Research
Understanding the Self-construction of Living Systems
The long-term goal of our laboratory is to understand the means by which living cells and tissues are constructed from a vast array of molecular components. To accomplish this goal, we work with model systems ranging from Drosophila embryos to stem cell-derived cardiomyocytes (see below). The ability of living cells to generate, sense, and respond to mechanical forces is a key aspect of their ability to self-organize on every length scale. For that reason, we often incorporate tools drawn from single-molecule and soft-matter physics into our research. We are also increasingly drawn to cryo-electron tomography (cryo-ET) as a tool for directly visualizing how cells are constructed at the molecular level. Over the next decade, we anticipate that these efforts will contribute to a comprehensive and quantitative understanding of how the forms of living cells, tissues, and organisms arise from the interactions of their molecular components.
Planar Cell Polarity
During embryonic development, many epithelial cells must be oriented relative to their surroundings. Directing the cells requires activity of the planar cell polarity (PCP) signaling pathway. This pathway provides directionality within the layer of cells and distinguishes one end of the developing tissue from the other. The molecular mechanism underlying PCP signaling remains poorly understood. Therefore, we use advanced microscopy techniques to study how molecular interactions are responsible for the PCP pathway inside living organisms. In collaboration with the the Axelrod Lab, we use fruit flies that have a suite of highly developed genetic tools and exhibit a well-characterized PCP patterning in their wings. Acquiring an increased understanding of the PCP pathway may one day contribute to preventing developmental anomalies such as heart and neural tube defects.
Force-mediated Gene Regulation
Cells are subjected to a wide variety of chemical and physical cues from their microenvironment that signal for shifts in gene expression to coordinate specific cellular responses. Significant focus has been placed on how biochemical signals impact fundamental cellular processes such as stem cell differentiation and cell migration, yet mechanical forces are known to impact these same processes and their roles remain poorly understood. In collaboration with the Wysocka Lab, we are utilizing various imaging techniques (such as live-cell imaging and correlative light and electron microscopy) in concert with biophysical and biochemical approaches to determine how mechanical force impacts chromatin structure and organization in both mouse and human embryonic stem cells.
Cryo-ET of Cardiomyocytes
Cryogenic electron tomography (cryo-ET) is the highest resolution 3D imaging technique available to the life sciences, capable of visualizing molecular complexes in their native cellular environments at nanometer resolution. We previously developed a technique to improve the throughput of cryo-ET data acquisition at cell-cell junctions through micropatterned electron microscopy grids, and we are currently applying this technique to study heart muscle cells, or cardiomyocytes (CMs). Mutations that affect the contractility of CMs are implicated in numerous cardiomyopathies, but the basis of pathogenesis is poorly understood. In collaboration with the Dahlberg and Bernstein Labs, we are determining the ultrastructural ramifications of disease-associated mutations and investigating how such structural changes could lead to disease.
High-Throughput Screening of Catch Bonds
Many proteins involved in cell adhesion, motility, and contraction are sensitive to mechanical forces. In fact, a subset of proteins experience stronger interactions with their binding partners under load, exhibiting properties of a “catch bond.” Conventionally, catch bond behavior is assessed through optical tweezers and other single-molecule assays, which limits the throughput of molecular discovery. In collaboration with the Fordyce Lab, we will design and implement custom microfluidic devices to spatially array, purify, and measure 1,500 unique proteins under force. These high-throughput measurements of protein function under force will empower the discovery, engineering, and characterization of force-sensitive proteins, and mutational scanning may reveal molecular determinants of catch bonds and their biological relevance in vivo.
Engineering a New Class of Molecular Tension Sensors
While existing FRET-based force sensors have yielded many insights into the ways by which forces are transduced across proteins, the complex optical setups and data analysis required for measuring and interpreting FRET have limited their use. We have engineered a compact, 11 kDa molecular tension sensor termed STReTCh (Sensing Tension by Reactive Tag Characterization) that does not rely on experimentally demanding FRET-based measurements and whose use follows typical fix-and-stain protocols. STReTCh reliably detects forces above 2 pN, making it one of the most most sensitive molecular force sensors described to date. As proof-of-concept, we have demonstrated that an extracellular STReTCh-based sensor reliably visualizes cell-generated forces at integrin-based adhesion complexes. In addition, we have incorporated STReTCh into vinculin, a cytoskeletal adaptor protein, as a genetically-encoded sensor of intracellular force and show that STReTCh reports on forces transmitted between the cytoskeleton and cellular adhesion complexes. We are currently building upon this work by engineering new variants of STReTCh to enable streamlined, live imaging of force in cellular systems to probe the spatiotemporal dynamics of force transmission and improve the accessibility of force measurements in biological systems.
