Networks are imaged using a Nikon A1R laser scanning confocal microscope with a 60× 1.4 NA objective (Nikon). Actin filaments are imaged using a 561 nm laser with 561 nm excitation and 595 nm emission filters. Microtubules are imaged using a 488 nm laser with 488 nm excitation and 525 nm emission filters. Myosin II motor activity is controlled by deactivating blebbistatin with the same 488 nm excitation used to image microtubules (continuous 360 ms pulses of 488 nm light over the duration of each experiment). While blebbistatin deactivation is typically achieved using 405 nm light,17 (link) previous studies have shown successful photoinactivation of blebbistatin using 488 nm light.37 (link),55 (link) 256 × 256 pixel images (212 μm × 212 μm) are taken in the middle of the ~70 μm thick chamber for 45 min at 2.78 fps. Examples of time-series are shown in Movie S1 (ESI†), and representative images are shown in Fig. 1. Experiments are performed on 3–5 different replicates.