Centrosome recentering following cell enucleation. PtK2 cells expressing GFP-tubulin were enucleated in the presence of Nocodazole (10 µM), Y27632 (10 µM), and Cytochalasin D (3 µg/ml). After enucleation and before the removal of the enucleation drugs, all cytoplasts displayed a damaged actin network and an off-centered centrosome biased in the direction of the enucleation pull. The contributions of actin and microtubules in the recentering processes were investigated by washing out one or the other drug. (A) The image sequence shows microtubule network recovery upon Nocodazole, Y27632, and Cytochalasine-D washout (n = 70). The dotted red curve highlights the motion of the centrosome. The graph shows the distance separating the centrosome and the centroid before and 4 h after drug washout. (B) Image sequence showing microtubule network recovery upon Y27632 and Cytochalasine-D washout (n = 70). Nocodazole is still present. The dotted red curve highlights the motion of the centrosome. The graph shows the distance separating the centrosome and the centroid before and 4 h after drug washout. (C) Image sequence showing microtubule network recovery upon Nocodazole washout in the presence of Y27632 (20 µM) and Jasplakinolide (600 nM) (n = 66). The graph shows the distance separating the centrosome and the centroid before and 4 h after drug washout. (D) Images illustrate a well-centered microtubule aster 4 h after Nocodazole, Y27632, and Cytochalasine-D washout (top), and a non-centered aster upon Nocodazole washout in the presence of Y27632 (20 µM) and Jasplakinolide (600 nM) (bottom). (E) Schematic depiction of the nonspecific centration of the centrosomal aster through the centripetal actomyosin flow and its latter maintenance near the center of the cell through its embedding inside the actomyosin network. (F) Vectorial plots depicting the displacement of the centrosomes toward the centroid of the cytoplasts in the three different centrosome recentering conditions (left). Graph representing the total displacement of the centrosomes in the three different centrosome recentering conditions (right). (G) Graph representing the recentering ratio (i.e., the ratio between the final and initial distances separating the centrosome and the centroid) in the three different centrosome recentering conditions. (A–C) Live images were processed following the described pipeline in the Materials and methods section. (D) Images are max projections that were further processed using an unsharp mask and a gamma filter. In the graphs, horizontal bars represent the mean. P represents the P values, which were obtained from Kruskal–Wallis non-parametric tests.