Respective role of actin filaments and microtubules networks in centrosome positioning. (A–I) Mini-aster positioning in PtK2 cells (A–C and F–I) and cytoplasts (D and E) expressing GFP-tubulin. (A) Images show the actin filaments (magenta), microtubules (green), and centrosome (white) in a non-treated PtK2 cell (left), a cell treated with 6 µM of Nocodazole and 1 µM of Y27632 (middle) or 30 µM of Y27632 (right). (B) Graph representing the distance between the centrosome and the centroid of the cell in the control (n = 40), the highly (n = 40), and poorly (n = 41) contractile cells. (C) Representative live acquisitions depicting the stationary positions of the centrosome and the centroid during the establishment of mini-asters inside PtK2 cells. Generation of a mini-aster in a strongly contractile cell (Nocodazole 6 µM and Y27632 1 µM) (left); generation of a mini-aster inside a poorly contractile cell (Nocodazole 6 µM and Y27632 30 µM) (right). (D) Images show the actin filaments (magenta), microtubules (green), and centrosome (white) in a PtK2 cytoplast (left), a cytoplast treated with 6 µM of Nocodazole and 1 µM of Y27632 (middle) or 20 µM of Y27632 (right). (E) Graph representing the distance between the centrosome and the centroid of the cytoplast in the control (n = 55), the highly (n = 45), and poorly (n = 51) contractile cytoplast conditions. (F) Images show the actin filaments (magenta), microtubules (green), and centrosome (white) in a PtK2 cell treated with Nocodazole (6 µM) and Cytochalasin D (0.5 µg/ml). (G) The graph shows the distance between the centrosome and the centroid of the cell in the control (n = 40) and Cytochalasin D and Nocodazole cotreated condition (n = 44). (H) The graph shows the centrosome distance to the centroid normalized by cell size (i.e., divided by the square root of the area of the cell) in the control (n = 40) and Cytochalasin D and Nocodazole co-treated condition (n = 44). (I) Live acquisition depicting the independent drifts of the centrosome and the centroid during the generation of a mini-aster within a cell with a disrupted actin network (Nocodazole 6 µM and Cytochalasin D 0.5 µg/ml). (A, D, and F) All images are max projections, further processed using an unsharp mask and a gamma filter, and in C and I, this process was followed by background subtraction. In the graphs, horizontal bars represent the mean. P represents the P values, which were obtained from Kruskal–Wallis non-parametric tests (B and E) or Mann–Whitney non-parametric tests (G and H). Scale bars are 10 μm.