Figure 3
From: Acetyl-CoA flux from the cytosol to the ER regulates engagement and quality of the secretory pathway
Aberrant AT-1 models demonstrate altered protein trafficking through the secretory pathway. (a) Schematic of three experiments used to assess protein trafficking in the secretory pathway. Experiment 1 assesses protein-laden carriers following release from the ER; solubilizer-dependent cargo fluoresces for visualization. Experiment 2 quantifies the time for ER cargo to accumulate in the Golgi apparatus and then egress; solubilizer-dependent DsRed cargo traffics to GFP-labeled Golgi. Experiment 3 quantifies the time for ER cargo to reach the cell surface; solubilizer-dependent cargo with a HaloTag ligand is tracked for 70 min with HaloTag cell impermeable dye added prior to imaging. (b,c) Maximum speed of cargo release (b), and the percentage of cargo release (c), are tracked for 3 min after solubilizer induced cargo release in MEFs (MEFs from multiple cells from 3 biologically independent animals for each genotype n = 14 WT; n = 20 AT-1 sTg; n = 18 AT-1S113R/+; MEF cells from 3 biologically independent animals for each group for (b,c). Student’s T-test. (d) Representative cargo (red) accumulation in the Golgi (green) every 2 min, for 30 min in MEFs from WT, AT-1 sTg, and AT-1S113R/+ (scale bar, 40 µm). (e) Cargo accumulation in the Golgi over 30 min in primary-cultured MEFs (MEFs from multiple cells from 3 biologically independent animals for each genotype at each time point, n = 27 WT; n = 22 AT-1 sTg; n = 23 AT-1S113R/+). Mean with SEM are represented at each time point. (f) Representative surface trafficking of L1CAM in MEFs from WT, AT-1 sTg, and AT-1S113R/+ (scale bar, 40 µm). (g) L1CAM intensity at the cell surface in MEFs (MEFs from multiple cells from 3 biologically independent animals for each genotype at each time point, n = 42–76 WT; n = 10–27 AT-1 sTg; n = 19–34 AT-1S113R/+). Mean with SEM are represented at each time point. Two-way ANOVA with multiple comparisons at each time point for (e,g). Purple and orange statistics indicate differences between AT-1 sTg and WT, and between AT-1S113R/+ and WT, respectively. Grey statistics indicate difference between AT-1 sTg and AT-1S113R/+. *P < 0.05; **P < 0.005; #P < 0.0001.
