Quantifications of centrosome recentering experiments. PtK2 cells expressing GFP-tubulin, which had endocytosed 500 nm fluorescently labeled non-functionalized polystyrene beads, were enucleated in the presence of Nocodazole (10 µM), Y27632 (20 µM) and Cytochalasin D (3 µg/ml). To challenge the contributions of actin and microtubules in the recentering process, we designed three washout experiments that were recorded over a period of 4 h. (A and B) Images of fixed cytoplasts. (C and E) Images from live cytoplasts. (A) Top: Images show well-centered microtubule asters after 4 h of all drugs washout. Middle: Images show small microtubule asters after 4 h of Y27632 and Cytochalasin D washout, i.e., in the presence of Nocodazole. Bottom: Images show off-centered microtubule asters 4 h after the washout of Nocodazole but in the presence of Y27632 and Jasplakinolide. (B) Images show the actin networks after 4 h of recentering. Left: Control conditions, i.e., Nocodazole washout, Y27632 washout, and Cytochalasin D washout. Middle: Nocodazole present, Y27632 washout, Cytochalasin D washout. Right: Nocodazole washout, Y27632 present, Jasplakinolide. (A and B) All images are max projections, further processed using an unsharp mask and a gamma filter. (C) Temporal projection of the successive positions of the centrosome during a representative recentering event in the presence of dynamic actin and microtubule networks. (D) Quantification of centrosome trajectories in the presence of dynamic microtubules (NZ washout, Y27632 washout, Cytochalasin D washout, n = 70) and in the absence of microtubules (Nocodazole present, Y27632 washout, Cytochalasin D washout, n = 81). From left to right, graphs represent the total distance traveled by the centrosomes during recentering (i.e., the sum of all their displacements and not the linear distance joining their initial and final positions), the average speeds at which the centrosomes moved during recentering, the frame-to-frame angular variation in centrosome motion during recentering, and the linearity of centrosome trajectories during recentering. Centrosome trajectories and their characteristics were extracted using the simple LAP tracking method in Trackmate. (E) Time-lapse sequences show the motions of centrosomes and endocytosed 500 nm beads over 4 h in the various recentering conditions. Kymographs show the coordinated displacements of the centrosome and the beads inside the zoomed-in region. The linescans were performed along a line following the trajectory of the centrosome and joining its initial and final positions. Images are max projections; the centrosome is further processed with an unsharp mask and a Gaussian blur. (F) Graphs comparing the centration of the centrosomes to that of fluorescent non-functionalized polystyrene beads after 4 h of recentering. Centrosomes (n = 70) and beads (n = 471) final centering in the presence of dynamic actin and microtubule networks (left). Centrosomes (n = 81), and beads (n = 380) final centering in the presence of a dynamic actin network but without microtubules (right). In the graphs, horizontal bars represent the mean. P represents the P values, which were obtained from Mann–Whitney non-parametric tests.