Fig. 2

Expression of LD2-LD3-LD4 leads to the dose-dependent displacement of FAK from FAs without affecting overall FA composition a) Confocal images of methanol/acetone fixed HeLa cells, transiently transfected with GFP LD2-LD3-LD4 and immunostained for FAK and Talin. In control cells, FAK strongly localizes at FAs, labeled with Talin. In cells expressing LD2-LD3-LD4 (marked with asterisk) there is no detectable enrichment of FAK at FAs. In contrast, Talin localization at FAs is unaffected. b-c) Quantification of the % change in the mean FA/cytosolic FAK (b) and Talin (c) intensity ratios. FA/Cytosolic ratio of FAK in control cells is ~ 4.2 fold higher (100 ± 2.86, n = 469 FAs from 30 cells) compared to cells expressing GFP LD2-LD3-LD4 (23.78 ± 1.25, n = 409 FAs from 30 cells), indicating that LD2-LD3-LD4 leads to the displacement of FAK from FAs. In contrast, the FA/cytosolic ratio of Talin is higher in cells expressing GFP LD2-LD3-LD4, possibly due to increased FA size (109.7 ± 3.22 in expressing, compared to 100 ± 2.59 in controls). d) Quantification of the % change in the mean FAK/Talin intensity (based on b and c) reveals a ~ 5-fold drop of FA localized FAK in cells expressing GFP LD2-LD3-LD4 (19.01 ± 0.91 in expressing, compared to 100 ± 4.8 in control cells). e) Dose-dependent displacement of FAK from FAs as indicated by quantification of the % change in the mean FAK/Talin intensity in control compared to expressing cells (high and low) (15.55 ± 1.25, n = 188 FAs from 13 cells, expressing higher, and 28.52 ± 1.14, n = 218 FAs from 17 cells, expressing lower amount of GFP LD2-LD3-LD4, compared to 100 ± 3.1, n = 229 FAs from 28 control cells). To discriminate between high and low expressing cells, we initially determined the mean GFP intensity of all expressing cells, and then compared this to the GFP intensity of individual cells, so as to classify them as high or low expressors. (f-i) Confocal images of PFA-fixed control and GFP LD2-LD3-LD4 (marked with asterisk) HeLa cells immunostained for Vinculin (f), Paxillin (g), av. Integrin (h) and Tensin (i) showing that localization of these proteins is not affected by the expression of GFP LD2-LD3-LD4. (j-m) Corresponding quantification of the % change in the mean FA/cytosolic intensity for each protein presented in f-i. FA/cytosolic ratio of Vinculin (j) and Paxillin (k) is higher in cells expressing GFP LD2-LD3-LD4, possibly due to increased FA size (111.8 ± 2.86, n = 460 FAs from 30 expressing, compared to 100 ± 2.57, n = 455 FAs from 30 control cells for Vinculin; 107.5 ± 2.29, n = 402 FAs from 30 expressing, compared to 100 ± 2.64, n = 432 FAs from 30 control cells for Paxillin). There is no significant difference in the FA/cytosolic ratio of av. Integrin (l) and Tensin (m) (102.5 ± 1.94, n = 568 FAs from 30 expressing, compared to 100 ± 2.16, n = 485 FAs from 30 control cells for av. Integrin; 105 ± 2.24, n = 532 FAs from 30 expressing, compared to 100 ± 2.09, n = 568 FAs from 30 control cells for Tensin). Scale bars: 10 μm. The error bars represent standard error of the mean (S.E.M). ***; p < 0.0001, **; p < 0.005, *; p < 0.05