Synthetic cargo adaptors reveal molecular features that enhance dynein activation.

Cytoplasmic dynein-1 (dynein) facilitates the microtubule-based retrograde trafficking of all cellular cargo. To become active, dynein binds dynactin and one of many cargo-specific adaptors forming the dynein-dynactin-adaptor transport complex. Despite having similar structures, transport complexes assembled with different adaptors move with different properties in vitro. To explore how adaptors activate dynein, we engineered a library of synthetic adaptors and characterized their ability to activate dynein using in vitro reconstitution and cell-based trafficking assays. We found that dynein motility is highly plastic and tunable by the adaptor, and that it is possible to engineer adaptors that outperform endogenous adaptors' ability to generate highly motile transport complexes. We also found that different adaptors support distinct cellular trafficking behavior and cargo movement. In this study, we provide insight into how dynein motility is modulated to meet the unique trafficking requirements of all cellular cargo.