Aster relaxation upon microtubule ablation. (A) Schematic representation of dynein-generated tensional stresses along microtubules leading to centrosome centration and centrosome maintenance at the center of the cell when a mechanical force balance is reached. In this context, ablating microtubules on one side of the centrosome will lead to a disruption of the mechanical force balance and recoil of the centrosomal aster away from the ablated area in the direction of the remaining net pulling force. (B) On the left, first, and last time points of a laser ablation experiment in a cytoplast from a PtK2 cells expressing GFP-tubulin. Actin was stained using SiR actin. Images are max projections, further processed using an unsharp mask, a gamma filter, and a subtract background function. Microtubules were repeatedly ablated on one side of the centrosome for 5 min. The white triangular marks indicate the position of the centrosome. On the right, the graph representing the average relaxation curve shows the mean relaxation profile of the centrosomes (n = 22) during the 5 min laser ablation experiment. The displacement of the centrosome was projected along an axis connecting the centroid of the ablated area and the centrosome (for a graphical representation see Fig. S1 C). The circles represent the average displacement of the centrosome at each time point, the continuous lines represent the standard deviation. (C) Zoomed-in view of the centrosomal area of the cytoplast shown in B. White arrows indicate from left to right a recoiling actin structure and the centrosome position. Kymograph representation of the centrosome and actin relaxations inside the zoomed-in area. The kymograph (scaled three times to smoothen the signal) was performed along a straight line connecting the centrosome with the ablated area and spanning the entire length of the zoomed-in area. (D) Representative live image of a control PtK2 cell microinjected with both actin and tubulin on a 3,500 µm² H-shaped micropattern. On the right, two magnified regions show actin and tubulin speckles in higher detail. For details regarding actin and tubulin speckle processing, see the dedicated section in the Materials and methods. (E) Representative responses of single microtubules in the 15 s that followed laser ablation. Left images show microtubule depolymerization after laser ablation with no mechanical relaxation. Right images show actin stress fiber recoiling after laser ablation accompanied by a local microtubule buckling and recoiling. (F) Graphs show the displacements of tubulin speckles before and after ablation (n = 121, left), and the displacements of tubulin and actin speckles after laser ablation in the cases of large relaxation events (displacements >400 nm) (n = 15, right). (G) Microtubule displacement events when only actin was ablated. The graph shows the motion of tubulin speckle and actin speckle after ablation. The dotted line marks the 400 nm threshold used to define large relaxation events (n = 10). Vector map depicting the coordinated displacements of actin and tubulin speckles after an actin ablation event (over a 15 s period). Images of the actin and tubulin speckles localization before the laser ablation event. (H) Same as B in a PtK2 cytoplast expressing GFP-tubulin treated with Jasplakinolide (600 nm) and Y27632 (20 µM) for 4 h (n = 24).