Project Summary Leukocytes must be able to infiltrate and migrate within essentially all tissues of the body in order to deal with infections and damage that occur throughout the host. Molecular signals like chemoattractants and adhesive ligands are critical for this process, but immune cells also sense and respond to mechanical cues. While most tissues of the body are relatively static, the intestines are mechanically dynamic due to the repetitive contractions of the smooth muscle layers, which apply compressive, stretch, and shear forces to the tissue. These forces are altered during intestinal infections and chronically dysregulated in inflammatory bowel disease, pointing towards a relationship between intestinal mechanics and inflammation. Leukocytes are exquisitely mechanosensitive, but it is presently unknown if they sense or respond to mechanical cues in the intestines directly. Investigating these forces in rodent models is challenging since the intestinal tissue needs to be physically immobilized for intravital imaging. Here, we propose to investigate the role intestinal forces on immune cell function by using the zebrafish system. Intestinal T lymphocytes can be directly visualized in this system without any surgical manipulation or tissue immobilization. With pharmacological and genetic tools that interfere with smooth muscle function, we can study intestinal T cell behavior in the presence and absence of mechanical deformation. In preliminary data, we have found that intestinal T cells migrate by a distinct strategy in the intestines relative to static tissues like the skin or gills, one characterized by thin, filopodia-like protrusions that undergo successive branching to propel the T cell forward. Blocking intestinal movement with smooth muscle inhibitors severely impairs T cell motility within the intestines, but not in static tissues like the skin. Collectively, these results suggest that mechanical cues dictate T cell migration strategies in the intestines. This proposal will investigate how T cells sense and respond to deformation in the intestines. Specifically, we will test the hypothesis that intestinal deformation activates the ion channel Piezo1 to promote filopodia-like migration. To our knowledge, this will be the first study to investigate how intestinal forces influence gut immunity and our findings could have broad implications for the diagnosis and treatment of inflammatory disorders of the gut.