Figure 3.

SNX3 binds to PI(3)P and colocalizes with PI(3)P at B. burgdorferi–containing phagosomes. (A–L) Confocal micrographs of macrophages coexpressing fluorescent probes for PI(3)P (A), PI(3,4)P₂ (E), or PI(4)P (I), and coexpressing RFP-SNX3 (B, F, and J), with internalized borreliae stained by antibodies (C, G, and K). Yellow boxes indicate areas shown enlarged as insets. Arrows indicate enrichments of PI(3)P and RFP-SNX3. Scale bars: 10 µm, and 1 µm for insets. (M and N) Binding of GST-fused SNX3 WT and SNX3-Y71A mutant on phosphatidylinositol-spotted strips incubated with GST alone or GST-SNX3 WT or GST-SNX3-Y71A mutant and developed with anti-GST antibody (M) or anti-SNX3 antibody (N). LPA, lysophosphatidic acid; LPC, lysophosphocholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; S1P, sphingosine 1-phosphate. (O–T) PI3K activity regulates compaction of borreliae. (O, P, R, and S) Confocal micrographs of macrophages expressing PI(3)P sensor p40phox-GFP (O and P) or GFP-SNX3 (R and S), with internalized borreliae stained by antibodies, with merges. Cells were treated with DMSO (0.1%) as control (O and R) or with PI3K inhibitor Wortmannin (1 µM; P and S); yellow boxes indicate areas shown enlarged as insets. Scale bars: 10 µm, and 1 µm for insets. (Q and T) Statistical evaluation of borreliae compaction in cells expressing p40phox-GFP (Q) or of GFP-SNX3 localization to borreliae phagosomes (T); cells were treated with Wortmannin or DMSO as control. n = 3 (3 × 30 cells). For specific values, see Table S1. Two-tailed Student’s t test; *, P < 0.05; ***, P < 0.001. Error bars: mean ± SEM.