Junctate expression increases the length of ER–phagosome membrane contact sites. Electron micrographs illustrating ER cisternae (arrows) juxtaposed to phagosomes (Ph) in wild-type (top panels) and Stim1^−/− (bottom panels) MEFs. Quantitative analysis shows that the length of ER cisternae is significantly longer in cells overexpressing junctate (middle panels) in Stim1^−/− MEFs (bottom right) but not in wild-type MEFs (top right). Qualitative differences of ER cisternae thickness can also be appreciated in Stim1^−/− cells overexpressing junctate (arrows). Scale bars: 200 nm. Data are means±s.e.m. of three independent experiments comprising the following number of phagosomes, contacts. Wild type: KDEL, 101, 28; junctate, 87, 20. Stim1^−/−: KDEL, 110, 24; junctate, 101, 24. **P<0.01.

Junctate increases SOCE, but only in the absence of both STIM proteins. (A) Representative changes in the ratio of Fura-2 fluorescence evoked by the addition of 2 mM Ca²⁺ to wild-type (WT, left panel), Stim1^−/− (right panel) or Stim1^−/−; Stim2^−/− MEFs that had been transfected with GFP–KDEL (black), YFP–junctate (‘J’, blue) and mCherry–STIM1 (‘S’, red) and treated with 1 µM thapsigargin (Tg) in Ca²⁺-free medium to activate SOCE. Traces showing the inhibition of SOCE due to the use of 50 µM lanthanum chloride (La³⁺) in Stim1^−/− and Stim1^−/−; Stim2^−/− MEFs transfected with GFP–KDEL (dotted grey), YFP–junctate (dotted light blue) and mCherry–STIM1 (dotted purple) are also shown (middle and right panels, respectively). (B) Representative changes in the ratio of Fura-2 fluorescence evoked by switching from 2 mM Ca²⁺ to Ca²⁺-free medium, followed by 2 mM Ca²⁺ re-addition in the absence of thapsigargin in Stim1^−/−; Stim2^−/− MEFs transfected with GFP–KDEL (black), YFP–junctate (‘J’, blue) and mCherry–STIM1 (‘S’, red) show that junctate expression does not lead to Ca²⁺ entry in the absence of store depletion. (C) Quantification of Ca²⁺ entry rates (slope) in Stim1^−/−, WT and Stim1^−/−; Stim2^−/− cells shows that junctate does not increase SOCE unless both STIM1 proteins are absent and that this influx is abrogated by the non-specific SOCE channel inhibitor La³⁺. Data are means±s.e.m. of three to eight independent experiments comprising 8–20 cells per experiment per condition. Ctr, control. *P<0.05, **P<0.01.

Junctate increases the phagocytic capability of cells in the absence of STIM proteins. YFP–junctate but not GFP–KDEL overexpression boosts phagocytosis in wild-type (WT, left), Stim1^−/− (middle) and Stim1^−/−; Stim2^−/− (right) MEFs as efficiently as mCherry–STIM1. Data are means±s.e.m. of four to six independent experiments comprising the following total number of cells, phagosomes. Wild type: KDEL, 200, 402; junctate, 200, 746. Stim1^−/−: KDEL, 279, 458; junctate, 257, 769; STIM1, 310, 1407. Stim1^−/−; Stim2^−/−: KDEL, 190, 270; junctate, 152, 623; STIM1, 136, 724. n.s., not significant. *P<0.05, **P<0.01.

Junctate boosts InsP3R-dependent Ca²⁺ release near phagosomes independently of STIM1. (A) Pseudo-colored (F/F_0ave) images of periphagosomal Ca²⁺ hotspots (arrows) in Stim1^−/− MEFs overexpressing RFP–KDEL (left panel), RFP–junctate (middle panel) or mCherry–STIM1 (right panel) and loaded with 4 µM Fluo-8-AM in Ca²⁺-containing medium. RFP–junctate and mCherry–STIM1 both doubled the occurrence of periphagosomal Ca²⁺ hotspots (bottom left panel). (B) Quantification of Ca²⁺-hotspot frequencies revealed that RFP–junctate generated significantly more hotspots than mCherry–STIM1 in Stim1^−/− MEFs when Ca²⁺ entry was prevented by Ca²⁺ removal or by the SOCE inhibitor La³⁺ (50 µM) but not when Ca²⁺ release was inhibited by the combination of La³⁺ and the InsP3R inhibitor 2-APB (50 µM). RFP–junctate is also able to generate significantly more hotspots than RFP–KDEL in Stim1^−/−; Stim2^−/− MEFs, an effect prevented by xestospongin-C (20 µM). (C) Pseudo-colored (F/F_0ave) images of periphagosomal Ca²⁺ hotspots (arrows) in Stim1^−/− MEFs loaded with 4 µM Fluo-8-AM, overexpressing RFP-junctate in Ca²⁺-free medium (left panel), or in medium containing La³⁺ (50 µM, middle panel) or La³⁺+2-APB (both at 50 µM, right panel). Colored bars, color-coded Fluo-8 fluorescence divided by the initial average cytosolic fluorescence (F/F_0ave). Scale bars: 3 µm. Data are means±s.e.m. of three to six independent experiments comprising the following number of cells, phagosomes. Stim1^−/−: KDEL, 54, 524; junctate, 78, 589; STIM1, 67, 648. Stim1^−/−+Ca²⁺-free medium: KDEL, 121, 417; junctate, 212, 840; STIM1, 130, 517. Stim1^−/− La³⁺: KDEL, 127, 272; junctate, 151, 487; STIM1, 99, 312. Stim1^−/− La³⁺+2-APB: KDEL, 93, 88; junctate, 91, 140; STIM1, 61, 89. Stim1^−/−; Stim2^−/− Ca²⁺-containing medium: KDEL, 50, 120; junctate, 37, 126; STIM1, 28, 113. Stim1^−/−; Stim2^−/−+xestospongin-C: KDEL, 35, 108; junctate, 37, 72; STIM1, 32, 104. n.s., not significant. *P<0.05, **P<0.01, ***P<0.001.