Fig. 7

STIM1 promotes phagosomal proteolysis and endomembrane fusion. a Proteolysis was measured in BMDCs exposed to DQ-OVA-Alexa-568 beads. STIM1-deficient cells showed lower levels of DQ-OVA fluorescence at 90 min (right panel). Pre-incubation with BAPTA-AM (40 μM) reduced WT proteolysis to levels similar to STIM1-deficient cells. Lines representing the BAPTA condition are omitted and 90 min points are displaced to the right for clarity. ConcA (0.2 nM) only partially inhibited proteolysis. N = 5/10/3/5/3 (coverslips) comprising a total of 236/1050/237/432/200 (WT) or 264/975/218/405/217 (Stim1 ^−/−) cells for 30/90/30 + ConcA/90 + ConcA/90 + BAPTA(min). b OVA degradation was measured with anti-OVA immunostainings of isolated phagosomes by flow cytometry. Values are % OVA degradation. Full gating strategy is shown in Supplementary Fig. 7a. Proteolysis was decreased at 30 and 60 min after ingestion. N = 3. c Phago–lysosome (P–L) fusion was measured by exposing Alexa-488-OVAb to cells loaded with lysosomal FRET acceptor Alexa-594-HA. Colour-coded images (left) show the FRET signal at 90 min for WT and STIM1-deficient cells. P–L fusion indices are matched to the colour-coded bar. P–L fusion was decreased in STIM1-deficient cells as compared to WT at 90 min, whereas BAPTA-AM loading further decreased P–L fusion and eliminated differences between WT and Stim1 ^−/− cells. ConcA decreased P–L fusion to similar levels as BAPTA-loaded cells. N = 3/4/4/3 (coverslips) comprising 470/1009/1691/797 (WT) or 643/1314/1617/503 (Stim1 ^−/−) phagosomes for 30/90/90 + ConcA/90 + BAPTA (min). d Phago–endosome (P–E) fusion was measured by exposing Alexa-488-OVAb to cells loaded with endosomal FRET acceptor Alexa-594-dextran. P–E fusion was decreased in STIM1-deficient cells as compared to WT. Addition of GSK (10 μM) eliminated differences in P–E fusion. N = 3 (coverslips) comprising a total of 213/244/144/146 (WT) or 210/154/233/253 (Stim1 ^−/−) cells for 15/30/15 + GSK/30 + GSK(min). Phagocytic targets added at 20:1. e Leu-AMC fluorescent substrate cleavage was reduced in phagosomes isolated from STIM1-deficient cells but not in whole-cell lysates, N = 3. f Periphagosomal IRAP, quantified from single confocal slices, was reduced in STIM1-deficient cells. N = 4 (coverslips). White bars = 10 μm. Error bars are means ± SEM. *p < 0.5, **p < 0.01 using a two-way ANOVA and Sidak’s post test for a–d and a Student’s t-test for e and f