Coupled myosin VI motors facilitate unidirectional movement on an F-actin network

This sample metamorph '.STK' file contains 4200 frames acquired at 10Hz. It shows 200-nm fluorescent nanospheres coated with dimers of the molecular motor myosin VI landing from solution and moving on the dense interlaced actin filament network of an extracted keratocyte (see Figure 1f of manuscript).
Image sequence was acquired on a Nkion Eclipse 80i microscope with a Nikon Plan Apo VC, 60x, 1.40NA Oil immersion objective at 20 C. The imaging medium was 25 mM imidazole HCl (pH 7.4), 25 mM KCl, 4.5 μM calmodulin, 1 mM EGTA, 10 mM DTT, 4 mM MgCl2, an oxygen-scavenging system to retard photo damage (25 μg/ml glucose oxidase, 45 μg/ml catalase, 31 mM β-mercaptoethanol, 0.5% glucose), and an ATP-regeneration system (0.1 mg/ml creatine phosphokinase, 1 mM creatine phosphate), 2 mM ATP, 1 mg/ml BSA. Images were acquired using a Roper Scientific, Photometrics, CoolSnap HQ camera with Metamorph software.
Note the small stage drift over the 7 min image sequence. In tracking the movement of the fluorescent nanosphere (see Methods in manuscript), nanospheres that stick without moving to either the keratocyte or the coverslip surface are tracked and their movement is subtracted to obtain the movement trajectories and velocities (Figure 1 of manuscript) of the myosin VI driven nanospheres.