2008 Progress Report – Executive Summary
The Nanotechnology Center for Mechanics in Regenerative Medicine has focused on the mechanisms by which nanoscale changes in cellular environment alter cell fate in health and disease. Several principles are emerging: 1. The stretching of proteins provides a primary cue for force-induced signaling. 2. Immune synapse formation involves mechanically directed molecular sorting. 3. Membrane geometry and cell shape play a major role in the signals that the cell generates at membrane-bound adhesion complexes 4. Organizing large multiprotein networks into functional modules can be performed with a new algorithm capable of sorting functionally meaningful proteins and pathways in the adhesome network. 5. Extracellular matrix deposition and mechanical stretching can alter the fate and behavior of attached cells.
Integration of nanoscale technologies, combined with cell biology and computational modeling has provided new quantitative insights into environmental regulation of cell function. These advances were made possible by the interaction of Center members working toward the common goal of understanding important aspects of cellular mechanics at the nanometer scale. The tools that have been created are now ready to be applied to the engineering of specialized environments for directing growth, differentiation and fate of immune cells, fibroblasts and stem cells.
Recent publications and meetings have highlighted the importance of a finely-tuned combination of physical and biochemical factors in tissue regeneration, cancer and a variety of diseases. In cancer, for example, there is evidence that the metastatic seed cells have a greater probability of growing in the right tissue environment, and that physical factors co-regulate their proliferation and differentiation. A similar impact of physical factors has been described for stem cells.
An integrated interdisciplinary approach to understand how physical factors are sensed and produce appropriate responses over time to develop (regenerate) and maintain the proper tissue shape and function is urgently needed. The read-out of physical factors by cells needs a transduction machinery that operates synergistically with, yet in many aspects rather differently from, biochemically triggered cell signaling responses. Our goal is to discover and quantitatively describe the underlying mechanisms, from the molecular transduction mechanisms to coordination of coherent responses at the cellular level. A number of stereotypical cell motility processes were described that constitute subcellular modules. To measure and generate forces at the specific molecular-scale locations during these functions, a number of nanostructured devices were designed and built by a new high throughput process. Similarly, novel microscopy-based screening technologies were established for identifying genes involved in regulating cellular mechanics. Bioinformatics analyses of the many proteins involved in cytoskeleton, membrane and mechanosignaling provide important clues about interacting partners and RNAi screens for defects in rigidity sensing and matrix fiber deposition are providing targeted lists of proteins potentially involved in specific mechanical functions. New data gathering and analysis techniques enable rapid quantification of force, texture and geometry parameters that can be analyzed with new simulations. The convergence of these technologies offers novel tools for regulating cell activities and fate.
Nanoimprint lithography (NIL) has become a key ingredient in our preparation of surfaces for studying cellular behavior, allowing us to fabricate samples in far greater quantities (and far less time) than would be possible by direct-write electron beam lithography. All members of our nanofabrication team incorporate NIL into their fabrication schemes, and we have developed some new techniques and optimized others, making our NIL fabrication more robust and repeatable. We have developed processes for both shallow and deep (~ 10 μm) NIL; a new bilayer NIL resist scheme facilitates lift-off of metal patterns down to ~ 30 nm resolution; introduction of a new NIL template material, fluorinated diamond-like-carbon, enable easy separation of the template from the imprinted sample, a critical element of NIL; and a new self-aligned process allows us to routinely access sub-10 nm dimensions with NIL. In addition, we have developed new techniques for fabricating elastomeric surfaces with multi- and variable rigidity with micron-scale and nanometer-scale precision. These are critical to understanding the role of force transduction in cell function and behavior.
Force transduction in long-term cell culture has been linked to the mechanical unfolding of proteins from extracellular fibronectin (Vogel) to intracellular p130Cas (Sawada et al., 2006). A number of paradigms for the transduction process have now been identified. In the case of fibronectin, domain unfolding can expose a number of different activities from hidden epitopes to enzyme functions (ref). The definition of the stresses in cell-derived matrices has been shown to be changed over time as a function of both the matrix rigidity and the cell contractile behavior (REF). As we move to the cell membrane, we find that the integrins show force-dependent changes in affinity for fibronectin that appear somewhat as catch bonds, holding onto fibronectin for much longer times under stress than when relaxed. On the inside surface of the membrane, the integrin-binding protein, talin has been shown to have hidden helices that can form complexes with vinculin exposure. Steered molecular dynamics calculations indicate that those epitopes can be exposed by stretching the molecule and would stimulate vinculin binding (REF). The substrate priming of p130Cas and potentially other tyrosine kinase substrates provides a means by which the mechanical process of rigidity sensing could be transduced into changes in cell growth through small G protein activation and tyrosine kinase cascades. All of these systems can be activated in cell functions but we have evidence that specific processes are selectively activated as a cell receives specific mechanical cues that could then explain how physical environment is transduced into biochemical changes that alter cell function.
The immune synapse is an important process whereby immunological signals are converted into the programming of immune function. Once engaged, the movements of the receptor-ligand pairs follows the pattern of actin movements but there appears to be a weak coupling of the actin to the membrane proteins. 2. Immune synapse formation involves mechanically directed molecular sorting.
Modeling is an important part of developing an understanding of how physical factors can control cell function. The actin assembly machinery is involved in the movement of most plasma membranes either through the assembly of filopodia or lamellipodia. The assembly complexes push out the membrane with considerable force and also are linked to signaling pathways in the rest of the cell. We have developed a relatively complete model of the assembly process that is involved in early cell spreading. At another level, the geometry of the cell could be a critical factor in the signaling generated by membrane complexes, such as matrix adhesions. The surface area to volume is much greater in cell extensions; consequently, membrane generated signals can exhibit positive feedback and tip the balance toward a critical signal. With the finer definition of the cellular changes in response to geometry and matrix forces, we can better model how the cell will generate force and activate signals for the rest of the cell.
For many aspects of the analysis of force-dependent signaling, we need to be able to quantify images as well as identify linkages between functional protein modules and motile processes at the light microscopic level. An algorithm was developed to enable us to take the images of fluorescent proteins and convert them into quantitative data based upon sorting of linked image parameters. That same algorithm organizes large multiprotein networks into functional modules, by sorting functionally meaningful proteins and pathways in the adhesome network. With the definition of specific proteins involved in particular functions through our RNAi screens that are currently underway, we will be able to rapidly move to target the components of the functional modules. Knowing the full list of proteins involved in specific mechanical functions will make it much easier to work out the mechanisms of mechanotransduction and signal propagation.
Cells grown in culture for many days remodel their environment in many ways that can alter their behavior. Our studies indicate that some of the early matrix signals have long-term consequences on the cell behavior as well as the cell fate. Thus, it is important to properly set initial conditions to properly develop the desired cellular response. Systematic testing of the types of environments that will produce the optimal signals for different pathways is underway with the development of a test chip that will enable the survey of many environments simultaneously.
The Nanotechnology Center for Mechanics in Regenerative Medicine has begun to apply these technologies to the question of how does a stem cell or t-cell respond appropriately to mechanical aspects of their environment. Such a coordinated analysis of the physical parameters in conjunction with the biochemical changes has only been possible through such a center, where the many aspects can be approached in parallel and different types of expertise can be applied to the same cell system simultaneously. Our plan is to address the practical questions of how to program memory t-cells as a way of applying our tools to solve a clinical problem. Presentation of the signals in physically different patterns changes the cell response and we plan to develop the proper pattern for the appropriate signal generation.
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