Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion - PubMed (original) (raw)

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion

Jen Liou et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca(2+) sensor that responds to ER Ca(2+) store depletion and activates Ca(2+) channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca(2+) store depletion. These oligomers rapidly dissociated when ER Ca(2+) stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER-PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca(2+) store depletion but failed to form puncta at ER-PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 mum to reach ER-PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca(2+) gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (i) dissociation of Ca(2+), (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER-PM junctions, and (iv) activation of PM Ca(2+) channels.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

ER Ca2+ controls STIM1 oligomerization and puncta formation. (A) Schematic representation of a FRET-based assay for STIM1 oligomerization. (B) STIM1 oligomerization and puncta formation in YFP-STIM1 and CFP-STIM1 cotransfected RBL cells. Confocal images were acquired near the adhesion surface before and 90 s after antigen (2 μg/ml dinitrophenol-BSA) stimulation. (Scale bar, 10 μm.) (C) The average STIM1 FRETE trace of 31 RBL cells. The average and SE of _t_1/2 are shown. (D) Confocal images of YFP-STIM1 and CFP-ER marker cotransfected RBL cells were acquired near the adhesion surface before and 90 s after antigen stimulation. The overlay images show the colocalization of STIM1 (green) and the ER marker (red). (Scale bar, 5 μm.) (E) YFP-STIM1 and CFP-STIM1 cotransfected HeLa cells were stimulated with 100 μM histamine (Hist.) plus 5 μM BHQ in a Ca2+-free buffer for 4 min. Cells were then washed three times, and 10 mM Ca2+ was added back. Confocal images were acquired near the adhesion surface before (Basal), 75 s after stimulation (Store depleted), and 70 s after Ca2+ readdition (Store refilled). (Scale bar, 10 μm.) (F) The average STIM1 FRETE trace of 10 HeLa cells. The average and SE of _t_1/2 are shown.

Fig. 2.

Fig. 2.

STIM1 oligomerization and translocation to ER–PM junctions are sequential processes. (A) YFP and FRETE images of a YFP-STIM1 and CFP-STIM1 coexpressing HeLa cell acquired near the adhesion surface after 10 μM ionomycin stimulation in a Ca2+-free buffer. (Scale bar, 20 μm.) (B) A kinetic comparison of STIM1 FRETE increases and puncta formation in the same cells. The average FRETE responses of 28 ionomycin-stimulated cells are shown. STIM1 puncta formation was monitored in these cells by measuring the average granule intensity in each cell by using a Gaussian filter (see Materials and Methods). SEs are shown. (C) The average FRETE trace of 29 CFP-STIM1-ΔK and YFP-STIM1-ΔK cotransfected HeLa cells was compared with the wild-type STIM1 FRETE trace shown in B. (D) Confocal images of a YFP-STIM1-ΔK transfected HeLa cell acquired near the adhesion surface after 10 μM ionomycin stimulation in a Ca2+-free buffer. There was no puncta formation in all ionomycin-stimulated YFP-STIM1-ΔK-transfected HeLa cells examined (>100 cells from 30 experiments). (Scale bar, 20 μm.)

Fig. 3.

Fig. 3.

STIM1 ER-to-PM signaling is a local process. (A) YFP-STIM1 diffusion in basal and ionomycin-treated cells monitored by fluorescence recovery after photobleaching measurements. (Scale bar, 10 μm.) (B) The average YFP-STIM1 intensity ratio of two sites inside and outside of the photobleached region (5 μm apart; n = 8 for each condition). (C) A series of relative Gaussian intensity profiles were fit through images such as those shown in A, and the increase in the square of the Gaussian peak radius (_a_2) was tracked over time to derive an apparent diffusion coefficient. (D) The average diffusion coefficients of YFP-STIM1 before and after Ca2+ store depletion (n = 5 for each condition; SEs are shown).

Fig. 4.

Fig. 4.

Schematic representation of the four steps that define the STIM1 ER-to-PM signaling relay. (Left) When ER stores are loaded with Ca2+, Ca2+ is bound to the EF hand of STIM1, preventing STIM1 oligomerization. (Center) In the first two activation steps, a reduction in luminal ER Ca2+ leads to Ca2+ dissociation from the EF hand, which triggers a rapid oligomerization of STIM1. The oligomerization of STIM1 exposes the C-terminal polybasic PM-targeting motif. (Right) In the next two steps, STIM1 oligomers are recruited via diffusion along the ER network to nearby ER–PM junctions where STIM1 interacts with the PM Ca2+ channel Orai1 and activates SOC influx.

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