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. 2015 Jan 29;5:8124. doi: 10.1038/srep08124

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Copyright © 2015, Macmillan Publishers Limited. All rights reserved

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(A,D) Left, Montages of time-lapse TIRF images for an assembling (panel A) and a disassembling (panel D) adhesion (white arrows) in cells expressing GFP-paxillin are shown in pseudo-color coding. Warm colors correspond to higher fluorescence intensity values whereas cool colors represent lower fluorescence intensity values. Right, Outlines of the adhesions as segmented by PAASTA. (B,E) Graphs of the fluorescence intensities for the assembling (panel B) and disassembling (panel E) adhesion are shown for both Manual and PAASTA tracking. (C,F) Plots of the natural log of the fluorescence intensity of the adhesions at given time points (I) relative to the initial fluorescence intensity (I₀) are shown. Trendlines with the corresponding equation (y = mx + b) and R² values are shown for fluorescence intensities attained manually (Manual) and with PAASTA. The slopes of these graphs (m) are the apparent rate constant for adhesion assembly (panel C) or the rate constant for adhesion disassembly (panel F). (G) The average t_1/2 values for adhesion assembly and disassembly for cells expressing GFP-paxillin (FL-Pax) or GFP-paxillin with a truncated CMV promoter (Spec-Pax) are shown for Manual and PAASTA tracking. S.E.M. was calculated from: 21–24 adhesions (11–14 assembly, 10 disassembly) for each construct. A total of 14 cells were analyzed for adhesion assembly and disassembly. (H) The average t_1/2 values for adhesion assembly and disassembly for cells expressing GFP-vinculin (FL-Vinc) or GFP-vinculin with a truncated CMV promoter (Spec-Vinc) are shown for Manual and PAASTA tracking. S.E.M. was calculated from: 20–21 adhesions (10–11 assembly, 10 disassembly) for each construct. A total of 13 cells were analyzed for adhesion assembly and disassembly. A Wilcoxon rank sum test showed no statistically significant difference between Manual and PAASTA tracking for FL-Pax assembly (Z = −1.689, p = 0.091), disassembly (Z = −0.153, p = 0.878); Spec-Pax assembly (Z = −0.941, p = 0.347), disassembly (Z = −1.580, p = 0.114); FL-Vinc assembly (Z = −0.561, p = 0.575), disassembly (Z = −0.255, p = 0.799); or Spec-Vinc assembly (Z = −1.122, p = 0.262), disassembly (Z = −1.172, p = 0.241). For panels A and D, Bar, 1 μm.