Panel A shows fluorescence lifetime imaging of Halo-Flipper and ManII-Halo (JF646) in Dimethyl sulfoxide and Latrunculin A treated cells, with zoomed regions and a fluorescence lifetime color scale. Panel B shows a superplot of Halo-Flipper lifetime (nanoseconds), with the x-axis labeled Control and 10 micromolar Latrunculin A and the y-axis labeled Halo-Flipper lifetime (nanoseconds). Panel C shows fluorescence lifetime imaging of Halo-Flipper and ManII-Halo (JF646) during 0.5 hour, 1 hour, 2 hours, 3 hours, and 4 hours of cell spreading, with zoomed regions and a fluorescence lifetime color scale. Panel D shows a superplot of Halo-Flipper lifetime (nanoseconds), with the x-axis labeled cell spreading time and the y-axis labeled Halo-Flipper lifetime (nanoseconds). Panel E shows fluorescence lifetime imaging of Halo-Flipper and ManII-Halo (JF646) on substrates of 2 kilopascals, 30 kilopascals, and Glass, with zoomed regions and a fluorescence lifetime color scale. Panel F shows a superplot of Halo-Flipper lifetime (nanoseconds), with the x-axis labeled substrate stiffness and the y-axis labeled Halo-Flipper lifetime (nanoseconds).
Golgi membrane tension responds to mechanical forces. (A) Images of live RPE1-ManII-Halo stably expressing cells before and after treatment with 50 nM Latrunculin A. The FLIM signal is displayed. The Golgi apparatus was post-labeled using JF-646. Higher magnification is shown on the right. (B) SuperPlot of FLIM value expressed as fluorescence lifetime in ns was quantified for each experimental condition (n = 3 replicates; N > 15 cells per replicate). A two-sided parametric ratio paired t test was used. The P value is indicated in the plot. (C) Images of live RPE1-ManII-Halo stably expressing cells spread for 30 min to 4 h on FN-coated glass coverslips. The Flipper average lifetime obtained by FLIM is displayed (color scale). To specifically measure FLIM at the Golgi, ManII-Halo was post-labeled with JF-646 (intensity in gray scale shown). For each time point, a higher magnification of the boxed area is shown on the right. (D) FLIM value expressed as Flipper average lifetime (in ns) in the Golgi-positive area (delineated by the dashed white lines, zoom in images) was quantified at the indicated time points. Statistical analysis was performed using a mixed-effects model (REML) allowing for missing data points assuming sphericity and matching across biological replicates, correcting for multiple comparisons using Dunnett’s test (n ≥ 3 replicates; N ≥ 10 cells per replicate). (E) Images of live RPE1-ManII-Halo stably expressing cells plated on FN-coated glass coverslips, or stiff (30 kPa) or soft (2 kPa) FN-coated PAA gels. Flipper lifetime was measured as in C. The Golgi apparatus was post-labeled using JF-646 (gray scale). (F) Flipper average lifetime in the Golgi-positive area was quantified and shown for the different conditions. Repeated-measures one-way ANOVA tests were performed using Tukey’s post hoc multiple comparison test (n = 3 replicates; N∼30 cells per replicate). All scale bars, 30 µm.