three-dimensional-culture-murine-colonic-crypts-to-study-intestinal

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function <em>Ex Vivo</em>

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

East Carolina University

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin-matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z-axial view of noncancerous MDCK-II and PZ-HPV-7 cell-cell junctions resolved β-catenin and p120 localizations to TJs and AJs, with p120 apical to β-catenin and colocalizing with TJ protein claudin-7. More basally, p120 and β-catenin become colocalized. This topography was lost in isogenic Ras-transformed MDCK cells and cancerous PC3 cells, where p120 becomes basally localized in relation to β-catenin. Claudin-7 gene conditional knockout (cKO) in mice also have altered polarity of p120 relative to β-catenin, like that seen in normal-to-cancer cell phenotypic transformation. Additionally, claudin-7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin-1, zonula occludens-1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical-lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans-compartmental modulation of protein functions.

Amna N. Naser

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