Patterning Microtubule Network Organization Reshapes Cell-Like Compartments

Eukaryotic cells contain a cytoskeletal network comprised of dynamic microtubule filaments whose spatial organization is highly plastic. Specialized microtubule architectures are optimized for different cell types and remodel with the oscillatory cell cycle. These spatially distinct microtubule networks are thought to arise from the activity and localization of microtubule regulators and motors and are further shaped by physical forces from the cell boundary. Given complexities and redundancies of a living cell, it is challenging to disentangle the separate biochemical and physical contributions to microtubule network organization. Therefore, we sought to develop a minimal cell-like system to manipulate and spatially pattern the organization of cytoskeletal components in real-time, providing an opportunity to build distinct spatial structures and to determine how they are shaped by or reshape cell boundaries. We constructed a system for induced spatial patterning of protein components within cell-sized emulsion compartments and used it to drive microtubule network organization in real-time. We controlled dynamic protein relocalization using small molecules and light and slowed lateral diffusion within the lipid monolayer to create stable micropatterns with focused illumination. By fusing microtubule interacting proteins to optochemical dimerization domains, we directed the spatial organization of microtubule networks. Cortical patterning of polymerizing microtubules leads to symmetry breaking and forces that dramatically reshape the compartment. Our system has applications in cell biology to characterize the contributions of biochemical components and physical boundary conditions to microtubule network organization. Additionally, active shape control has uses in protocell engineering and for augmenting the functionalities of synthetic cells.