Synthetic Approaches to Investigate Intracellular Transport and Cytoskeletal Regulatory Systems

Eukaryotic cell physiology is deeply dependent on the spatial control of intracellular chemistry. The regulation of cytoskeletal filament architecture and the activated transport of material within these networks therefore have a profound influence over a wide range of biophysical processes and cell signaling responses. Moreover, the development of methods to modulate cytoskeletal structure and intracellular transport are uniquely important to gaining synthetic control over many eukaryotic cell functions.  Herein, we describe synthetic methods to engineer activated intracellular transport behaviors in living cells. This is accomplished via the genetic-level programming of various molecular motors, their coupling to vesicular cargos and cargo sizes. We also demonstrate how this procedure can be adapted to examine functional relationships among multiple interacting regulatory proteins that influence cytoskeletal architecture, cell morphology and cell-cell adhesion. In each case, these methods offer opportunities to dissect fundamental dynamic responses of coupled macromolecular systems and characterize their collective response to specific regulatory factors and perturbations.