Panel A shows a schematic timeline of cell seeding on Fibronectin or Poly L Lysine, cargo release from the Endoplasmic Reticulum, D/D plus Cycloheximide treatment, fixation, and total spreading time at 37 degrees Celsius. Panel B shows fluorescence images of Green Fluorescent Protein membrane, mKate2 FM4 PAUF, and merged images after 1 hour and 4 hours on Fibronectin or Poly L Lysine. Panel C shows a superplot of the number of CARTS per cell, with the x-axis labeled 1 hour, 1.5 hours, 2 hours, 4 hours, 24 hours on Fibronectin and Poly L Lysine, and the y-axis labeled Number of CARTS per cell. Panel D shows a superplot of cell adhesion area (square micrometers), with the x-axis labeled 1 hour, 1.5 hours, 2 hours, 4 hours, 24 hours on Fibronectin and Poly L Lysine, and the y-axis labeled Cell adhesion area (square micrometers). Panel E shows a superplot of the number of CARTS per cell adhesion area (square micrometers), with the x-axis labeled 1 hour, 1.5 hours, 2 hours, 4 hours, 24 hours on Fibronectin and Poly L Lysine, and the y-axis labeled Number of CARTS per cell adhesion area (square micrometers). Panel F shows a superplot of the number of CARTS per cell versus cell adhesion area (square micrometers) for cells on Fibronectin at different time points. Panel G shows fluorescence images of CD59 RUSH after 45 minutes Biotin treatment on 2 kilopascal, 30 kilopascal, and Glass substrates. Panel H shows a superplot of the number of CD59 positive vesicles per cell, with the x-axis labeled 2 kilopascal, 30 kilopascal, Glass and the y-axis labeled Number of CD59 positive vesicles per cell.
Golgi-derived carrier biogenesis scales with cell adhesion and FA formation. (A) Schematic representation of the spreading assay. D/D is D/D solubilizer, which dissolves mKate2-FM4-PAUF aggregates, allowing synchronized cargo release from the ER. (B) Representative images of fixed HeLa cells transfected with GFP-mem and the mKate2-FM4-PAUF, seeded over FN or PLL, and acquired by confocal microscopy after the indicated times. Scale bars, 10 µm. (C–E) SuperPlots showing individual cell measurements (small, light-colored symbols; N∼10 per biological replicate) and the mean value for each independent biological replicate (larger, black-outlined circles; n = 6 on FN, n = 3 on PLL). Each color represents a different experimental replicate. Plots represent (C) the number of CARTS per cell, (D) the cell adhesion area, and (E) the number of CARTS per cell adhesion area. Repeated-measures two-way ANOVA tests were performed, and P values were obtained using Tukey’s post hoc multiple comparison test. (F) Correlation plot showing the number of CARTS per cell and the corresponding cell adhesion area per each measured individual cell across all tested conditions (see legend, cells seeded on FN only). Normality of both variables was tested using the Shapiro–Wilk test, and the results showed non-normality. Each point represents an individual cell. A nonparametric Spearman correlation analysis was performed, with the correlation coefficient being ρ = 0.37 and P value <0.0001. (G) Spinning disk confocal microscopy images of fixed HeLa cells stably expressing RUSH-EGFP-CD59 plated on FN-coated glass coverslips, or on stiff (30 kPa) or soft (2 kPa) FN-coated PAA gels. 4 h after plating them, cells were incubated for 45 min with biotin before fixation to specifically visualize CD59-positive post-Golgi carriers. (H) SuperPlot showing quantification of the number of CD59-positive vesicles per cell in cells seeded on substrates of various stiffness as described in G, and a repeated-measures one-way ANOVA test was performed using Tukey’s post hoc multiple comparison test (N = 2–10, n = 3).