The β-catenin protein functions in regulating transcription activity following Wnt signaling. β-catenin recruits transcription factors from the TCF/LEF family to the promoter region of several genes to facilitate gene activation. We are investigating the intracellular dynamics and localization of β-catenin in response to Wnt signaling and during cell cycle progression. We use a cell system that enables the detection of β-catenin in vivo using live-cell imaging based on HEK293 and U2OS cells stably expressing YFP-β-catenin. Under steady state conditions, the YFP-β-catenin protein is expressed mainly in the cell membrane mimicking endogenous β-catenin, and following Wnt activation, significant accumulation in the nucleus and the cytoplasm are observed. Previous studies have shown that β–catenin co-localizes with the centrosome and we wish to further investigate these dynamics using live-cell imaging. Measurements of the β-catenin increase in the membrane, nucleus, cytoplasm and centrosome have revealed similar response dynamics to Wnt. While imaging cells under Wnt activation conditions we observed an interesting phenomenon where β-catenin puncta were detected moving from the cell membrane into the cytoplasm. The movement appeared to be in a directed manner, towards the centrosome area. Since β-catenin puncta movement seemed to be oriented and since microtubules are concentrated at the centrosome, we hypothesized that β-catenin might be traveling on microtubules, and are conducting experiments along these lines. In addition, in order to investigate the influence of the cell cycle on the β-catenin response, we created cells that in live-cell imaging could be identified according to their stage in the cell cycle. Fucci is a reporter gene system containing mCherry-Cdt1 and CFP-Geminin. Cdt1 is a protein that is mainly expressed during G1/S phase while Geminin is a protein that is mainly expressed during G2/M. Experiments performed with the Fucci system show that the β-catenin response to Wnt signaling varies from cell to cell. Namely, cells at the same stage of the cell cycle will respond differently to Wnt signaling. Altogether, we are able to show for the first time the response dynamics of β-catenin to Wnt signaling in real time.
