Fig. 5: Tpm3 is critical for proper microtubule organization and focal adhesion targeting.

a Wide-field images of wild-type and Tpm3 knockout U2OS cells stained for F-actin (phalloidin) and microtubules (α-tubulin antibody). The panels on the right are magnified images of the regions at the cell periphery indicated with black boxes in the whole cell images. Scale bars, 10 µm and 5 µm, respectively. Experiments were repeated three times. b Blinded analysis of the percentage of cells displaying aligned, intermediate and tangled microtubule networks in wild-type (n = 219) and Tpm3 knockout (n = 344) cells. The data are from three independent experiments and represent mean ± SE. c TIRF microscopy images of wild-type U2OS and Tpm3KO cells expressing miRFP670-Paxillin and GFP-α-tubulin demonstrating diminished targeting of microtubules to focal adhesions. Scale bars, 5 µm. d Wide-field images of wild-type and Tpm3 knockout U2OS cells stained for microtubules plus-end tracking protein EB1. The panels on the right (1-4) are magnified images of the regions indicated with black boxes in the whole cell images. Scale bars, 10 µm 5 µm, respectively. Experiments were repeated three times. e TIRF time-lapse images of wild-type and Tpm3 knockout cells expressing EGFP-EB1 and miRFP670-Paxillin. Selected time-lapse frames from the magnified areas (indicated by white boxes) are shown on the right as merged frames. Yellow arrows highlight the point of initial contact of EB1 with focal adhesions. Scale bars, 5 µm and 1 µm, respectively. f Analysis of the EB1 resident times in focal adhesions analysed from TIRF time-lapse movies of cells expressing EGFP-EB1 and miRFP670-Paxillin. Wild-type (n = 40 EB1 foci from 5 movies) and Tpm3 knockout (n = 26 EB1 foci from 5 movies) cells. The data represents mean ± SE. The exact p-value (two-tailed Student’s t-test): p = 0.000015. g Schematic representation of the organization of microtubule plus-ends in wild-type and in Tpm3 knockout cells based on the data presented here.