The lateral membranes between adjacent epithelial cells are critical for establishment and maintenance of cell morphology, adhesions and cell-cell communication. It has been suggested that there is directed actin filament flow beneath the lateral membranes of cultured epithelial cells, raising possibilities that the flow generates force that is implicated in regulation of above important cellular phenomena at epithelial lateral membranes. However, little is known about properties of the actin flow and its physiological function. In this project, I characterize dynamics of actin flow in cultured epithelial cells by single-molecule speckle (SiMS) analysis. I also determine the flow’s physiological functions by combining SiMS analysis with cell biological approach.

To characterize the actin flow at lateral membranes, I determine the speed and direction of the flow by SiMS analysis using dual-focus imaging optics. I will observe the cultured epithelial cells in monolayer, cells undergo migration of epithelial cell sheets and cells establishing cell adhesion. The roles of the actin flow will be examined by: a) characterizing effects of actin- and myosin-perturbing drugs on the actin flow and the cellular phenomena at lateral membranes; and b) comparing the molecular dynamics from the actin flow to the lateral membrane components including cell-cell adhesion molecules.

While molecular mechanisms of lateral membrane organization of epithelial cells have been well characterized, the possibility that force generated by the actin flow might regulate cell morphology, adhesion or cell-cell communication has never tested. In addition, I newly develop three-dimensional SiMS analysis by using dual-focus imaging optics in this project. This work will be the first to show actin dynamics in 3D characterized by the fluorescent speckle analysis. This project of actin flow dynamics and function at lateral membranes is unique in the epithelial tissue morphology field, and is pxpected to provide novel and fundamental regulatory mechanisms of lateral membrane organization.