Mg2+‐free ATP regulates the processivity of native cytoplasmic dynein

Cytoplasmic dynein, a microtubule‐based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 μm. We also identified the ratio of Mg2+‐free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.