Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca ²⁺ store depletion

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 m 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 ERto-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.Ca 2ϩ release-activated Ca 2ϩ ͉ fluorescence recovery after photobleaching ͉ FRET ͉ store-operated Ca 2ϩ influx C a 2ϩ signals are used by cells for transducing receptor or electrical inputs into functional outputs such as gene expression, contraction, and secretion (1). Although endoplasmic reticulum (ER) Ca 2ϩ stores are the main source for inositol trisphosphate-induced transient Ca 2ϩ signals, a necessary step for activation of T lymphocytes, mast cells, and many other cells is a persistent increase in Ca 2ϩ concentration (2). These sustained Ca 2ϩ signals require retrograde signaling from the lumen of the ER to Ca 2ϩ channels in the plasma membrane (PM) in a process called store-operated Ca 2ϩ (SOC) influx [also termed Ca 2ϩ release-activated Ca 2ϩ (CRAC) in immune cells] (3). The Stauderman/Cahalan groups and our group have recently identified the single transmembrane protein stromal interaction molecule 1 (STIM1) as an ER Ca 2ϩ sensor that responds to the depletion of ER Ca 2ϩ and activates SOC/CRAC channels in the PM (4-6). STIM1 senses Ca 2ϩ by an EF hand Ca 2ϩ -binding site in the lumen of the ER (7). A recombinant fragment of the luminal region of STIM1 has been shown to form dimers and oligomers in the absence of Ca 2ϩ in vitro (8). Overexpression of STIM1 mutants with a disrupted EF hand Ca 2ϩ -binding motif resulted in constitutive activation of Ca 2ϩ influx (5, 6, 9). It was also observed that depletion of ER Ca 2ϩ stores induced STIM1 translocation to punctate structures near the PM that correspond to ER-PM junctions (5, 10). STIM1 colocalizes at these junctions with the CRAC channel Orai1 (also known as CRACM1) and synergistically activates SOC/CRAC infl...

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

“…Orai1-YFP was clearly visible in the PM of spreading lamellipodia, and was also seen in moving puncta near the stimulatory surface ( Figure 1A, Movie 1). In Jurkat T-cells expressing STIM1-CFP plated onto coverslips coated with anti-CD45 antibodies that do not activate the TCR, STIM1-CFP was seen in an ER-like pattern that colocalized with ER markers such as calnexin (Rajagopalan et al, 1994) as expected from previous studies ( Figure 1B; Wu et al, 2006;Liou et al, 2007;Ross et al, 2007). Activation of these cells resulted in formation of puncta containing STIM1-CFP (Supplemental Figure S3).…”

Attenuated mesangial cell proliferation related to store-operated Ca2+ entry in aged rat: the role of STIM 1 and Orai 1

“…Recently, STIM 1 has been identified as a Ca 2+ sensor in the ER, activating SOCE in the plasma membrane upon store depletion (Liou et al 2005;Roos et al 2005). STIM 1 that is diffusely distributed in the ER membrane translocates to the ER-plasma membrane junctions upon store depletion, where it triggers SOCE (Lewis 2007;Baba et al 2006;Liou et al 2007). Thus, SOCE can be considered as channels that are regulated by STIM 1 and require store depletion that leads to clustering of STIM 1.…”

“…It is well known that store depletion will trigger STIM 1 and Orai 1 to form puncta (Zhou et al 2010;Liou et al 2007). STIM 1 and Orai 1 puncta formation directly correlates to SOCE activity (Liou et al 2005).…”

Calcium signaling in lymphocytes

“…Following store depletion, STIM1 aggregates into small clusters ("puncta") in the ER membrane [37,55]; STIM1 with an EF-hand mutation that impairs Ca 2+ binding is constitutively present in puncta even in resting cells with replete Ca 2+ stores [37]. The propensity of STIM1 to aggregate when its EF-hands are not bound by Ca 2+ can be demonstrated using just a short recombinant N-terminal fragment of STIM1, containing only the EF-hands and the adjacent SAM (sterile α-motif) domain [56,57].…”