GBF1 Protein Levels and Phosphorylation Control its Function in VWF Golgi Trafficking(A) Representative blot showing the amount of GBF1 protein in cells treated with varying levels of siRNA targeting GBF1 (applies to experiments shown in (B)–(I)).(B) Quantification of the amount of GBF1 protein i...
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GBF1 Protein Levels and Phosphorylation Control its Function in VWF Golgi Trafficking(A) Representative blot showing the amount of GBF1 protein in cells treated with varying levels of siRNA targeting GBF1 (applies to experiments shown in (B)–(I)).(B) Quantification of the amount of GBF1 protein in cells treated with varying levels of siRNA targeting GBF1 (quantification of WB). n = 3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗∗∗p < 0.0001.(C) Mean number of WPB per cell. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗∗∗p < 0.0001.(D) Mean number of VWF quanta per cell. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.(E) Proportion of WPBs longer than 2 μm in the entire WPB population. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.(F) Amount of unprocessed VWF, quantified by image analysis (see STAR Methods). n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.(G) Mean number of VWF exocytic sites per cell upon 15 min of histamine stimulation. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗∗∗p < 0.0001.(H) Proportion of secreted VWF from total VWF upon 30 min of histamine stimulation, relative to mock cells. n = 3 independent experiments, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.(I) rtPCR showing changes in mRNA levels for the indicated transcript relative to mock samples.(J) Representative blot showing the varying amounts of GBF1Thr1337 phosphorylation in HUVECs treated with varying concentrations of AICAR for 24 h.(K) Quantification of the amount of GBF1Thr1337 relative to loading control (quantification of WB). n = 2 independent experiments, SD, one-way ANOVA with Dunnett’s multiple comparisons test, ∗p = 0.038, ∗∗p = 0.0021, ∗∗∗p = 0.0003, n.s. = not significant.(L) Quantification of the total amount of GBF1 relative to loading control (quantification of WB). n = 2 independent experiments, SD, one-way ANOVA with Dunnett’s multiple comparisons test, n.s. = not significant.(M) Proportion of WPBs longer than 2 μm in the entire WPB population after 24 h AICAR treatment. n = 5 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗p = 0.0110, ∗∗∗∗p < 0.0001.(N) Control and GBF1-siRNA-treated cells were treated with 2 mM AICAR for 24 h. Proportion of WPBs longer than 2 μm in the entire WPB population. n = 7 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, two-way ANOVA with a Sidak's multiple comparisons test, ∗∗∗∗p = 0.0001, ∗∗∗p = 0.001.(O) The change in WPB size after AICAR treatment in control and GBF1-siRNA-treated cells. n = 7 independent experiments, median (with minimum and maximum), unpaired t test, ∗∗∗ p = 0.0006.(P) Proportion of WPBs longer than 2 μm in the entire WPB population after 24 h of varying glucose treatment. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, one-way ANOVA with Dunnett’s multiple comparisons test, ∗∗∗∗p < 0.0001.(Q) Control and GBF1-siRNA-treated cells were treated with 5 nM (“+”) or 0.1 mM glucose (“−”) for 24 h. Proportion of WPBs longer than 2 μm in the entire WPB population. n = 8 wells, where for each well the mean for each of the 9 fields of view were analyzed, SEM, two-way ANOVA with a Sidak's multiple comparisons test, ∗∗∗∗p = 0.0001, n.s. = not significant.(R) Model of the effect of GBF1 depletion in retrograde membrane retrieval and anterograde cargo trafficking. The levels of GBF1-dependent COPI-mediated Golgi membrane retrieval control the rates of anterograde cargo traffic through the ER-Golgi. Low levels of GBF1 in cells, reduce the amount of COPI vesicles, reducing the rate of Golgi membrane retrieval and hence the rate of Golgi maturation and anterograde trafficking. Phosphorylation of GBF1 via AICAR, 2DG, or glucose starvation increase GBF1 activity resulting in an increase in anterograde trafficking.See also Figure S4.
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