Journal: The Journal of Experimental Medicine
Article Title: Mutations in the exocyst component EXOC2 cause severe defects in human brain development
doi: 10.1084/jem.20192040
Figure Lengend Snippet: Impaired plasma membrane trafficking in fibroblasts from affected patients is rescued by lentiviral transduction of wild-type EXOC2 . (A) Representative 3D kymographs showing vesicle fusions over time in the entire cell for each condition indicated. Images were recorded over 2 min with 180-ms intervals and 70-ms exposure using TIRFM. (B) Vesicle fusion rates calculated for control (C1), Patient 2 (P2), and Patient 3 (P3) fibroblasts as fusion per unit area (µm 2 ) per second. Experiments were replicated at least three times, and a representative experiment is shown. Error bars indicate IQR, and line indicates median. P values were calculated using one-way ANOVA and Tukey post hoc analysis. P value for the statistics between C1 and P2 is 0.048; C1 to P3 is 0.423. Total number of fusions per cell in control, Patient 2, and Patient 3 fibroblasts was plotted against cell surface area. (C) Expression level of lentivirally expressed EXOC2 protein in fibroblasts from Patient 2 compared with wild-type cells. (D) Vesicle fusion rates calculated for Patient 2 fibroblasts that were mock transfected or overexpressing EXOC2. P value was calculated using Student t test. Experiments were repeated three times, and pooled data are shown. P = 0.0094. (E) Control fibroblasts and fibroblasts derived from an affected individual from Family 1 and Family 2 were transfected with a VSVG-GFP ts045, then immunostained with the VSVG surface marker 8G5 and counterstained with the nuclear marker DAPI. VSVG-GFP ts045 relative intensity to 8G5 was calculated to represent exocytosis. There was a significant decrease in the exocytosis rate in fibroblasts from Patient 2 (P2) and Patient 3 (P3) compared with control (C1) fibroblasts. (F) Representative images of untreated and EXOC2-rescued fibroblasts from Patient 2: blue, DAPI; green, VSVG-GFP ts045; red, 8G5. (G) Control fibroblasts were either untreated or stably transduced with wild-type EXOC2 lentivirus (EXOC2). There was no difference in exocytosis between naive control cells and those transduced with EXOC2 lentivirus. (H) Exocytosis in fibroblasts from Patient 2 in Family 1 was restored back to levels of control fibroblasts upon transduction with wild-type EXOC2 lentivirus. (I) Exocytosis in fibroblasts from Patient 3 was unchanged upon transduction with wild-type EXOC2 lentivirus; however, endogenous protein levels were normal . Data in E and G–I are median ± IQR. Significance was measured by two-way ANOVA with Sidak multiple comparisons correction. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Scale bar = 20 µm. Representative of three experiments.
Article Snippet: The following day, cells were transfected with the VSVG-GFP ts045 construct (a gift from Jennifer Lippincott-Schwartz, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA; Addgene plasmid #11912) using Lipofectamine 3000 (Thermo Fisher Scientific).
Techniques: Clinical Proteomics, Membrane, Transduction, Control, Expressing, Transfection, Derivative Assay, Marker, Stable Transfection
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