Stanford University

Understanding the 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 endeavor will be highly relevant to many aspects of basic research and 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.

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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 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.

People in this line of research: Silas


CryoET

Cryo-electron tomography (cryoET) is the highest resolution method to visualize molecular complexes in their native cellular environments. We developed a technique that improves the throughput of data acquisition by positioning regions of interest of cells and cell doublets in an imageable regions of the electron microscopy grid. We are using cryoET to visualize the molecular-scale architecture of intact, flash-frozen cell-cell junctions. Ongoing work will uncover the identity, composition, and organization of the cellular structures that mediate intercellular adhesion. We hope to create the first molecularly detailed picture of the organization of a cell-cell junction.

People in this line of research: Claudia

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Physical basis of gliding motility

We are working to understand the physical basis of gliding motility, a type of cell locomotion used during host infection by single-celled parasites like Toxoplasma gondii or Plasmodium species, which cause malaria. Gliding differs fundamentally from other known mechanisms of eukaryotic cell motility, like paradigmatic cilia-dependent swimming and cell-shape-change-dependent crawling. We know that gliding depends on actin filaments and the molecular motor myosin, but how are these molecules organized in order to generate directional force and actually move the cell? In particular, we wondered how the stereotypic rigid body motions seen during gliding arise from the collective organization of actin filaments and myosin motors at the parasite surface. To help solve this puzzle, we are imaging single actin and myosin molecules in live gliding Toxoplasma gondii parasites and developing theoretical and computational infrastructure for predicting cytoskeletal self-organization on complex shapes. We have found that two distinct classes of emergent actin flows – driven by an anchored carpet of myosin motors and tuned by filament turnover rate – can explain all four observed Toxoplasma gliding modes. This project is a collaboration with Li-av Segev Zarko, John Boothroyd, and Rob Phillips.

People in this line of research: Christina


Mechanism of catch bond interactions

Many adhesion proteins sense and transmit mechanical load by binding to F-actin, which mediates long range organization of epithelial tissues. In several types of proteins, their binding interaction to actin is strengthened under load - a property known as a catch bond. We study the mechanism behind the catch bond property in the cadherin-catenin complex with optical tweezers, and build kinetic models to simulate how these complexes may be further stabilized by neighbors and binding partners.

People in this line of research: Amy

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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.

People in line of research: Brian

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