Engineering Cellular Control: Synthetic Signaling and Motility Systems
Specific Aims & Significance. Our long-term goal is to understand the fundamental design principles of cellular control systems and to apply these principles to engineer “smart” cells or cell-like devices with novel therapeutic functions. This goal is extremely challenging, as cells are built from large numbers of molecules that interact with one another to form complex, dynamically regulated, self-organizing systems. Our ability to engineer cells and their constituent molecular systems is extremely primitive. Our goal is to develop the foundation for precision cellular engineering, analogous to the foundations that already exist in other highly developed engineering disciplines.
In the long-term, the ability to precisely manipulate cells or synthesize molecular assemblies with cell-like behaviors would have revolutionary therapeutic potential. Engineered therapeutic cells, much like endogenous immune cells, would be “smart” — they could diagnose a lesion or threat, move to that site, and respond with a precise, feedback controlled treatment. Smart cells could hunt and destroy microscopic cancers or cardiovascular lesions. Their responses could be complex: they might directly release a therapeutic agent, or they might act as intermediaries, mobilizing and redirecting the immune system. The designed cells could act as a network, with distinct cells carrying out specific steps in a complex therapeutic cascade. Engineered cells with sophisticated homeostatic control circuits could precisely readjust hormonal and metabolic imbalances. Engineered control of cell motility could be used for repair of nerve and other tissue damage. Modified cells could be from the patient, or from compatible matches, thus eliminating or alleviating problems of immune response. The range of therapeutic potential would be staggering. In the shorter-term, this center may attempt to generate cells that mimic the regulated shape change of platelets or cells that can move in response to heterologous inputs.
To achieve our goal, we are initially focusing on actin-based cell motility as a testbed system. Many cells have the remarkable ability to detect environmental cues and to precisely move or alter their shape through spatiotemporal regulation of the actin cytoskeleton. Our primary goal is to learn how to program molecular systems to achieve this type of precise morphological control. Development of a fundamental cellular engineering framework within this testbed will then allow expansion and application to engineering other cellular behaviors. We have formed an interdisciplinary Center which we refer to as the Cell Propulsion Lab (CPL).
To understand how cell motility systems are built, our team is focusing on three engineering Grand Challenges:
- Reprogram cell guidance/signaling systems (e.g., convert a non-motile cell into a motile cell)
- Build alternative force generating polymer systems that can perform work in a cell (either from non-actin polymers or from nanomaterials)
- Build synthetic cell-like assemblies capable of induced shape change or movement
Organization of the Center. We have made significant progress in the organization of our center. The full center (12 Pis) meets once a month, with various subgroups meeting twice a month. This high level of communication has led tot the establishment of an extensive network of highly integrated interlab collaborations. Our lab website and wiki sites are at: www.qb3.org/CPL . The Center has served as a highly effective means to attract outstanding new graduate students and postdocs who are excited to be part of this highly integrated and creative network. In all, 17 new individuals joined member labs this year in order to work on a Center-related project.
In this first year, our early experimental results demonstrate the potential feasibility of re-engineering signaling and cytoskeletal protein networks in order generate novel, precision biological function. Key results are summarized below.
Re-engineering GTPase components and pathways. One significant area of progress involves efforts to manipulate and engineer Rho family GTPase signaling. These proteins play a central role in controlling cell morphology. The state and extent of GTPase activity is controlled in large part by a set of upstream activators known as guanine nucleotide exchange factors (GEFs). A major goal of our center is to learn how to engineer regulatory control and output of GEF proteins. Focusing on the DH-PH family of GEF proteins, We have demonstrated that we can use modular regulatory domain recombination to engineer GEFs that show novel input gating. We have also demonstrated that these synthetic proteins can be used to activate the Rho family GTPases Cdc42 and Rac in vivo, thereby generate new morphological regulatory pathways in living cells.
In addition we have used a combined computational and experimental approach to successfully redesign the interface of a GEF-GTPase complex. This has allowed us to design new GEF-GTPase pairs that are “orthogonal” — they only interact with a mutant partner and not with endogenous GTPases. Thus these orthogonalized proteins can be used as new control channels to mediate new, highly specific outputs. These early findings suggest that it is possible to rationally alter some of the signaling linkages that control cell morphology through cellular systems engineering.
We have also made progress is developing total internal reflectance fluorescence (TIRF) microscopy based assays for following Rho family GTPase activation and downstream effector activation in living cells. We have also developed a new lattice-based computational framework for simulating and understanding the process of Rho GTPase driven cell polarization.
Force Generation/Cytoskeletal Components. Another significant area of progress is in understanding the regulatory and force generating behavior of non-actin polymer systems. We have extensively characterized the nucleation/polymerization behavior of the bacterial actin homology ParM, which is normally used to segregate drug resistance plasmids to the opposite poles of bacteria. We demonstrate that we can reconstitute ParM nucleation in vitro (using the native nucleator, a plasmid binding protein called ParC), and that this system can be used to move loads such as polystyrene beads in solution or in microfabricated chambers, if a nucleation complex is properly tethered to the beads. Specific dynamic instability properties of the ParM polymer (both ends need to be attached to a nucleator complex to get stable polymer growth) allow it to push two objects to the long axis of an exclosed space – explaining how it is used to segregate two copies of a plasmid in a dividing bacteria. We have recently shown that ParM polymerization can occur when the protein is microinjected into mammalian cells. Thus we hope to use this alternative system to perform new motility functions in these mammalian cells. The ParM polymer system should be completely orthogonal to the host cytoskeleton -- it should not interfere with actin and other endogenous cytoskeletal systems. We have also made significant progress in developing assays for characterizing the mesoscopic properties of specific actin networks.
We can measure the distinct elasticities of different networks using a differential force microscopy technique. We have also begun reconstituting the growth of actin networks on phase separated vesicles. These are early models for artificial systems that show synthetic morphological control, such as an artificial platelet.