STIM1/2 maintain signaling competence at ER-PM contact sites during neutrophil spreading

Abstract

Neutrophils are highly motile leukocytes that migrate inside tissues to destroy invading pathogens. Ca2+ signals coordinate leukocytes migration, but whether Ca2+ fluxes mediated by Stim proteins at ER-PM contact sites regulate neutrophil actin-based motility is unclear. Here, we show that myeloid-specific Stim1/2 ablation decreases basal cytosolic Ca2+ levels and prevents adhesion-induced Ca2+ elevations in mouse neutrophils, reducing actin fiber formation and impairing spreading. Unexpectedly, more ER-PM contact sites were detected on the actin-poor adhesive membranes of Stim1/2-deficient neutrophils, which had reduced inositol-1,4,5-trisphosphate receptor (IP3R) immunoreactivity on confocal and immunogold micrographs despite preserved IP3R levels on western blots. Remarkably, Stim1/2-deficient neutrophils regained signaling and spreading competence in Ca2+-rich solutions and were recruited more effectively in mouse inflamed cremaster muscles in vivo. Our findings indicate that Stim1/2 preserve IP3R functionality in neutrophils, generating adhesion-dependent Ca2+ signals that control actin dynamics during neutrophil spreading. Stim proteins thus maintain IP3R signaling competence at adhesive membranes, enabling Ca2+-dependent actin remodeling during spreading in mouse neutrophils.

Figure S1.

Validation of Salsa6f and of myeloid Stim1/2 ablation. (A) Flow cytometry gating strategy to isolate tdT⁺Ly6G⁺CD115⁻F4/80⁻ neutrophils from flushed mouse BM. The two FACS profiles at bottom left are replicated in Fig. 1 A. Cartoon shows the LSL-Salsa6f cassette with its Ca²⁺-insensitive tdTomato (tdT⁺) and Ca²⁺-sensitive GCaMP6f inserted at the Rosa26 locus. (B) Percentage of BM neutrophils isolated by negative selection from mice expressing or not Salsa6f. n = 6 pairs of mice, two-tailed Welch’s t test. (C) Simultaneous Fura-2 and Salsa6f recordings in cells exposed to increasing extracellular [Ca²⁺] in the presence of ionomycin (left) and steady-state Fura-2 and Salsa6f ratios as a function of intracellular [Ca²⁺], calculated from [Ca²⁺]_ext using the Kd of Fura2 (right). (D) TIRF kymograph of Ca²⁺ waves propagating in an adherent neutrophil (top) and changes in GCaMP6f intensity in three aligned regions (bottom, regions are indicated by dotted circles on the TIRF image) separated by 3.6 µm along the kymograph axis (dotted line). See Video 2. Representative of 10 recordings. (E) Flow cytometry Fluo-8 recordings and quantification of Ca²⁺ responses evoked by Ca²⁺ readmission to WT and Stim1/2^−/− neutrophils exposed to Tg (1 µM) in Ca²⁺-free medium. n = 3 recordings from one pair of mice. (F) Salsa6f recordings and quantification of the Tg-Ca²⁺ evoked responses in WT and Stim1/2^−/− neutrophils. n = 2 recordings from two pairs of mice. AUC, area under the curve. (G) Luminometric LO-12 recordings of the ROS production evoked by PMA in WT and Stim1/2^−/− neutrophils. n = 5/6 mice, in triplicate recordings.

Figure 1.

Ca ²⁺ elevations reported by Salsa6f in adherent murine neutrophils. (A) Representative micrograph and flow cytometry profiles of BM cells from CEBPa-Salsa6f mice. Myeloid Salsa6f⁺ cells have a high tdT fluorescence (red) and most express the granulocyte marker Ly6G (cyan). (B) Proportion of Ly6G⁺ cells in sorted and adhered TdT⁺ BM cells (left, n = 7/54, Student’s unpaired two-tailed t test) and proportion of confirmed neutrophils (CD115⁻F4/80⁻) in TdT⁺Ly6G⁺ cells purified with a neutrophil kit or flushed from BM (right, n = 5/7, Student’s unpaired two-tailed t test, cells termed “purified” and “flushed” thereafter). (C) Representative micrographs of the Ca²⁺ fluctuations reported by Salsa6f in purified neutrophils plated on PLL. Ca²⁺ elevations increase the green fluorescence of GCamP6f. See Video 1. (D) Representative Ca²⁺ recordings of flushed murine neutrophils crawling on PLL (left) and incidence and frequency of Ca²⁺ spikes. (right, n = 25 recordings from three mice). (E) Ca²⁺ fluctuations recorded by Fura-2 and Salsa6f in purified neutrophils loaded or not with Fura-2 (left) and kinetic properties of the Ca²⁺ transients recorded in each condition (right, n = 49/90/122 cells from three mice). (F) Representative micrographs of Fura-2–loaded Salsa6f⁺-purified neutrophils, showing the cellular distribution of the TdT and Fura-2 fluorescence. (G) In situ calibration of Salsa6f and Fura-2 in cells equilibrated at increasing extracellular [Ca²⁺] with ionomycin. n = 3 mice.

Figure 2.

Stim1/2 ablation prevents repetitive Ca ²⁺ signals during neutrophils spreading. (A) Fluo-8 recordings and quantification of Ca²⁺ responses evoked by Tg and fMIVIL in WT and Stim1/2^KO-purified neutrophils suspended in physiological saline. n = 6/5 recordings from three pairs of mice, Student’s unpaired one-tailed t test. (B) Single-cell Salsa6f recordings and quantification of Ca²⁺ responses evoked by Tg and fMIVIL in flushed WT and Stim1/2^KO neutrophils adhered to PLL. n = 6/5 recordings from three pairs of mice, mean ± SEM, Student’s unpaired one-tailed t test. (C) Salsa6f micrographs and recordings of flushed cells adhered to PLL coatings (left, see Video 3 and Video 4) and incidence of Ca²⁺ transients and integrated Ca²⁺ responses (right, n = 25/19 recordings from three mice pairs, two-tailed Welch’s t test). fMIVIL; N-formyl-Met-Ile-Val-Ile-Leu.

Figure 3.

Effect of Orai1 and PLC inhibition on Ca ²⁺ signals during neutrophils spreading. (A) Salsa6f recordings (left), Ca²⁺ transient frequency (middle), and integrated Ca²⁺ responses (right) of flushed neutrophils adhered to PLL and exposed to DMSO (0.1%), the Orai1 inhibitor GSK-7975a (10 μM), or the PLC inhibitor U73122 (1 μM) in 2 mM [Ca²⁺]_ext. n = 436/83/56 cells in 10/4/4 recordings from four to eight mice, Student’s paired two-tailed t test. (B) Time-course micrographs of bottom recordings in A. See Video 5.

Figure S2.

Effect of pharmacological inhibitors on neutrophil Ca ²⁺ responses. (A) Salsa6f recordings (left), Ca²⁺ transient frequency (middle), and integrated Ca²⁺ responses (right) of adherent neutrophils exposed to CM4620 (10 μM), Gd³⁺ (10 μM), or to the PKC activator PMA (1 μM). n = 27/42/35 cells in 4/4/3 recordings from two mice, Student’s paired two-tailed t test. (B) Entire recordings of Fig. 3 A showing the subsequent sequential addition of fMIVIL (10 nM) and Tg (1 μM) to cells treated with DMSO, GSK-7975a, and U73122. (C) Effect of the indicated inhibitors on Ca²⁺ responses evoked by fMIVIL and Tg. n = 92/40/35/45 cells for fMIVIL with DMSO/DPI/PMA/U73 and 68/44/31 cells for Tg with DMSO/GSK/EGTA from two to eight mice. Two-tailed Welch’s t test. (D) Effect of DMSO, GSK7975a, and CM4620 on the Ca²⁺ responses evoked by Ca²⁺ readmission to cells treated with Tg in Ca²⁺-free medium. n = 125/126/116 cells in 4/4/4 recordings from two mice. Ordinary one-way ANOVA with Šídák multiple comparison test. Gd^3+: gadolinium; fMIVIL: N-formyl-Met-Ile-Val-Ile-Leu; AUC: area under the curve.

Figure 4.

Effect of Stim1/2 ablation on the spreading of neutrophils. (A) Average tdTomato fluorescence area and circularity of WT and Stim1/2^KO-flushed neutrophils plated on PLL during the 3-min recordings in Fig. 2 and relative changes in the fluorescence area of these cells (n = 127/134 cells in 7/7 recordings from three WT/Stim1/2^KO mice pairs, Student’s paired one-tailed t test on mice pairs. Small and large symbols show data from individual cells and mouse average, lines show median cellular values). (B) DIC micrographs (left) and fraction of spread WT and Stim1/2^KO-flushed neutrophils plated on PLL for ∼20 min at 37°C and at RT (right, n = 37/39 and 11/11 recordings from 3 WT/Stim1/2^KO mice pairs, Mann–Whitney U test. Student’s paired two-tailed t test on mice pairs). See Video 6 and Video 7. (C) TIRF micrographs of flushed neutrophils labeled with the PM dye CellMask adhering to PLL-coated glass (left, Video 8 and Video 9) and change in the size of neutrophil footprints after PLL contact (right, n = 25/25 graphed and 75/41 analyzed cells from four WT/Stim1/2^KO mice pairs, two-tailed Welch’s t test on cell values).

Figure S3.

Effect of Stim1/2 ablation on the spreading of neutrophils. (A) Effective and cumulative distance covered by the centroids of WT and Stim1/2^−/− neutrophils tracked in Fig. 4 A. Panels at right show the cross-correlation of these parameters. Bottom graphs show the centroid location and position relative to the initial landing spot and centroids probability distributions. (B) DIC micrographs (left) and fraction of spread WT and Stim1/2^−/− neutrophils treated or not with U72133 following ∼20-min plating at 37°C (right, n = 37/20 and 39/9 recordings from three WT/Stim1/2^−/− mice pairs, two-tailed Welch’s t test). (C) Change in the size of TIRF footprints of WT neutrophils treated or not with U72133 (n = 25/25 graphed and 34/19 analyzed cells from three WT/Stim1/2^−/− mice pairs, two-tailed Welch’s t test).

Figure 5.

Effect of Stim1/2 ablation on the ultrastructure of mouse neutrophils. (A) Representative electron micrographs of suspended (top) and adherent (bottom) neutrophils purified from WT (left) and Stim1/2^KO (right) mice. Arrowheads indicate ER structures located <35 nm from the PM at unsettled (black) or adherent (white) membranes. Ѳ: adjacent cell. Scale bar: 1 µm. Insets: Higher magnification micrographs showing cER sheets (asterisks) apposed to the PM (closed circle). Black arrow denotes the dish bottom. Scale bar: 40 nm. Micrograph of adherent Stim1/2^KO neutrophil is replicated in Fig. 7 A. (B) Percentage of PM decorated by cER and quantification of cER length and ER-PM gap distance in suspended (Susp) and adherent (Adh) WT and Stim1/2^KO neutrophils. n = 15/15 Susp, 26/19 Adh cells with 148/95 cER sheets and 150/124 cER sheets from two mice pairs, Mann–Whitney U test. (C) Percentage of adhesive membrane with cER. n = 26/19 cells. Lines show median values, larger symbols mouse average.

Figure S4.

Effect of Stim1/2 ablation on the ultrastructure of mouse neutrophils. (A) Perimeter and shape factor of WT and Stim1/2^−/− neutrophils fixed in suspension or after adhesion to PLL in the absence or presence of Tg (Adh+Tg). (B) Number of contact sites detected in the indicated conditions along the entire cell perimeter (left) and on the adhesive and nonadhesive membranes (middle and right). (C) cER length and gap distance at adhesive PM of WT and Stim1/2^−/− neutrophils. n = 26/19 cells with 40/60 cER sheets from two mice pairs, Mann–Whitney U test. (D) cER proportion, length, and gap distance in adherent WT and Stim1/2^−/− neutrophils treated with Tg. n = 25/25 cells with 139/209 cER sheets pfrom two mice pairs, Mann–Whitney U test. Adh: adherent; Susp, suspended.

Figure S5.

Effect of Stim1/2 ablation on Ca ²⁺ handling proteins and actin dynamics. (A) Representative micrographs of PLAs between the indicated molecular targets in WT and Stim1/2-deficient neutrophils and in the absence of primary antibodies (left, scale bars: 10 µm) and quantification of the interactions (right, n = 11/10, 10/10 and 3/3 micrographs with >10 cells for each condition from 2/2/1 mice pairs, Mann–Whitney U test). (B) Confocal micrographs of WT and Stim1/2^−/− neutrophils plated on PLL for 30 min, fixed and stained with an anti-PMCA antibody (left, nuclei stained blue with DAPI) and averaged cell-associated fluorescence area (right, n = 32/27 cells from one mice pair, Student’s unpaired t test). (C) Basal Salsa6f ratio of WT and Stim1/2-deficient neutrophils recorded with two different imaging cameras. n = 108/236 and 37/87 cells from three mice pairs. Lines show median values, larger symbols mouse average. Data from camera 1 were converted to [Ca²⁺]_cyt in Fig. 6 B. (D) Proportion of WT neutrophils exhibiting Ca²⁺ transients before and after switching from 2 to 10 mM [Ca²⁺]_ext (left) and quantification of the spreading area of WT neutrophils adhered in 2 and 10 mM [Ca²⁺]_ext (right, n = 22/33 cells in 4/5 recordings from two WT/Stim1/2^−/− mice pairs, exact two-tailed Mann–Whitney U test on cell values).

Figure 6.

Effect of Stim1/2 ablation on IP ₃ R expression and basal [Ca ²⁺ ] _cyt levels and effect of Ca ²⁺ supplementation on Ca ²⁺ activity and spreading of Stim1/2-deficient neutrophils. (A) Representative z-stack maximal intensity projection micrographs of PLAs between IP₃R3 and the PM Ca²⁺ pump PMCA in WT and Stim1/2-deficient purified neutrophils (left, red dots on the confocal micrographs, nuclei stained blue with DAPI, scale bars: 10 µm) and quantification of the interactions (right, n = 10/10 micrographs with >10 cells for each condition from two mice pairs, Mann–Whitney U test). (B) Western blot showing IP₃R1 protein levels in WT and Stim1/2-deficient purified neutrophils. (C) Representative immuno-electron micrographs (left) of WT and Stim1/2-deficient purified neutrophils exposed to an anti-IP₃R1 antibody coupled to 10-nm-diameter gold particles (arrows) and quantification of the number of gold particles in the cytosol and in regions <50 nm from the PM in WT and Stim1/2^KO neutrophils (right, n = 20/20 cells from one pair of mice, exact two-tailed Mann–Whitney U test. Scale bar: 500 nm). (D) Resting cytosolic Ca²⁺ concentration of WT and Stim1/2-deficient flushed neutrophils, measured with Salsa6f in non-spiking cells. Salsa6f ratios were converted to [Ca²⁺]_cyt using the calibration curve of Fig. 1 G. n = 108/230 cells from two pairs of mice, unpaired two-tailed t test on cell values, lines are median values, larger symbols mouse average. (E) Salsa6f recordings (left), basal Ca²⁺ levels (middle), and proportion of flushed Stim1/2^KO neutrophils exhibiting Ca²⁺ transients before and after switching from 2 to 10 mM [Ca²⁺]_ext (right, n = 151 cells in eight recordings from two pairs of mice, Student’s paired two-tailed t test). (F) Change in the size of CellMask TIRF footprints of flushed Stim1/2^KO neutrophils adhering to PLL-coated glass in 2 and 10 mM [Ca²⁺]_ext (left) and quantification of the spreading area (right, n = 22/33 cells in 4/5 recordings from 2 WT/Stim1/2^KO mice pairs, exact two-tailed Mann–Whitney U test on cell values). Source data are available for this figure: SourceData F6.

Figure S6.

The effect of Stim1/2 ablation on IP ₃ R mRNA levels and on neutrophil actin dynamics, related to Fig. 6 . (A) Real-time qPCR of neutrophils purified from WT and Stim1/2-deficient with primers designed against the indicated targets. n = 3 mice pairs, paired t test. (B) Quantification of adhesive membranes length (left) and actin coating thickness (right) in the electron micrographs of Fig. 7 A. n = 26/19 cells from two mice pairs, exact two-tailed Mann–Whitney U test. (C) Sir-actin kymographs of WT and Stim1/2-deficient neutrophils during spreading. Dotted lines on micrographs at left indicate the kymograph axis. Scale bar: 10 µm. (D) Representative micrographs of spread WT and Stim1/2-deficient neutrophils stained with phalloidin (left) and quantification of phalloidin staining intensity (right, n = 44/45 cells in four experiments from two mice pairs, exact two-tailed Mann–Whitney U test).

Figure 7.

Effect of Stim1/2 ablation on actin dynamics at neutrophils adhesion sites. (A) Representative electron micrographs of purified WT and Stim1/2^KO adherent neutrophils (left) and quantification of the organelle-free area (presumably rich in actin) covering their adhesive membranes (right, n = 26/19 cells from two mice pairs, exact two-tailed Mann–Whitney U test). Adhesive (Adh) and nonadhesive membranes are outlined in white and green, respectively, cytosolic pad area in violet. Scale bar: 1 µm. Micrograph of Stim1/2^KO neutrophil is duplicated from Fig. 5 A. (B) Time-lapse micrographs of flushed WT and Stim1/2-deficient neutrophils stained with the cell-permeable F-actin probe SiR-actin (left, Video 10 and Video 11) and change in SiR-actin fluorescence intensity during spreading (right, n = 31/18 cells from 2 WT/Stim1/2^KO mice pairs, exact two-tailed Mann–Whitney U test). Scale bars: 5 µm. AUC, area under the curve.

Figure 8.

Effect of Stim1/2 ablation on neutrophil extravasation during inflammation. (A) Fluorescence micrograph of cremaster tissue from WT and Stim1/2^KO mice, injected i.p. with 2.5 µg/ml TNF for 2 h prior to sacrifice and surgical extraction of the cremaster muscles. Granulocytes are stained white with GR-1, blood vessels blue with CD31 antibodies. (B) Quantification of interstitial GR-1⁺ cells within a distance of 75 µm from postcapillary venules in tissue sections from WT and Stim1/2^KO mice. n = 57/60 vessels from four mice pairs, exact two-tailed Mann–Whitney U test. Small and large symbols show data from individual vessels and mouse average, lines show median values by vessel.