Nanomedicine Development Centers
Cell Propulsion LabCenter for Cell ControlCenter for Protein Folding MachineryNanomedicine Center for Nucleoprotein MachinesNanotechnology Center for Mechanics in Regenerative MedicineNational Center for Design of Biomimetic NanoconductorsOptical Control of Biological FunctionPhi29 DNA-Packaging Motor for Nanomedicine

Nanomedicine Center for Mechanics in Regeneration

Executive Summary

At the present time, there is a major gap in our understanding of molecular complexes in the critical size range between molecules and cells. Both intracellular and extracellular components, in the nanoscale range from about 20 to 800 nm, determine many of the functions that define cell shape, as well as forces exerted by cells on matrices and other cells, that are critical for life. Basically, we have very little knowledge of how cells (about 20 microns in diameter) can create all of the diverse biological forms that range from tiny insects (less than a millimeter long) to huge whales. It is well known that operation of the nanomachinery inside cells is critically dependent on factors in the extracellular environment such as hormones, cytokines, and other cell products. However, beyond single molecule signals from other cells and tissues, we are beginning to understand the importance of the environment surrounding cells that is critical to their function. In other words, the structure, composition and geometry of the extracellular environment influences intracellular molecular function and cell behavior.

Introduction

We have little knowledge of how cells, which are about 20 microns in diameter, can create all of the diverse biological forms that range from tiny insects to huge whales. It is well known that operation of the nanomachinery inside cells is critically dependent on factors in the extracellular environment, such as hormones, cytokines, and other cell products. The structure, composition, and geometry of the extracellular environment influences intracellular molecular function and cell behavior. We know that mechanical processes affecting cells helps define the shape of tissues and underlies proper regeneration and repair. We believe that all cells use a common set of motile functions to determine the morphology of tissues. Our research can provide new strategies for treating, cancer, hypertension, cardiovascular diseases, osteoporosis, nerve regeneration, and manipulating the immune response. For example, we recently discovered that mechanical unfolding of proteins inside and outside the cell transduces force into biochemical signals. The unfolding inside the cell exposes new amino acid sequences for phosphorylation that were not available before the mechanical perturbation. The newly phosphorylated sites cause alterations in cell migration, growth and differentiation suggesting a new drug target for selectively blocking cell migration in metastatic cancers.

Since so many critical cellular functions are mechanical and occur rapidly, our center is developing new tools to quickly make measurements below the resolution limit of the light microscope and existing force measurement devices. To work at the nanoscale, we began with the knowledge and tools of material science and the growing field of nanotechnology in order to enhance our toolset to specifically manipulate the mechanical properties of biological tissues and make nanomedicine a reality.

NDC Goals

We are approaching the problem of understanding cell mechanical functions by determining first what are the mechanical as well as biochemical bases of cell functions at a sub-micron (nanometer) level (see Figure 1). When we know the basic steps and the molecular processes involved, we can hope to intervene to correct or alter cell behavior. It is useful to consider how automobiles work as an analogy to understand how cells work. Similar to the array of cells in the human body, there is a diverse array of automobiles that share many functions, but the individual components involved in a function may be different. A major step forward to controlling operation of a machine is to fully describe each step of the various functions performed. For example, cars have windows that go up and down by a similar mechanism, but the specific parts may be totally different. To describe how turning the window crank causes the window to go down, as in an engineering operations manual, is an important step forward. This requires taking apart the door and identifying which gears, pulleys and other components move, which are stationary, and how they are coupled to accomplish the function. Once this process is described for one car, it will be much easier to understand the mechanism in another even if the components are not identical.

Figure 1
Figure 1. Mechanism of Periodic Contraction Involves Mechanically Driven Steps not Biochemically. Periodic Contractions in spreading or motile cells are driven by a mechanical process that has several important steps that are mechanically linked. Taken from Giannone et al., 2007.

Recent analyses of subcellular motile functions suggest that they involve many similar steps in different cell types (Giannone et al., 2007). Thus, it is useful to describe these functions in detail to understand how cells generally perform mechanical functions. Linking molecular complexes and mechanics to the individual steps of cell behavior is just as important as understanding each step required to put a car's window assembly together in order to fully understand how to control cell behaviors. Indeed, this is the process that we are undertaking to describe how a cancer cell can metastasize to specific tissues and how a mesenchymal stem responds to different mechanical environments after differentiating into a bone versus a muscle cell.

We feel that the best way to understand mechanotransduction quantitatively is to develop an integrated approach by examining multiple scales ranging from individual molecules to nanoscale intracellular machinery to whole systems. Biologists, engineers, computer scientists and modelers are all focused on the same questions such as the biophysical and biochemical steps in the genesis of focal contacts, the cellular response to substrate compliance, or the assembly of extracellular matrix fibers. Our team has a wide range of expertise including Cell Biophysics and Nanofabrication, Advanced Fluorescence Microscopy and Image Analysis, Immunology and Supported Bilayer Fabrication, Developmental Biology and Signaling Systems Modeling.

Our preliminary results indicate that many different cells are capable of utilizing similar force-sensing, force-generating and force-bearing systems, suggesting that, for many tasks, cells can use a common tool set and phenotypic differences result from differences in the extent and location of use of one tool versus another. Common tool sets and functional complexes offers the possibility of developing engineering capabilities for intracellular components that could be useful for multiple cells types and tissues which is the part of the promise of Nanomedicine. For example, movement of the T cell receptor and ICAM during immune synapse formation involves an active movement, and mechanically perturbing the process leads to dramatically different synapse morphology and function (Figure 2). Understanding this function in one situation should provide insights into similar functions in other situations. We will develop and test models of mechanotransduction at the cell and molecular level. Nanotechnology developed in our center will enable us to rapidly screen for the optimal matrix rigidity, form, and spacing needed to elicit a given function. Our plan is to customize these technologies to test bioengineering models of cell processes. We are addressing these questions at the single cell level, at the level of cell-cell interactions with a focus on the immune synapse, and in stem cell growth or differentiation. We are just beginning to examine clinically relevant problems of immune stem cells, metastasis, and cardiac hypertrophy.

Figure 2
Figure 2. Immune synapses take on dramatically different shapes when the lateral transport of T-cell receptors and ICAM molecules in supported bilayers is disrupted by barriers (seen in bright field). Patterns belie active transport processes that underlie them. Taken from Groves and Dustin, 2003.

Intracellular systems sense force and/or nanometer level geometry, transduce the cues into biochemical signals that are then processed over space and time to give mechanoresponses that then cause the cell to move and alter the mechanical cues, producing a new set of signals (Vogel and Sheetz, 2006). The long-term effects of these cycles determine whether cells grow or die, the shape of the organism, and whether many tissue functions are effective. Intricate intracellular protein networks integrate mechanical cues over many length scales. We will need an understanding of how forces regulate signaling pathways and gene expression. For nanomedicine to become a reality, our goal is to fully understand and characterize these physical phenomena. Generating a well-defined, and extensive knowledge base of cellular forces and employing an engineering approach allows us to view the cell as a "complex machine" that can activate highly specialized tool sets to accomplish the necessary tasks at a number of different hierarchical levels.

Accomplishments

Matrix engineering

Cellular remodelling of extracellular matrix fibers is a major determinant of the structure of tissues and organs. We currently have few tools to measure the forces and the details of the fiber movements In particular, collagen fiber remodeling is critical during wound repair. We have described a mechanism of collagen fiber transport in three-dimensions, however, quantification of the force generated in this phenomenon has not yet been undertaken. Further, movements of fibronectin and other matrices have not been studied. Elucidation of average forces generated by various cell types on collagen fibers could then provide a simple method for assaying deficiencies in tissue repair and organogenesis. The goals of this project revolve around developing an efficient and robust method for determining the amount of force generated by cells on ECM fibers in a three-dimensional environment. In the past, studies of this type were conducted in two-dimensions, but recent studies showed marked differences in cell morphology and motility when exposed to two-dimensional and three-dimensional environments. For this reason, it is necessary to establish a novel three-dimensional environment to undergo relevant quantification of force generation.

In our studies, nanofabricated molds (Fig. 3) were used to produce PDMS nanofences substrates that were subsequently electrospun with silk fibers. These silk fibers were later coated with ECM proteins (collagen in Fig. 4). Another strategy has been to mechanically generate fibronectin fibers on the fences. Once the nanofence substrates have been covered with silk fibers coated with ECM proteins or with fibronectin fibers, the force generated on fibers can be measured as well as the effect on cell behavior. A variety of different strategies can be used to measure the cellular manipulation of the fibers or movement on the fibers. Stem cell movements and differentiation on the fibers will tell us how these highly engineered matrix environments can alter cellular behaviors and forces. For example, cells on a substrate can be brought to the fibers on the fences and once a cell attaches to a fiber, laser scissors can be use to free the fence to which the cell is attached thus enabling the measurement of the force that the cell generates on the fiber. This project is now well engaged since issues of nanofences' production have been overcame with the use of the recently acquired Nanonex model NX-B200 nano-imprint machine. Many relate studies with altered matrix geometries are showing important aspects of the cell morphology as well as fiber composition and geometry on the cellular behavior.

Figure 3
Figure 3. Nanofences produced by KOH etching of silicon wafers are exactly reproduced in PDMS when negative molds obtained by nano-imprint techniques are used to cure PDMS.
Figure 4
Figure 4. DIC/fluorescence microscopy overlay image of PDMS nanofence substrates with electrospun silk fibers. Silk fibers were subsequently labeled with Cy5-collagen shown in red.

Immune Synapse

The movements of the membrane components to form the immune synapse clearly constitute an integral part of the t-cell-antigen recognition process (Figure 2). We have begun to unravel how specific genes control generation of forces that regulate the immune response. We have found that a gene that is critical for generation of allergic responses in animal models increases cellular forces in such a way as to break off immune cell communication. A separate gene product that is responsible for a human immunodeficiency when mutated allows immune cells to reduce forces to enter into new communication with neighbors. The activity of these genes is analogous to providing punctuation for cellular communication- starting and stopping the flow of information that is necessary for effective immunity. Incorrect punctuation can also lead to autoimmune disease and allergic responses. This process of regulating communication is dependent upon action of forces at the nanoscale.

We have also started to exploit our understanding of the immune cells force vocabulary and punctuation to print surfaces with instructions at the micro and nano-scale. Early efforts have results in novel surfaces that can instruct immune cells to activate molecules that help them survive, which is important for immunotherapy of diseases like cancer and chronic viral infections. We are in the process of evaluating how these novel surfaces can be incorporated into existing immunotherapy protocols with clinicians at the bedside.

ECM adhesion/ migration

Complexity of the Focal Adhesion Network: the "Adhesome"

A main goal of the Center is to elucidate the dynamics of the multiple components of cell adhesion sites, and their coordinated modulation during force generation or application. Focal adhesions are large, multiprotein complexes that provide a mechanical link between the cytoskeletal contractile machinery and the extracellular matrix. They exhibit mechanosensitive properties; they self-assemble and elongate upon application of pulling forces and dissociate when these forces are decreased.

Members of our center provided new insight into the molecular complexity of the "integrin adhesome". In a comprehensive literature survey, combined with critical evaluation of the data, an updated "connectivity map" was established (Fig. 5), presenting the molecular and functional inter-relationships between 153 focal adhesion components connected via ~650 links. Each link was further characterized as a binding interaction or enzymatic modification. The complex adhesome network could be subdivided into different functional views ("subnets") within this network that are involved in switching on/off many molecular interactions, affecting cell adhesion, migration and cytoskeletal organization. Examining the adhesome network subnets revealed a relatively small number of key motifs, dominated by 3-component-complexes where a scaffolding molecule recruits both a signaling molecule and its downstream target to the adhesion site. In our recent paper (Zaidel-Bar, et. al., 2007), we discuss the role of the different network subnets in regulating the structural and signaling functions of cell-matrix adhesions.

Future Applications

The development of novel procedures for modulating the growth of mesenchymal stem cells for cell-based therapies is a major challenge in current biomedical research. We need to define culture conditions that instruct stem cells to remain pluripotent and avoid differentiation, or conversely, to drive these cells to undergo specific differentiation processes. Most efforts are geared toward discovery of specific soluable ligands that determine the fate of particular cell types. We plan to develop specialized adhesive "scaffolds" that can affect stem cell development. The concept is to develop an ability to screen many different architectures (bumps, holes, lines (fibers), matrix patterns, and rigidities). We will determine the better surface for control of cell behavior, then design a second generation of surfaces based on the first screen to yield the best surface for the desired parameter, e.g. growth in an undifferentiated state. Different media will be tested to determine whether the media affected the optimal architecture. In vitro studies are planned, but many of the findings could affect how patients are treated during the wound-healing process.

Consider the example of immune stem cells. Following maturation in the thymus, T cells enter a series of differentiation choices, leading to production of many classes of cells, including memory, regulatory, and effector as well as numerous subtypes. Memory cells exhibit extended life-spans which can include long periods of senescence, combined with the ability to respond to appropriate stimulus with rapid proliferation and further differentiation, and thus act as stem cells in the context of the immune system. The ability to control the differentiation process ex vivo, including the use of beads or other engineered surfaces to direct cells, has wide impact on a range of immunotherapy approaches.

There are many new tools to measure and generate forces at the submicron level, to organize molecules at the nanometer level, and to simulate the effect of force on protein structure. We have plans for additional tools to measure new parameters that are biologically relevant; these tools are conceptually feasible but still require significant development. In addition, we plan to extend our well-established techniques to new dimensions and new systems. For example, we have implemented devices for measuring submicron 2-D forces and have plans for modifying those devices to enable 3-D force measurement (aided by magnetic beads and wires as well as molecular force probes integrated into force-bearing elements). The tools that we are developing are flexible, and we are committed to helping other labs apply them to their specific systems. Further, the software that we are generating has general application to other cell systems and we will make that available. Finally, the modeling of the systems will be done in a format that can be easily applied to other systems. Since we are only addressing a small fraction of the force-dependent effects, we expect that there will be many more analogous systems where the technologies that we develop will find application.

References

Background

Current NDC supported papers

Contact Information

Center Website

Directors

Key Investigators