A Cell-Free Platform to Characterize and Engineer Genetically Encoded Motor Proteins

Kinesin motor proteins interact with microtubule tracks to drive essential cell processes such as locomotion and division. Eukaryotes contain genes encoding diverse kinesin subtypes, which are deployed to perform specific tasks determined by the structural features of each subtype. Due to the limitations of traditional methods, understanding how cells generate behaviors utilizing their kinesin toolbox remains challenging. Here, we introduce ActiveDROPS (Dynamic Reprogrammable self-Organizing Protein System), a cell-free platform to quantitatively characterize the cytoskeletal dynamics of genetically encoded motor proteins by real-time imaging of lysate-in-oil emulsion droplets. Using ActiveDROPS, we show that motors display two distinct evolutionarily conserved dynamic modes: sustained-like and burst-like. By recombining specific domains with computational and molecular biology approaches, we show that the dynamic mode of a motor can be switched from sustained-like to burst-like, shedding light on how motor protein structure translates to function. ActiveDROPS shortens the gap between in vitro and in vivo approaches by offering a tool for mining kinesin sequence space and identifying the evolutionary pressures shaping their diversity of functions. This work has a significant bearing on the problem of how motor proteins generate various robust structures and opens up new avenues to investigate and generate biological behavior using cell-free technologies.