Figure 4. ACBD5/VAPB interaction... | Rockefeller University Press

Figure 4.

$Figure 4. ACBD5/VAPB interaction influences PO migration in gradients and PO motility in human fibroblasts. (A) ACBD5/VAPB coexpression alters PO distribution in density gradients. PO-enriched fractions, prepared from HepG2 cells (Control) and cells cotransfected with GFP-rACBD5 and Myc-VAPB, were separated in continuous Nycodenz-gradients. Distribution of organelle markers was assessed by immunoblotting of fractions. Coexpression of ACBD5 and VAPB shifts POs to lower densities, similar to ER markers (compare boxed regions). Pex14 (PO); ACOX1, acyl-CoA oxidase 1 (PO); ATP synt. a, ATP synthase α subunit (MITO); PDI, protein disulfide isomerase (ER). (B–H) Loss of ACBD5 increases PO movement. Human fibroblasts were treated with control (Cont) or ACBD5 siRNA, transfected with GFP-PTS1, and analyzed by live cell imaging (Videos 1 and 2). (B–D) Trajectory plots. 100 PO trajectories were retrieved for each condition and the first 20 time frames plotted starting at a center. (E–G) Density plots. The x and y coordinates of all trajectories ≥20 time frames were pooled and binned in the interval −3,3 µm in x and y directions, using 50 bins. The log-scaled 2D histogram of these points was plotted using “jet” color map. (H) ECDF plots. Instantaneous trajectory speed profiles were estimated by calculating distance moved between each time point in the trajectory. These speeds were pooled and converted to an empirical cumulative distribution function (ECDF). By pooling speeds for all datasets for a given condition, a single ECDF was generated for each (minimum of 38,175 trajectories from 24 videos per condition).$

ACBD5/VAPB interaction influences PO migration in gradients and PO motility in human fibroblasts. (A) ACBD5/VAPB coexpression alters PO distribution in density gradients. PO-enriched fractions, prepared from HepG2 cells (Control) and cells cotransfected with GFP-rACBD5 and Myc-VAPB, were separated in continuous Nycodenz-gradients. Distribution of organelle markers was assessed by immunoblotting of fractions. Coexpression of ACBD5 and VAPB shifts POs to lower densities, similar to ER markers (compare boxed regions). Pex14 (PO); ACOX1, acyl-CoA oxidase 1 (PO); ATP synt. a, ATP synthase α subunit (MITO); PDI, protein disulfide isomerase (ER). (B–H) Loss of ACBD5 increases PO movement. Human fibroblasts were treated with control (Cont) or ACBD5 siRNA, transfected with GFP-PTS1, and analyzed by live cell imaging (Videos 1 and 2). (B–D) Trajectory plots. 100 PO trajectories were retrieved for each condition and the first 20 time frames plotted starting at a center. (E–G) Density plots. The x and y coordinates of all trajectories ≥20 time frames were pooled and binned in the interval −3,3 µm in x and y directions, using 50 bins. The log-scaled 2D histogram of these points was plotted using “jet” color map. (H) ECDF plots. Instantaneous trajectory speed profiles were estimated by calculating distance moved between each time point in the trajectory. These speeds were pooled and converted to an empirical cumulative distribution function (ECDF). By pooling speeds for all datasets for a given condition, a single ECDF was generated for each (minimum of 38,175 trajectories from 24 videos per condition).