The GEF Tuba is specifically implicated in linear invadosome formation. (A and B) From left to right, MDA-MB-231 and A549 cells were transfected with control (siCT) or two independent Tuba siRNAs (Tuba #1 and #2). Tuba protein expression was analyzed by immunoblotting. GAPDH was used as a loading control. The graphs show quantification of the percentage of cells able to form linear invadosomes. Error bars represent the SEM (n = 900, three independent experiments; ***, P < 0.001 as compared with the control siRNA condition). (C) MDA-MB-231 cells were treated as in A and seeded for 4 h on collagen I. Shown are representative confocal images of MDA-MB-231 cells. Tks5 (green) and F-actin (red) are shown. Panels on the right show enlarged views of the boxed regions. Similar results were obtained with siTuba #2. Tuba extinction decreases the capacity of cells to form linear invadosomes. (D) MDA-MB-231 cells stably expressing DDR1-GFP were seeded for 4 h on collagen I and fixed. Immunofluorescence with endogenous Tuba (green) and DDR1-GFP (red) was performed and reveals a colocalization of the GEF Tuba with DDR1. Right panels show enlarged views of the boxed region. Correlation coefficient of colocalization: DDR1/Tuba r = 0.15 (n = 10). (E) MDA-MB-231 cells were seeded on gelatin (top) or mixed matrix (collagen I/gelatin) for 24 h and 4 h, respectively, then processed for immunofluorescence staining (cortactin, red; Tuba, green). While Tuba is not present on classical invadosomes, it colocalizes with cortactin on linear invadosomes. Bars: (C, left) 5 µm; (C, enlarged panels on the right) 2 µm; (D, left) 10 µm; (D, enlarged panels on the right) 3 µm; (E) 3 µm.