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. Author manuscript; available in PMC: 2025 Aug 16.
Published in final edited form as: ACS Chem Biol. 2024 Jul 18;19(8):1683–1694. doi: 10.1021/acschembio.4c00345

Figure 3. An artificial E-Syt1 tethered to ER-mitochondria contact sites redirects IMPACT-derived lipid transport toward mitochondria.

Figure 3.

(A) Domain maps of WT E-Syt1 and engineered constructs wherein a functional (WT-tether) or mutant (Mutant-tether) E-Syt1 lipid transfer module (SMP domain) is tethered at ER-mitochondria contact sites. (B) Confocal microscopy images of HeLa cells transfected with WT and mutant tether and then stained with ER-Tracker Green (ER) and MitoView 405 (Mitochondria), showing colocalization of tether with both organelles. Merged images show overlay of all three channels: ER (cyan), tether (yellow), and mitochondria (magenta), with colocalization appearing as white. (C) Confocal microscopy analysis showing colocalization of RT-IMPACT-derived lipids with mitochondria. HeLa cells were transfected with WT-tether (top row), Mutant-tether (middle row), or no transfection (bottom row). After 24 h, cells were labeled via IMPACT by treatment with PMA and oxoTCO in the presence of HaloTag-JF635 and MitoView 405 to label WT-tether and stain mitochondria, respectively, for 5 min. After a brief rinse, cells were labeled with Tz-BODIPY for 1 min, followed by immediate imaging. Merged images show overlay of IMPACT (green) and MitoView 405 (magenta), with colocalization appearing as white. Pearson correlation coefficients (R2) were calculated for the two channels in the merged images. (D) Quantification of effects of WT-tether and Mutant-tether expression on trafficking of IMPACT-derived fluorescent lipids to mitochondria. Scatter plot shows quantification of Pearson coefficients from (C). For the WT-tether and Mutant-tether groups, n = 25–46 individual transfected cells from three dishes acquired on separate days. For the nontransfected group, each data point represents the average fluorescence of 15–30 cells from a single dish (n = 17 dishes). Shown are mean and standard deviation. One-way ANOVA, Games-Howell post hoc test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Scale bars: 10 μm.