Aster relaxation upon microtubule ablation in different cell types. (A) First and last time points of a 2-min laser ablation experiment in PtK2 cells expressing GFP tubulin. A few microtubules were ablated on one side of the centrosome. The white triangular marks indicate the positions of the centrosomes. (B) First and last time points of a 2-min laser ablation experiment in PtK2 cytoplasts expressing GFP tubulin. A few microtubules were ablated on one side of the centrosome. The white triangular marks indicate the positions of the centrosomes. (C) Schematic representation of the quantifications of centrosome relaxation (left) and of the microtubule depletion ratio upon microtubule ablation (right). (D) The graph shows the proportion of depleted microtubules on the side of the centrosome, where the laser ablations were performed in the control condition (n = 22) and in the case of Jasplakinolide (600 nM) and Y27632 (20 µM) treatment (n = 24). Horizontal bars represent the mean. P values were obtained from a Mann–Whitney non-parametric test. (E) Images of the microtubule network in fixed PtK2 cells (left); MEF cells (middle); RPE1 cells (right). (F) Relaxation of the actin network (magenta) and microtubule network (green) upon repeated laser ablations during 5 min in RPE1 cytoplasts (top) and in MEF cytoplasts (bottom). White arrowheads indicate the positions of the centrosomes. Graphs show the mean relaxation profile of the centrosomes (30 RPE1 cytoplasts and 36 MEF cytoplasts). Circles represent the average displacement of the centrosome at each time point, the continuous lines represent the standard deviation. (G) Magnified view of the centrosomal area of an REP1 cytoplast (top) and a MEF cytoplast (bottom) depicted in Fig. S1 F. White arrows indicate the position of the centrosome. Kymograph representations of the centrosome and actin relaxations inside the zoomed-in areas. The kymographs (scaled three times to smoothen the signal) were performed along a straight line connecting the centrosomes with the centroids of the ablated areas and spanning the entire length of the zoomed-in areas. (H) Magnified views of the centrosomal areas during repeated laser ablation experiments in RPE1 and MEF cytoplasts. The temporal sequences are made of consecutive images covering a total period of 40 s. Small centrosomal translocation events can be visualized in parallel to local relaxations of the surrounding actin meshwork. (A, B, E, and F) All images are max projections, further processed using an unsharp mask and a gamma filter. (H) All images are max projections, further processed using a Gaussian blur filter. The LUT of the SPY FAST actin was inverted to facilitate the visualization of the local destruction and relaxation of the meshwork.