To understand the changes in adhesion and motility, we used confocal R428 fluorescence microscopy to monitor the localization and expression of the adherens junction proteins E-cadherin, b-catenin and Zo-1 in matched samples of parental, LGR5 knockdown or LGR5 overexpressing LIM1899 cells. Fixed cells were incubated with the appropriate INCB28060 antibodies and fluorescent secondary antibodies, and co-stained with rhodamine-phalloidin to visualize actin. Neither the levels nor distribution of E-cadherin changed significantly with up-or down-regulation of LGR5, however there was a consistent recruitment of b-catenin to the cell junctions in LIM1899 overexpressing LGR5. This was surprising as the LIM1899 cell line carries an activating b-catenin mutation resulting in a predominantly cytosolic bcatenin, with some weak association to the membranes but rarely seen at the cell-cell junctions; this distribution is insensitive to wnt signalling stimulation or inhibition. The tight-junction molecule Zo-1 was also increased at the cell-cell contacts in LIM1899 cells overexpressing LGR5. The relative amounts of these proteins in cells under- or over-expressing LGR5 were also assessed by immunoblotting, confirming little change in total b-catenin levels, marginal increase in Ecadherin, and significant increase of Zo-1. Overall, these results are consistent with an enhancement of cell-cell adhesion in cells overexpressing LGR5. Given the effects of LGR5 modulation on cell migration, we hypothesized that LGR5 levels might affect, directly or indirectly, the expression or localization of adhesion molecules. CD44, CD133 and CD166 are adhesion molecules expressed on intestinal stem cells and colorectal cancer stem cells and therefore can be expected to have overlapping expression patterns to LGR5. As these molecules have been used extensively as stem cell markers, we also wanted to assess whether changes in LGR5 expression resulted in altered patterns of expression for these markers. CD133 is not expressed on LIM1899, as assessed by flow cytometry, while CD44 and CD166 are expressed at high levels. Only CD44 surface expression is weakly enhanced by upregulation of LGR5 in these cells, but there is no appreciable change in total cellular CD44 as assessed by immunoblotting. Confocal microscopy revealed that the cell surface distribution of CD44 is subtly altered in LGR5 knockdown cells. In parental LIM1899 cells and in cells overexpressing LGR5, CD44 associates with actin rings as assessed by morphology and colocalization with actin. When LGR5 expression is reduced or abolished by inhibitory RNAs, CD44 is more uniformly distributed on the cell surface and is missing selectively from the focal actin rings. This phenomenon was observed consistently with knockdown of LGR5, either by siRNA or shRNA, in both Lim1899 and LIM1215 cells. The distribution of CD44 in LIM 1899 cells overexpressing LGR5 was unaltered.