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Engineering Cellular Control: Synthetic Signaling and Motility Systems

 
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Executive Summary

Engineering Cells and Cell-Like Devices as Next Generation Therapeutic Agents
 

Living cells are extremely sophisticated sensor-actuator devices that detect specific environmental cues, process this information, and generate specific mechanical responses such as growth, shape change, or directed movement. These processes are controlled by networks of signal transduction and cytoskeletal proteins that form a dynamic self-organizing system. The long-term goal of our center is to engineer cells and artificial cell-like devices that can be flexibly programmed to carry out a wide range of specific diagnostic and therapeutic tasks. Such cellular “nanorobots” would in principle be able to carry out combined diagnostic (sensing) and therapeutic (delivery) functions in a patient (“theranostics”). As a testbed, we are focusing on understanding how cells achieve signal-guided movement (chemotaxis) and exploring how we can harness the cell’s intrinsic motility and signaling machinery to generate cellular search-and-delivery platforms. In principle, the motility machinery could also be harnessed to guide neurons and stems cells to specific sites for regenerative medicine. We hope to demonstrate the broad application of synthetic biology approaches to reprogram complex cellular behaviors like motility and signal integration, and to develop the resulting cells and cell-like devices as next generation therapeutic agents. Moreover, we also believe that through the cycles of design and refinement that will be required to achieve this goal, we will learn more deeply about the fundamental mechanisms and organizational principles of living cells and how they are able to execute complex sensor-actuator functions.

Figure 1 - Our vision is to engineer “smart” therapeutic cells and cell-like assemblies programmed to perform diverse search and delivery functions in a patient

Introduction

Fantastic Voyage
 

One of the most popular and captivating visions of nanomedicine is that epitomized by the 1966 film "Fantastic Voyage," in which nanorobots could be injected into our bodies to hunt down pathogens and tumors, or restore damaged organs and nerves. Of course, this was science fiction, but is this really the future? Perhaps, but probably not as envisioned in this film. For example, it is unrealistic to build a miniature robot operating at the nanoscale that exactly resembles the macroscopic mechanical machines that we encounter everyday (Figure 2a). This is because the behavior of materials at these dimensions differs significantly from behavior at larger scales -- molecular structures operate in unique and interesting ways. Nonetheless, something akin to therapeutic microscale robots does already exist — our own cells are efficient mechanical and sensing devices that can carry out highly complex tasks (Figure 2b). For example, our immune cells can detect infection, move to these sites, and respond by destroying the infectious agent either by phagocytosis or by secreting countermeasures such as antibodies. Cells can form new repair structures, such as clots and bone. These are only a few of the many tasks that our cells, which are about 10-40 microns in diameter, are genetically programmed to carry out.
 

Figure 2 - Visions of nanomedicine

Employing cells for targeted therapeutic purposes, however, faces several fundamental barriers. Real robots are useful because we can program them to carry out specific tasks that we desire. We can also program them with highly specific sets of instructions, so that this task is carried out only when, where, and how we specify. What we lack in the case of cellular "robots" is this ability to reprogram and precisely control cellular behavior, as we do not fully understand the design principles that define the modularity, interplay, and control of cellular components. Our inability to program cells also prevents us from specifying that a cell only carryout its function at a very precise time and under a defined set of conditions; the execution of a task at the wrong time and place might be ineffective and could be harmful. Natural cells also do not always function in desired ways — our immune cells can be tricked by evasive tumors or pathogens; nerve cells may fail to regenerate — and it is these missing or misdirected cellular functions that can lead to or exacerbate disease. Thus, the overall goal of our center is to learn how cellular sensor-actuator systems are designed, and how they could be reprogrammed through synthetic biology to meet diagnostic and therapeutic medical needs.
 

Directed cell movement and signaling responses as a testbed

As a testbed for understanding how to reprogram cells we are focusing on directed cell motility and signaling. Many cells can detect specific extracellular signals, process this information, and use the information to activate complex mechanical programs, such as directional cell movement, morphing into a new shape, or initiating gene expression. These programs are critical in immune cell movement and function; they are also used in developing cells like neurons as they send out axons over great distances toward specific targets.

 Pathway to Medicine: Programmed search and delivery cells

If we could reprogram cells to detect and move to novel signals, we could generate powerful search and delivery "robots". It would be extremely useful to have cells that could search the body for an elusive target, such as a microscopic tumor or metastatic cells. Engineered smart cells could in principle be designed to detect and integrate a host of different information to pinpoint and diagnose the tumor and the local tumor environment, including the detection of generic environmental conditions (such as low oxygen, which is typical in tumors) and the detection of tumor specific molecules. This sensing could be integrated and linked to the cellular signaling machinery to allow for efficient diagnosis of specific tumor states or alternatively to dictate a local response. Once at site, the cells could be used to deliver a number of payloads, ranging from dyes or molecules for imaging the tumor, chemotherapeutics that would kill the tumor, to cytokines that can influence cellular responses. These applied investigations are envisioned to enable the creation a new platform of cell-based theranostic devices (Fig 3.).

Figure 3 - Smart cells and cell-like devices as a novel cell-based theranostic platform
 

How can we program cellular robots?
Macroscopic robots are controlled by electronic and mechanical subdevices that are linked by wires (Fig. 4a). What controls cellular behavior is, at the surface, very different. Cells contain a complex, self-organizing network of regulatory and mechanical molecules. Thus, to reprogram cells, we need to understand how this intracellular nanoscale machinery works, how it is wired, and how it can be manipulated. The overriding goal of our center is to learn how to program cellular systems, or more appropriately, cell-like systems, so that we can build novel and precisely controlled therapeutic agents. Ultimately, to fulfill this vision, we need to learn how to build and program cells or cell-like assemblies in which we can flexibly and precisely tune what they sense and how they respond.

While understanding how to program cells is a daunting task, genomic research over the last decade has revealed that biological control circuits are surprisingly modular — the same molecular toolkits are used repeatedly to generate diverse biological functions. This suggests that there is a fundamental logic to how complex biological tasks are programmed, and that we have the potential to understand and exploit this programming logic. For a behavior like directed cell motility, environmental cues are sensed by receptor molecules, this information is processed by signaling networks — in this case primarily by kinase/phosphatase and GTPase networks — which, in turn, engage and activate the actin cytoskeleton, which functions as a mechanical actuation system that ultimately moves the cell. Thus these cellular systems have functional modules that are analogous to those found in macroscopic robots (Fig. 4b), although these cellular control modules are composed of nanoscale molecules. Our goal is to understand how these molecular modules work and how they are linked together. Then, perhaps we can link them in novel ways to build cells that show novel, targeted behavior. Interestingly, this kind of relinking of components resembles a major mechanism in the natural evolution of new cellular responses.

Figure 4 - Both robots (a) and cells (b) are built from components and subsystems with distinct modular functions

 

NDC Goals
 

We are pursuing the following specific aims:
 

Understand the design principles underlying the natural guidance and force-generating systems used in directed cell motility
 
We are studying fundamental mechanisms of the signaling and actin cytoskeletal systems that are used to build cell movement and cell shape change circuits. We are interested in understanding how subtle gradients of signals are interpreted by the cell to yield polarized activation of the actin cytoskeleton.

 Determine methods and tools to relink and reprogram cellular signaling systems 

Cell signaling systems are built from receptors, kinases, phosphatases, GTPases, and other regulatory components. How are these components wired in specific ways, and how can we rewire them? A growing body of evidence suggests that many of these components are functionally linked via specific protein interaction modules. These modules can organize multiple proteins into physical complexes and allosterically gate the activity of signaling enzymes. Thus we are trying to use a toolkit of protein interaction domains to link signaling components in new ways. These new circuits may yield new cellular behaviors. We are also developing engineered protein-protein interaction modules that are optimized for cellular engineering (i.e. have minimal cross-talk with endogenous molecules).

Determine ways to regulate force generating molecular systems 

Ultimately to bring about movement, the signaling systems must activate the actin cytoskeleton in a spatially precise manner, and in a way that yields force and protrusion of the membrane. We are investigating how regulated polymerization systems can be used to perform spatially directed work in a cell-like environment. We are investigating the use of non-actin polymers to perform work in a cell. We are also investigating whether hybrid materials (synthetic nanomaterials linked to biological regulatory components) could be used to generate nanostructures that assemble in a regulated manner to perform mechanical work at the nanometer to micron scale.
 NDC accomplishments
 

To date we have made significant progress in characterizing the design principles of natural motility control systems, and have made significant first steps in learning how to manipulate the relevant sensing, signaling, force generating, and response subsystems in vitro (biomimetic devices) and in cells. We are developing and applying this knowledge for in vivo use.
 

Selected Major Advances
 

  • Engineered novel input control of neutrophil chemotaxis using engineered GPCR’s. Demonstrates ability to engineer cell motility system and provides a defined platform in which we can further manipulate parameters to identify those critical for engineering in vivo cell migration and response.
  • Developed methods to rewire kinase and GTPase signaling circuits in vivo using synthetic protein-protein interactions. Also developed computational methods to design interaction modules optimized for rewiring.
  • Developed a genetically encoded light-switchable interaction module (plant phytochrome-based system). This module is a general tool for fine spatiotemporal manipulation of any cell process that is controlled by a protein assembly reaction.
  • Discovery, molecular characterization, and mechanical analysis of diverse force-generating polymer systems (actin, bacterial actin homologs ParM and AlfA, hybrid abiotic/biotic polymers). Elucidation of mechanisms used control these systems (natural and pathogen nucleators).
  • Developed new methods for efficient and reproducible vesicle encapsulation for building biomimetic devices by microfluidic jetting.
     

Pathway to Medicine,

Apply our knowledge to build cells and cell-like particles with novel motility and effector control that can be used for targeting within an animal
 

We are taking both a top-down and bottom up approach towards building engineered devices that can be used as search and delivery vehicles. In the top-down approach we are trying to reprogram motile cells such as neutrophils so that they move towards novel inputs and respond to specific cues. We are also trying to generate cytoplasts (cell fragments lacking nuclei) that can respond to and chemotax toward specified targets. In the bottom-up approach, we are trying to reconstitute minimal cell-like molecular assemblies that can show regulated movement or shape change, from either biological molecules or from nanomaterial components that are regulated in a biologically inspired manner. These cells, cell fragments, or assemblies could potentially be used as flexible search-and-delivery vehicles in the body; alone or by being employed as a multi-component system for greater design flexibility and control. We will test our engineered devices for targeting and effector functions within a mouse.
 

References

Background
 

  1. Bio FAB Group , Baker D, Church G, Collins J, Endy D, Jacobson J, Keasling J, Modrich P, Smolke C,Weiss R. "Engineering life: building a fab for biology. Sci Am. 2006 294:44-51.
  2. Brian J Yeh and Wendell A Lim, "Synthetic biology: lessons from the history of synthetic organic chemistry, " Nature Chemical Biology 3, 521-525 (2007).
  3. Dueber JE, Yeh BJ, Bhattacharyya RP, Lim WA. "Rewiring cell signaling: the logic and plasticity of eukaryotic protein circuitry." Curr Opin Struct Biol. 2004,14(6):690-9.
  4. Fletcher DA, Theriot JA. "An introduction to cell motility for the physical scientist," Phys Biol. 2004 Jun;1(1-2):T1-10.
  5. Welch MD, Mullins RD. "Cellular control of actin nucleation." Annu Rev Cell Dev Biol. 2002;18:247-88.
     

Recent NDC papers
 

  1. Bashor CJ, Helman NC, Yan S, Lim WA. (2008) “Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics.” Science. 319(5869):1539-43.
  2. Stachowiak JC, Richmond DL, Li TH, Liu AP, Parekh SH, Fletcher DA. (2008) “Unilamellar vesicle formation and encapsulation by microfluidic jetting.” Proc Natl Acad Sci U S A, 105(12):4697-702.
  3. Choi CL, Claridge SA, Garner EC, Alivisatos, AP, Mullins RD. (2008) “Protein-nanocrystal conjugates support a single filament polymerization model in R1 plasmid segregation.” Journal of Biological Chemistry, 283 (42), 28081-28086.
  4. Liu AP, Richmond DL, Maibaum L, Pronk S, Geissler PL, Fletcher DA. (2008) "Membrane-induced bundling of actin filaments." Nature Physics Oct; 4(10):789-793.
  5. Pronk S, Geissler PL, Fletcher DA. (2008) "Limits of filopodium stability." Phys Rev Lett. 100(25):258102.
  6. Sallee NA, Rivera GM, Dueber JE, Vasilescu D, Mullins RD, Mayer BJ, Lim WA. (2008) “The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.” Nature. 454(7207):1005-8.
  7. Akin O, Mullins RD. (2008) “Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex.” Cell. 133(5):841-51.
  8. Lauffer BE, Chen S, Melero C, Kortemme T, von Zastrow M, Vargas GA. (2009) “Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs.” J Biol Chem. 284(4):2448-58.
  9. Good M, Tang G, Singleton J, Reményi A, Lim WA. (2009) “The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation.” Cell. 136(6):1085-97.
  10. Levskaya, A, Weiner, O, Lim, WA, and Voigt, CA. (2009) “Spatiotemporal control of cell signaling and morphology using a genetically-encoded light-switchable interaction”, Nature. 461(7266):997-1001.
  11. Tabor, JJ, Salis, H, Simpson, ZB, Chevalier, AA, Levskaya, A, Marcotte, E, Voigt, CA, and Ellington, AD. (2009) “A Synthetic Genetic Edge Detection Program.” Cell. 137(7):1272-81.
  12. Stachowiak JC, Richmond DL, Li TH, Brochard-Wyart F, Fletcher DA. (2009) “Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation.” Lab Chip. 9(14):2003-9.
  13. Liu AP, Fletcher DA. (2009) “Biology under construction: in vitro reconstitution of cellular function.” Nat Rev Mol Cell Biol. 10(9):644-50.
  14. Ma W, Trusina A, El-Samad H, Lim WA, and Tang C. (2009) “Defining network topologies that can achieve biochemical adaptation” Cell. 138(4):760-73.
     

Contact Information
 

Center Website: http://www.qb3.org/cpl/ Exit Disclaimer
 

Key Investigators
 

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