Abstract
Zonula occludens‐1 (ZO‐1) is a membrane‐associated tight junction protein that has been observed to participate in ATP‐dependent exchange via an intracellular pool. We hypothesized that a diffusion model can explain the ZO‐1‐C‐terminal domain exchange dynamics at the tight junction. We developed stable cell lines with GFP‐ZO‐1 carboxy‐terminus fusion constructs in Madin‐Darby canine kidney cells. Fluorescence recovery after photobleaching (FRAP) experiments were conducted using a Nikon A1R laser scanning confocal microscope to photobleach the localized construct and acquire intensity data over time. Recovery was analyzed by partitioning the bleach region of interest into one‐square micron sections or the entire bleach ROI. The local fluorescence recovery in the photobleached area yields curves that are modeled to be solutions of the diffusion process. Fitting experimental data to a one‐ or two‐dimensional diffusion model will quantitatively reveal if the exchange occurs within the membrane or via an intracellular pool, respectively. Future research will compare the behavior of different ZO‐1 constructs and relate the quantity of ZO‐1 expression to its recovery behavior.Grant Funding Source: Supported in part by NSF‐MRI #1229702 and NSF Award #0926702.
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