The questions
Our bodies are made up of trillions of cells that somehow manage to stick together while carefully controlling what passes between them. This remarkable feat is achieved through specialized structures called tight junctions (TJs)—intricate protein assemblies that act as molecular “zippers” and selective gatekeepers between adjacent cells. But TJs do more than just control traffic of water and solutes—they help cells and tissues polarize in order to tell top from bottom and distinguish inside from outside, and contribute to the monitoring of contacts with neighbors to detect tissue damage or need for expansion. Found primarily in epithelial and endothelial tissues throughout the body, but also in between neuronal axons and the glial cells that insulate them, TJs are essential for maintaining distinct compartments within our organs. We are fascinated by how these microscopic barriers encode what to let through differently in various tissues throughout the body, while also serving as sophisticated cellular sensors that monitor tissue integrity, geometry, and proliferation. How do they do it?
The approach
We are taking a two-pronged approach to understanding TJs. We study them “top-down” by examining how they function within the complex environment of living cells and tissues. This allows us to see how the entire system works together, like watching a city’s entire transportation network in action. At the same time, we are taking a “bottom-up” approach, building TJs from purified components, akin to taking apart an engine reassembling it piece by piece, allowing us to understand how each molecular interaction contributes to the TJs’ architecture and functions of tight junctions, down to the level of individual atoms and chemical bonds. By combining these complementary approaches, we can bridge our understanding from the atomic scale to the tissue level.
The tools
We are bringing together an arsenal of cutting-edge techniques to crack this molecular puzzle. In the lab, we produce and purify TJ proteins in large quantities, allowing us to measure how they interact and function in carefully controlled conditions. We then use leverage powerful electron microscopes and synchrotron X-ray sources at in the Chemistry and Biochemistry Department and the California NanoSystems Institute (CNSI), and national facilities—to elucidate the 3D-structures of these protein assemblies all the way to atomic detail by single particle cryo-electron microscopy (cryo-EM) and X-ray crystallography or reconstruct 3D-shapes of TJs in their native cellular environment by cryo-electron tomography (cryo-ET). To connect our molecular discoveries to living systems, we engineer cells using CRISPR technology to watch and manipulate TJs in action: we measure how TJs control what passes between cells, maintain cellular organization, and regulate tissue growth, while using proteomic tools to track the precise the TJs’ composition and dynamics.
The relevance
Understanding these cellular gatekeepers could revolutionize how we treat disease. When TJs malfunction or are hijacked by pathogens, cells can lose their ability to control what passes between them, their sense of direction, and their awareness of tissue organization—a cascade of problems that can lead to various health issues, including widespread gastrointestinal and neurological disorders. The breakdown or modification of TJs plays a crucial role in cancer progression, allowing malignant cells to lose their organized structure and spread throughout the body. During heat strokes, loss of TJ barrier in the intestines can lead to lethal evasion of the gut bacteria into the bloodstream. By understanding how these barriers work, we might find ways to repair them when they are broken, or temporarily open them to deliver therapeutics exactly where they are needed.