Team:Harvard/Description
Project Inspiration and Description
Why Shear-Stress Sensors?
Shear stress acts tangentially upon all endothelial cells in mammalian vasculature when exposed to blood flow. As such, endothelial cells must respond to different levels of shear stress in order to properly remodel themselves and the tissue around them to roll with the strain. Various membrane mechanosensing proteins, including G-protein coupled receptors and ion channels, are activated by shear stresses, permitting signal transduction for this vascular remodeling.
Despite the many signaling pathways identified in the literature, little work has been done to develop synthetic transcription factor networks directly modulated by shear stress. The few but notable contributions include the work of the iGEM team at Southern University of Science and Technology in 2016, using mechanosensors Piezo1 and TCRP5 to detect sound, and the manuscript published by Kis et. al. in 2016, describing linkage of activation of shear stress sensing G-protein coupled receptor BKRB2 to GFP response.
How will we contribute?
We seek to build on these efforts with the help of a vector technology in its infancy: the human artificial chromosome. It will allow us to express several very large genes at once from a single vector that is exceptionally stable in human cells. Using this vector, we plan to engineer human cells to present distinct fluorescent protein responses to different magnitudes of shear stress. To do so, we will use three membrane proteins: BDKRB2, GPR68, and Piezo1. Each fluorescent protein response will itself be graded to proportion of receptors activated on the membrane, according to the receptor’s activation curve, giving a spectrum of signal indicating magnitude of shear stress within the physiologically relevant range of 0 to 6 Pascals.
More specifically, when the levels of shear stress are low (0 - 2 Pascals), only the most sensitive receptor on the cells, BDKRB2, will be activated, and this will initiate a pathway that leads to the expression of a green fluorescent protein (GFP). Similarly, when the levels of shear stress are between 2 and 4 Pascals, the GPR68 receptor will be activated and lead to the expression of a blue fluorescent protein (BFP), in addition to the GFP which is expressed as long as the level of shear stress is above 0 Pascals. If the shear stress levels are above 4 Pascals, both G-protein coupled receptors will be activated, as well as the ion channel Piezo1, which would initiate expression of a red fluorescent protein (RFP) in addition to the GFP and BFP.
Due to the nature of BDKRB2 and GPR68, which are both G-protein coupled receptors, we are able to take advantage of a linking system called the Tango assay, developed by Barnea et. al. in 2008, to directly link activation of the protein to fluorescent protein expression. For Piezo1, we will tie calcium influx into the cell to RFP expression.
Figure 1. In the Tango assay design, the conformational change of a GPCR initiated by shear stress is linked to a fluorescent protein response via a “linker.” This linker is composed of a viral protease, N1a, fused to β-Arrestin2. A “sensor” module is composed of the actual GPCR, a protease cleavage site, and a transcription factor. When activated, the GPCRs associate with β-Arrestin2 which leads to release of the transcription factor upon cleavage by the viral protease. (We are using N1a protease for both GPCR modules.) The transcription factor released upon activation of BDKRB2 is tTa, which binds to a promoter upstream of a GFP gene. Similarly, transcription factor GAL4 is linked to activation of GPR68, and it binds to a promoter region upstream of a BFP gene.
Figure 2. Upon activation by shear stress in excess of 4.5 Pascals, Piezo1 ion channels opens, allowing flow of extracellular Calcium ions into the cell. These ions activate the Calmodulin protein, which can then form a complex with Calcineurin. This protein complex catalyzes the dephosphorylation of NFAT proteins, which then localize to the nucleus and bind the NFAT promoter, driving expression of the RFP reporter.
Applications
Pathologies such as blood clots and atherosclerosis are identifiable by abnormally high and low shear stresses, respectively. High shear stress itself can also be thrombogenic. Shear stress in the vasculature is important in development, and also is necessary to promote maturation of endothelial cells into an elongated morphology.
We envision several possible applications to our shear stress sensing cells. Currently, shear stress in in vivo systems, such as avian embryos, is not directly measured but instead calculated by velocimetric analysis of microparticles injected into the blood. These calculations are intensive due to the odd vasculature geometries and local variation in viscosity from blood composition, and circulation time of the microparticles cannot be extended much beyond 10-16 hours, in the case of avian embryos, limiting study time. Our cells could directly measure in vivo shear stress on the vascular wall, potentially providing longer study times and ease of use for studies of vascular diseases, developmental biology, and drug design.
Another possible use for our cells is in the realm of tissue engineered vascular grafts, like those recently used for coronary artery bypass surgery. One method of producing these engineered grafts is by cell sheet self assembly, where a patient’s cells are cultured in vitro in a bioreactor. As exposure to laminar flow is important for endothelial cell maturation, aiding robustness of the grafts, our cells could serve as indicators for proper shear stress levels.
Additionally, in the future, when in vivo transfection of our gene constructs is easier, we envision the use of these mechanosensing systems to identify blood clots and thrombogenic regions for targeted secretion of tPA or other clot-busting proteins in real time. This treatment would significantly lessen a patient’s dependence on prophylactic blood thinners, which pose significant bleeding risks to their users.
References
- Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., Lee, K.J. The Genetic Design of Signaling Cascades to Record Receptor Activation. Proceedings of the National Academy of Sciences of the United States of America, 105 (1) 64-69 (2008).
- Kis, Z., Rodin, T., Zafar, A., Lai, Z., Freke, G., Fleck, O., Del Rio Hernandez, A., Towhidi, L., Pedrigi, R.M., Homma, T., Krams, R. Development of a Synthetic Gene Network to Modulate Gene Expression by Mechanical Forces. Scientific Reports 6 (2016).