1563 Role of Nance-Horan Syndrome protein in epithelial morphogenesis

Saturday, March 24, 2012: 9:45 a.m. - 11 a.m.
Presentation Type: Poster Session
R. SARMICANIC, T. WITTMANN, and P. KUMAR, University of California - San Francisco, San Francisco, CA
Null mutations in the NHS gene cause an X-linked genetic disorder, Nance-Horan Syndrome that is characterized by congenital cataract, dental abnormalities, and in few cases, mental retardation. Many of these phenotypes could be explained by defects in epithelial morphogenesis. NHS has been proposed to interact with the tight junction protein ZO-1 and regulate actin dynamics through an N-terminal WAVE domain. However, the function of NHS during epithelial morphogenesis or remodeling is unknown. Objective: To begin to understand NHS function in epithelial cells, the current study examined the localization and dynamics of full length NHS protein in a 3D epithelial cell culture model. Methods: An MDCK cell line stably expressing EGFP-NHS was generated and EGFP-NHS dynamics were investigated by live cell spinning disk confocal microscopy in 3D cell culture in which MDCK cells form apical-basal polarized, spherical cysts. Fluorescence Recovery After Photobleaching (FRAP) was used to determine the NHS turnover at cell-cell junctions. Results: EGFP-NHS localized to lateral cell-cell contacts in polarized epithelial cells, but was absent from the basal or apical cell surfaces. EGFP-NHS localization was distinctly different from ZO-1. This was different from previously reported colocalization of ZO-1 and NHS, and indicates that NHS is not involved in tight junction formation. However, EGFP-NHS co-localized with actin cables that run laterally between polarized epithelial cells. NHS turnover had an average t1/2 of 6.7±1.8s which was faster than t1/2 of ZO-1 with 119±21s. The recovery time for NHS further indicated that NHS is not a tight junction protein. Conclusion: Our results support the hypothesis that NHS is an integral part of cell-cell contacts in polarized epithelial cells, where it may regulate circumferential actin polymerization dynamics. This research was supported in part by the AADR Student Research Fellowship Program.
This abstract is based on research that was funded entirely or partially by an outside source: NIH/NIGMS R01GM079139

Keywords: Cell biology, Cell culture, Epithelium/epithelial, Molecular biology and Nance Horan