Double-speckle microscopy and speckle tracking analysis. (A) PtK2 cell expressing GFP-tubulin (red), 2 h after the microinjection of 1 µM purified Atto-555-tubulin (green). On the left, a representative live image of the microtubule network with the non-processed and processed tubulin speckles along the microtubules. On the right, kymographs (constructed over a period of 108 s) along the length of a few selected microtubules. Selected time points, extracted from the video used to build the kymograph, can be found below each corresponding kymograph. The reader can visualize a microtubule polymerizing, a microtubule depolymerizing, a static microtubule, and a microtubule gliding—kymographs are scaled by a factor of 3 for smoothness. (B) Control PtK2 cell stained using SIR-Actin (cyan), 2 h after microinjection of 1 µM labeled actin (Alexa 488) (magenta). On the left, a representative live image of the actin network with the non-processed and processed actin speckles along the actin structures. On the right, kymographs (constructed over a period of 30 min) along the length of two selected stress fibers. The reader can visualize contraction events with the presence of antiparallel speckles sliding along the length of both stress fibers—kymographs are scaled by a factor of 3 for smoothness. (C) Position of laser ablations in control PtK2 cells platted on 3,500 µm² patterns and co-injected 2 h earlier with purified and labeled actin and tubulin (1 and 1.5 μM respectively). Images show the tubulin (green) (top) and actin speckles (magenta) (bottom). The map shows the localizations of all the analyzed ablation events. (D) Analysis pipeline used to describe the behavior of a microtubule undergoing ablation. Briefly, the motions of the tubulin speckles were tracked 15 s before ablation and then again for 15 s after ablation using the simple LAP tracking method in Trackmate. When microtubules displayed significant relaxations, the motions of the surrounding actin speckles were tracked in parallel to that of the tubulin speckles. The amplitudes of the displacements of individual speckles were averaged to yield the mean amplitude of the displacement of the microtubule before and after ablation as well as the mean amplitude of the displacement of the surrounding actin meshwork after ablation. (E) Large microtubule relaxation events (motions >400 nm, depicted in Fig. 1 F). Kymographs showing the coordinated motions of the actin and tubulin speckles in the 15 s following laser ablation. The kymographs are constructed along the length of the moving microtubules and are scaled by a factor of 3 for smoothness. (A–E) For the details regarding actin and tubulin speckle processing, see the dedicated section in the Materials and methods.