Panel A shows fluorescence microscopy images of PAUF-mRFP in Dimethyl sulfoxide and CRT0066101 treated cells. Panel B shows a superplot of Number of CARTS per cell, with the x-axis labeled Dimethyl sulfoxide and CRT0066101, and the y-axis labeled Number of CARTS per cell. Panel C shows fluorescence microscopy images of Green Fluorescent Protein-membrane in Dimethyl sulfoxide and CRT0066101 treated cells after 1 hour and 4 hours on Fibronectin, with cell diameter measurements indicated. Panel D shows a superplot of Cell adhesion area (square micrometers), with the x-axis labeled Dimethyl sulfoxide and CRT0066101 at 1 hour and 4 hours, and the y-axis labeled Cell adhesion area (square micrometers). Panel E shows fluorescence lifetime imaging microscopy images of Halo-Flipper (Lifetime) and ManII-Halo (JF646) in Dimethyl sulfoxide and CRT0066101 treated cells, including enlarged views and a color scale for Halo-Flipper lifetime (nanoseconds). Panel F shows a superplot of Halo-Flipper lifetime (nanoseconds), with the x-axis labeled Dimethyl sulfoxide and CRT0066101, and the y-axis labeled Halo-Flipper lifetime (nanoseconds). Panel G shows fluorescence microscopy images of Green Fluorescent Protein-membrane in HeLa wild type and HeLa TGN46 knockout cells after 1 hour, 4 hours, and 24 hours on Fibronectin. Panel H shows a superplot of Cell adhesion area (square micrometers), with the x-axis labeled Wild type and TGN46 knockout at 1 hour, 4 hours, and 24 hours, and the y-axis labeled Cell adhesion area (square micrometers). Panel I shows a schematic diagram illustrating interactions among Focal adhesion assembly and cell spreading, Trafficking to focal adhesions, Transport carrier biogenesis, Extracellular mechanical cues, Golgi diacylglycerol, Protein kinase D, Tubulin acetylation, Increased Golgi tension, Poly-L-lysine, CRT0066101, and Tubacin.
Golgi-derived export is necessary for cell spreading and mechanoadaptation. (A) HeLa cells transfected with the PAUF-RFP plasmid were spread on FN for 4 h and subjected to a treatment with 5 µM or without CRT0066101, a PKD inhibitor, fixed, and imaged by confocal microscopy. Scale bars, 10 µm. (B) SuperPlot showing the number of CARTS per cell for each experimental condition. A two-sided parametric ratio paired t test was used (n = 3 replicates; N∼10 cells per replicate). (C) Representative confocal microscopy images of fixed HeLa cells transfected with the GFP-mem plasmid, subjected or not to a treatment with 5 µM CRT0066101. Scale bars, 10 µm; representative cell length scales shown. (D) SuperPlots showing individual cell measurements (N∼10 per biological replicate; n = 3 biological replicates). A repeated-measures two-way ANOVA test using Fisher’s LSD test was performed. (E) Images of live RPE1-ManII-Halo stably expressing cells before and after treatment with 5 µM CRT0066101. The FLIM signal and lifetime are displayed. The Golgi apparatus was post-labeled using JF-646. Scale bars, 30 µm. Higher magnification of the boxed areas is shown on the right. (F) SuperPlot showing Halo-Flipper lifetime values in the Golgi-positive area for each experimental condition (n = 3 replicates; N∼10 cells per replicate). A two-sided parametric ratio paired t test was used. The P value is indicated in the plot. (G) Representative confocal microscopy images of fixed HeLa cells (WT or TGN46 KO), transfected with the GFP-mem plasmid, and seeded over FN for the indicated times. Scale bars, 10 µm. (H) SuperPlot showing individual cell measurements (N∼10 per biological replicate; n = 3 biological replicates) quantifying adhesion area from images in G. A repeated-measures two-way ANOVA test was performed. P values using Tukey’s post hoc multiple comparison test are reported. (I) Schematic representation of our findings (see text for details).