Single-Molecule Studies of Motor Adaptors Using Cell Lysates

Long-range transport of organelles and other cellular cargoes along microtubules is driven by kinesin and dynein motor proteins in complex with cargo-specific adaptors. While some adaptors interact exclusively with a single motor, other adaptors interact with both kinesin and dynein motors. However, the mechanisms by which bidirectional motor adaptors coordinate opposing microtubule motors are not fully understood. While single-molecule studies of adaptors using purified proteins can provide key insight into motor adaptor function, these studies may be limited by the absence of cellular factors that regulate or coordinate motor function. As a result, motility assays using cell lysates have been developed to gain insight into motor adaptor function in a more physiological context. These assays are a powerful means to dissect the regulation of motor adaptors as cell lysates contain endogenous microtubule motors and additional factors that regulate motor function. Further, this system is highly tractable as individual proteins can readily be added or removed via overexpression or knockdown in cells. Here, we describe a protocol for in vitro reconstitution of motor-driven transport along dynamic microtubules at single-molecule resolution using total internal reflection fluorescence microscopy of cell lysates.