Each cell in the human body contains a structural framework that is composed of a network of actin (a protein) filaments. This framework acts like a skeleton. It is the basis of the cell's ability to resist deformation, to change shape, and to move. Our understanding of this actin cytoskeleton is based on studies of the behavior of whole cells. However, we do not fully understand how these filamentous networks assemble. This project will reconstitute actin networks and observe how they reorganize themselves under stress. Actin will also be synthesized and its self-organization into structures will be observed under many environmental conditions. The project will provide insight into the mechanics of actin cytoskeleton network construction for production of robust synthetic cells. The project will also provide research opportunities for students from high school through graduate school. Of specific emphasis will be outreach activities for girls at the K-12 level.
Very little is known about how the ability of cells to sense and respond to force and acquire mechanical stiffness arises from cytoskeletal building blocks. Understanding this would enable the design of mechanical functionality in synthetic cells. A reconstitution platform could elucidate how actin architectures affect the mechanical properties of a cell. Two model systems will be used in this project. One involves synthetic cells that encapsulate reconstituted cytoskeletal components. The second is a lipid bilayer vesicle that encapsulates a cell-free protein synthesis (CFPS) system that produces actin. These will be processed through a novel microfluidic device capable of exerting mechanical compression, and the responses of the cell/vesicle and of the actin network will be evaluated. The mechanical properties of synthetic cells with different actin network will be quantified. Based on those results, the engineering of a synthetic cell with reconfigurable actin networks will be undertaken and evaluated. This demonstration of a reconfigurable internal cytoskeleton will pave the way for engineering stimulus-responsive changes to synthetic cells. This work may reveal new rules about synthetic cell design by highlighting the mechanical requirement of a robust synthetic cell. If successful, the results will lead to the design of synthetic cells with a more robust response to external mechanical perturbations. This is currently a major hurdle in the bottom-up construction of synthetic cells.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
