The role of mechanical signaling in stem cell self-renewal and differentiation
In a seminal Cell paper in 2006, Engler et al. showed that mesenchymal stem cells can adopt different lineages (differentiate to various cell types) based on the stiffness of the underlying substrate. This work highlighted the notion that mechanobiology has a profound impact in stem cell differentiation. Yes-associated protein (YAP) regulates organ size in Drosophila and cell proliferation in mammalian epithelia, and YAP is also implicated to influence cell-fate decision during embryonic development. Nishioka (Developmental Cell, 2009) showed in mouse embryos that YAP is nuclear in trophectoderm (which becomes the placenta) and cytoplasmic in inner cell mass (which develops into the actual organism). Dupont et al. (Nature, 2011) showed that YAP localization is stiffness-dependent in epithelia.
We are poised to investigate whether mechanical forces also influrence YAP localization in embryonic stem cells (ESCs). We would like to identify how mechanical forces translate into chemical signals to influence YAP localization and stem cell differentiation. Additionally, we can utilize tools that we have developed to measure intracellular forces at the molecular scale and also have the breadth of stem cell expertise in the Stanford Hospital to aid us in our endeavors.
Measuring forces on adhesion complexes at the single molecule level
Living cells are extremely responsive to mechanical cues, yet how cells produce and detect force remains poorly understood due to a lack of methods that visualize cell-generated forces at the molecular scale. We have developed molecular tension sensors that allow us to directly visualize cell-generated forces with single-molecule sensitivity. We apply these sensors to determine the distribution of forces generated by integrins, a class of cell adhesion molecules with prominent roles throughout cell and developmental biology. The experimental setup and results of our studies can be seen in the figure shown here and in our published paper. Link
Uncovering previously unrecognized functions of mechanical force in extracellular matrix remodeling
The stiff, the hard, and the brittle are harbingers of death. – Tao Te Ching trans. Stan Rosenthal
The benefits of flexibility, both literal and metaphorical, have been recognized for over 2,500 years. The material properties of our own bodies is governed largely by the extracellular matrix, a complex protein and carbohydrate network that gives shape to tissues and organs. Previously, the ECM was dismissed as passive glue that simply held cells together. We now know that failure to maintain the ECM leads not only to aching knees and wrinkles, but also to atherosclerosis, aneurism, and other cardiovascular diseases. Furthermore, the key step in cancer metastasis is the dissolution of the local ECM so that the cancer cells can escape to colonize the rest of the body.
We are using techniques ranging from single-molecule assays to live cell imaging to test the hypothesis that mechanical force is an unrecognized regulator of extracellular matrix (ECM) remodeling. We are particularly interested in the possibility that mechanical force may increase the susceptibility of ECM proteins to proteolysis by matrix metalloproteinases, enzymes that are the subject of intense medical interest due to their roles in cancer metastasis. This idea has been difficult to test due to the challenge of visualizing ECM structure and mechanical forces at the cellular length scale. Novel instruments and assays developed in our research group allow us to explore the interplay of force and ECM remodeling for the first time.
Understanding how myosin uses chemical energy to generate force and motion
Conventional myosin generates force in muscle, but other myosins play diverse biological roles, including transporting cargo throughout the cell. We will use sophisticated biophysical techniques to directly observe the motion of single myosin molecules in order to better understand how myosin converts chemical energy into useful motion. This project also has a more universal applicability. Recent work suggests that enzymes in general may derive their incredible catalytic ability by coupling protein motion to bond making and breaking. Single-molecule measurements on myosin offer a potentially powerful way to test this idea.
At right: A three bead optical tweezer setup to study the mechanism and function of motor proteins with nanometer spatial resolution and millisecond temporal resolution. The trace shows an example of an event (black) when the tweezer was oscillated (grey).
Design of new motor proteins
Artificial motor proteins have wide-ranging potential applications in fields like bottom-up nanofabrication, medical diagnostics, and responsive “smart” materials. Our group will use iterative rounds of protein design, screening, and single-molecule characterization to generate molecular motors with novel capabilities. Our work in creating novel motor proteins will test the validity and usefulness of the models of protein physics developed previously.
At left: A de novo designed switch protein. The lever arm helix (red) packs across the scaffold helices (blue).
Role of intercellular forces in cell and developmental biology
The generation and detection of mechanical force is a central aspect of cell and developmental biology. Cells sense their physical surroundings by pulling on each other and the extracellular matrix (ECM). The resulting physical cues allow cells to communicate with each other, to coordinate complex collective movements, and to make critical decisions about cell growth and differentiation. Despite this central importance, the mechanisms by which cells exert and detect force remain poorly understood both in single cells and in whole organisms.
Our goal is to understand how cells generate, detect, and respond to tension at the molecular level. To do so, we are using new microscopy techniques that allow us to measure mechanical forces inside living cells, and even in whole organisms. The results from this project will be highly relevant to many aspects of human health, including heart disease, cancer metastasis, and the development of stem cell therapies, all of which are governed in part by the mechanical interactions of cells with their surroundings. (Above: An osmotic shock assay where a cell expands or contracts depending on the osmolarity of the solution it finds itself in. Cells use different mechanisms to protect themselves from situations of rapid increases or decreases in volume).