-OASF2-CFP+Orai1 (OASF2+Orai1), and YFP-OASF2-CCmt-CFP (OASF2-CCmt). All Ca2+ traces shown are average data from at least 50 cells. Bar graphs show mean ± SEM. (F-F0) from 3–5 different experiments with number of cells (n) is indicated on the graph; ∗∗∗p < 0.0001; unpaired Student’s t test, relative to as indicated or OASF (H). Scale bar is 10 μm in all images. Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions

Figure 2 Extended Conformation of STIM2 Promotes Recruitment of STIM1 and STIM1/Orai1 Coupling (A) Ca2+ influx in cells expressing siControl (siCtrl)+STIM2-WT(S2-WT), siSTIM1 (siS1)+S2-WT, siCtrl+STIM2-CCmt(S2-CCmt), or siS1+S2-CCmt. Representative traces and quantitative data are shown. (B and C) Co-IP of STIM1 and STIM2 in cells expressing CFP- and Myc-tagged S2-WT and S2-CCmt. Lysates were obtained from unstimulated or stimulated (CCh and CPA, as indicated) cells. IPs were done using anti-STIM1 antibody (B) or with anti-Myc antibody (C). Quantitation of the blots are shown in the bar graphs. (D) Ca2+ influx in cells expressing S2-WT, STIM2-ΔSOAR (S2-ΔSOAR), and STIM2-ΔK5 (S2-ΔK5) with or without CCmt inserted. Representative traces and quantitative data are shown. All Ca2+ influx traces are of average from >50 cells, and bar graphs are mean ± SEM. (F-F0) of cells of at least 3 independent experiments; ns (non-significant); ∗∗∗p < 0.0001; unpaired Student’s t test, relative to S2-WT or as indicated. All western blot densitometric analysis are expressed as mean ± SEM of data obtained from number of experiments (n) indicated; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; relative to S2-WT (unstimulated), unpaired Student’s t test. Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions

Figure 3 STIM2 Enhances Orai1+STIM1-Dependent SOCE and NFAT Nuclear Translocation at Low Stimulus Intensities (A–C) SOCE responses in HEK293 cells transfected with various constructs as indicated and stimulated with 10 μM CCh (arrow). Representative traces of the different patterns of [Ca2+]i are shown. (D) The bar graphs show the proportion (%) of a cell population displaying various patterns of [Ca2+]i changes. Overall patterns in Orai1+STIM1+STIM2 cells were significantly different from that in Orai1+STIM1 cells at 10 μM CCh (p < 0.0001, cells from 3–9 experiments; χ2 test). (E and F) Representative traces show increase in nuclear GFP-NFAT in control, Orai1+STIM1, or Orai1+STIM1+STIM2 cells after stimulation with CCh; 10 μM (E) or 100 μM (F). (G) Bar graph shows the summary of NFAT nuclear translocation from average of at least 3 independent experiments with indicated number of cells. (H and I) TIRFM images of cells co-expressed with (H) CFP-Orai1+YFP-STIM1 or (I) mCherry-STIM1+CFP-STIM2+YFP-Orai1 in unstimulated and CCh-stimulated conditions. Images of each protein in the cell are shown (scale bar, 10 μm). (J) Summary bar graph showing relative fluorescence in puncta of cells expressing different proteins as indicated in the absence and presence of 10 μM CCh. In (G) and (J), ∗∗p < 0.01; ∗∗∗p < 0.001; ANOVA and Sidak’s multiple comparisons test. Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions

Figure 4 STIM2 Traps STIM1 within ER-PM Junctions (A) TIRFM images of cell expressing mCherry-ER3 (ER)+YFP-STIM1 (upper panel) and YFP-STIM1+CFP-STIM2 (middle panel) and mCherry-ER3 (ER)+YFP-STIM1+CFP-STIM2 (lower panel); frames show each protein individually, and insets show enlarged images. (B) Enlarged images showing STIM1 tracks (green arrow) leading to STIM2 puncta (orange arrow). (C) Relative intensity of YFP-STIM1 in puncta of cells co-expressing CFP-tagged STIM2 proteins. (D) TIRFM images of cells expressing YFP-Orai1+mCherry-STIM1 (upper panel), and YFP-Orai1+mCherry-STIM1+CFP-STIM2 (lower panel). Frames show each protein individually. Line scans show the overlapping peaks for Orai1 with either STIM1, STIM2, or both (as indicated by ∗). (E) Western blots (left) and quantitative densitometry (right) showing co-IP of STIM1 with endogenous Orai1 in control (none) and CFP-STIM2 (CFP-S2-WT)-expressing cells. STIM1 and Orai1 antibodies were used to detect STIM1 and Orai1, respectively, whereas GFP antibody was used to detect CFP-STIM2. (F) Western blots (left) and quantitative densitometry (right) showing co-IP of endogenous STIM1 and transiently expressed CFP-tagged STIM2-WT (S2-WT), STIM2-CCmt (S2-CCmt) or YFP-tagged STIM2-ΔSOAR (S2-ΔSOAR), STIM2-ΔK5 (S2-ΔK5) with endogenous Orai1 in cells expressing these constructs as indicated. All western blot densitometric analysis are expressed as mean ± SEM of data obtained from number of experiments indicated; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; relative to control (none, E), S2-WT (F); unpaired Student’s t test. For TIRF image analysis: ∗∗∗p < 0.0001, relative to STIM1+S2-WT; unpaired Student’s t test. Scale bar is 10 μm in all images. Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions

Figure 5 STIM2 Triggers STIM1 Activation to Enhance Orai1 Function in Cells with High ER-[Ca2+] (A–D) Ca2+ influx in cells expressing 0.2 μg of STIM1 (S1-WT) and different amounts of STIM2 (S2-WT) alone or in combination as indicated (A–C). Inset in (D) Ca2+ entry in control and siOrai1-treated cells (Control, Ctrl; STIM1, S1; STIM2, S2; siControl, siC; siOrai1, siO1). (E) Ca2+ influx in cells expressing S2-WT, S2-R517L, and S2-CCmt alone or with STIM1 (S1-WT). (F) Ca2+ influx in cells expressing S2-WT, STIM1-R426L (S1-R426L), and S2-WT+S1-R426L. (G) Ca2+ influx in cells expressing S2-WT, STIM1-ES122 (S1-ES122), S2-WT+S1-ES122. (H) Summary bar graph of data from (F) and (G). Control indicates mock-transfected cells. Representative traces are average responses from >50 cells. Data presented in bar graphs are mean ± SEM of cells from at least 3 independent experiments; ns (non-significant); ∗∗∗p < 0.0001; unpaired Student’s t test, relative to STIM2-WT or as indicated. (I–K) Measurement of store-operated current (ICRAC) in HEK293T cells transfected with indicated plasmids. Representative traces and average of maximum current densities obtained from (number of cells) in each case (I and J) and current-voltage relationship (K) are shown. Data are mean ± SEM; ∗∗p < 0.01. Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions

Figure 6 STIM2 Induces the Activated Conformation of STIM1 (A) Illustration to show Orai1-induced changes in YFP-OASF-CFP (Y-OASF-C) FRET. (B) Images showing CFP, YFP, and FRET signals in cells expressing YFP-OASF-CFP with or without Orai1. (C) Bar graphs showing changes in OASF-FRET by Orai1. (D) Proposed STIM2-induced changes in intramolecular YFP-OASF-CFP FRET. (E) Confocal images showing CFP, YFP, and FRET signals in live cells expressing OASF conformational sensor with S2-WT or S2-ΔSOAR. (F) Bar graphs showing changes in OASF-FRET by S2-WT and S2-ΔSOAR. Inset shows reduction of OASF-FRET by Orai1, S2-WT, and S2-ΔSOAR (calculated from C and F). All data are expressed as mean ± SEM of FRET signals obtained from at least 3 independent experiments. ns (non-significant); ∗∗∗p < 0.0001, relative to OASF only or as indicated; unpaired Student’s t test. Scale bar is 10 μM throughout. (G) Proposed model. STIM2 clusters within ER-PM junctions in cells without depletion of ER-Ca2+ stores. Under these conditions, a fraction of STIM2 in the cells is in an activated conformation and recruit/activates Orai1. Ca2+ influx via the channel is low due to weak activation by STIM2. STIM2 recruits and remodels STIM1, mediating assembly of a STIM2/STIM1/Orai1 complex. Since ER-[Ca2+] is relatively high, STIM1 is likely to be recruited in its inactive conformation with Ca2+ bound to its N terminus. Our data show that STIM2-STIM1 interaction triggers conformational change in STIM1-C terminus to induce STIM1/Orai1 coupling and enhancement of channel function. For simplicity, only dimers of Orai1 and STIM proteins are shown. Cell Reports 2018 23, 522-534DOI: (10.1016/j.celrep.2018.03.065) Copyright © 2018 Terms and Conditions