Reverse Engineering the Programming Logic of Cytoskeletal Dynamics

Eukaryotic cells generate mechanical forces through cytoskeletal filaments actively reorganized by families of molecular motors. However, how motor sequence specifies filament organization dynamics remains unclear. We developed ActiveDROPS, a cell-free platform that expresses kinesin variants in bacterial lysate droplets and quantitatively maps the resulting microtubule dynamics into a common phenotype space. Across a library of twelve kinesin-1 homologs, we observed three phenotypes: "Slow-Sustained" flows that activate after 8 h, reach peak mean velocities of 24 nm/s and decay after [~]32 h; "Fast-Burst" flows that activate within minutes and dissipate within 2 h, reaching velocities up to 900 nm/s; and a [~]36-h "Multiphase" progression through nematic, rotational, and contractile states. Microtubule gliding assays and molecular dynamics simulations using AlphaFold-predicted structures linked the "Fast-Burst" phenotype to generally faster motility and more favorable motor-tubulin interactions than "Slow-Sustained". By replacing the microtubule-binding region of a "Slow-Sustained" motor with that of a "Fast-Burst" motor, we generated a "Fast-Sustained" chimera with flows that activate within 1 h, peak at 70 nm/s and persist for 16 h, showing that recombination can reprogram the parental relationship between speed and timing of microtubule-motor self-organization. These results reveal a constrained logic through which kinesin sequence shapes cytoskeletal dynamics, providing a framework for dissecting the mechanical repertoire available to cellular systems.