Fig. 9

Both FAK HPs can be effectively targeted with a single LD motif in the form of a dimer. a) Schematic representation of LD2 (composed of 420–440 amino acids of paxillin), LD4 (composed of 783–845 amino acids of paxillin), LD2-LD2 (composed of two LD2 motifs linked together through a flexible 30 amino acid linker) and LD4-LD4 (composed of two LD4 motifs linked together through a flexible 30 amino acid linker) constructs, fused to GFP. b) Representative Western Blots showing expression of stable proteins encoding GFP fused LD2 and LD4 (expected molecular weight ~ 31 kDa) in the left panel and GFP fused LD2-LD2 and LD4-LD4 (expected molecular weight ~ 35 kDa) in the right panel. c) Widefield images of HeLa cells, transiently transfected with GFP-fused LD2, LD4, LD2-LD2 or LD4-LD4 and immunostained for FAK and Vinculin. Expressing cells are marked with an asterisk. GFP LD2 and GFP LD4 expressing cells display strong localization of FAK at FAs, similarly to control cells. GFP LD2-LD2 and GFP LD4-LD4 expressing display reduction in FA-localized FAK, compared to control cells. d) Quantification of the % change in the mean FAK/Vinculin intensity at FAs in control cells (100 ± 8.43, n = 388 FAs from 25 cells) and cells expressing GFP LD2 (83 ± 6.97, n = 276 FAs from 25 cells), GFP LD4 (76.54 ± 12.88, n = 441 FAs from 32 cells) or GFP LD2-LD4 (22.51 ± 0.9, n = 406 FAs from 30 cells). Neither GFP LD2 nor GFP LD4 displace FAK from FAs, unlike GFP LD2-LD4. e) Quantification of the % change in the mean FAK/Vinculin intensity at FAs in control cells (100 ± 2.64, n = 542 FAs from 30 cells) and cells expressing GFP LD2-LD2 (35.18 ± 1.75, n = 334 FAs from 27 cells), GFP LD4-LD4 (28.37 ± 1.31, n = 322 FAs from 28 cells) or GFP LD2-LD4 (32.61 ± 1.76, n = 276 FAs from 27 cells). Both GFP LD2-LD2 and GFP LD4-LD4 displace FAK from FAs, as efficiently as GFP LD2-LD4. f) Expression of GFP LD2-LD2 or GFP LD4-LD4 leads to decrease in the migration rate of MDA MB-231 cells (57.99 ± 4.87, n = 73 cells expressing GFP LD2-LD4 and 37.52 ± 4.54, n = 33 cells expressing GFP LD4-LD4, compared to 100.0 ± 4.038, n = 89 control cells), similarly to GFP LD2-LD4 does (49.84 ± 5.43, n = 49 cells). Scale bars: 10 μm. The error bars represent standard error of the mean (S.E.M). ***; p < 0.0001