Project Summary/Abstract The goal of this project is to understand how large biomacromolecules, like DNA, move through the crowded intracellular environment and what features of that environment and of DNA lead to the complex dynamics and spatial organization observed in cells. A highly tunable in vitro cytoskeleton system consisting of varying amounts of actin and microtubules, light-activated motor proteins, and crosslinkers will be developed and characterized with a suite of optical microscopy and rheology methods that span from the molecular-level to macroscopic scales. Advanced microscopy and analysis methods will allow for the dynamics of DNA molecules of controllable size and topologies, embedded in the biomimetic active cytoskeleton, to be precisely quantified across an unprecedented range of spatial and temporal scales. Further, the encapsulation of the active cytoskeleton and DNA molecules within lipid membranes will allow for elucidating the role of confinement and cytoskeleton-membrane interactions in determining cytoskeleton and DNA dynamics and structure. Studies of how macromolecules move through and spatially distribute within crowded intracellular spaces are often hampered either by the complexity of in vivo systems or the simplicity of in vitro environments. The proposed work finds a unique balance between complexity and tractability. The in vitro platform will be highly tunable and allow cell-like conditions to be recreated by independently allowing for control over: the relative concentrations of actin, microtubules, molecular motors and crosslinkers; the types of motors and crosslinkers; the location, timing, and strength of motor activity; and the properties of confining vesicles. Characteristics of this in vitro system will be linked to the dynamics and distributions of DNA molecules, which will be imaged with a custom-built light-sheet microscope and quantified with single-molecule conformational tracking and differential dynamic microscopy analysis methods. These methods allow for DNA dynamics to be captured across an unprecedented spatiotemporal range: from milliseconds to hours and from submicron to 100s of microns. The specific aims of this project are to: (1) design in vitro active cytoskeleton networks that exhibit tunable activity for probing non-equilibrium dynamics and rheological properties; (2) determine transport and conformational dynamics of linear and circular DNA within these active cytoskeleton networks; (3) link the time-varying spatial distributions of linear and circular DNA to cytoskeleton network properties and activity; and (4) incorporate active cytoskeleton networks and DNA into cell-mimicking lipid bilayer vesicles to determine the role of confinement and membrane interactions on results of Aims 1-3. Success in these Aims will allow for the complex macromolecular dynamics and distributions observed within cells to be recreated and understood. Further, this project will reveal how cells use their d...