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Quantitative determination of β-catenin by immunofluorescence microscopy

par Bunel, Valérian ;Antoine, Marie-Hélène ;Nortier, Joëlle ;Duez, Pierre ;Stévigny, Caroline
Référence 12ème Journée des Doctorants en Sciences Biomédicales, Sciences Dentaires, Sciences Médicales & Sciences Pharmaceutiques (20/12/2012: Erasme, ULB)
Publication Non publié, 2012-12-20

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Résumé :

Introduction: Fluorescence image analysis of cells provides qualitative data that can help in assessing proteins expression and/or localization. However, the quantitative analysis of such images often remains unexploited. For example, the repartition of transcription factors between nucleus/cytoplasm is regularly checked, without making any assumption on the respective amounts. β-catenin is a protein playing a dual role in cells: (i) it takes part in cellular adhesion, by linking cadherins to the actin cytoskeleton; and (ii) it can translocate to the cell nucleus, associate with Tcf/Lef factor and specifically activate genes transcription [1]. The pivotal role of β-catenin has notably been studied in the context of kidney injuries especially involving renal proximal tubular epithelial cells (RPTECs), main targets of common nephrotoxic compounds [2]. The reduction of β-catenin amounts at the sub-membranous level and its nuclear relocalization may be responsible for a loss of cells along the tubule and for the activation of a set of genes involved in cellular dedifferentiation, respectively. This study aimed at setting up a method for the assessment of membranous β-catenin quantity.Material and methods: For the evaluation of fluorescence intensity on microscopy slides, calibration was achieved using InspeckTM fluorescently labeled microbeads [3]. The fluorescence intensity was evaluated by the measurement of luminance using ImageJ software.HK-2 cells, originating from human RPTECs, were treated with nephrotoxic substances and stained for β-catenin. The fluorescence attributed to the membrane-localized protein was recorded.Results: The microbeads calibrators allowed to control the linearity and reproducibility of the method. The signal proved to be dependent upon the fluorescent intensity and the density of beads. Preliminary application to cell cultures confirmed that common nephrotoxic compounds induce mesurable loss of β-catenin at the membranous level, indicating that it is possible to complement immunofluorescence staining qualitative observations with quantitative observations.Conclusion: This study confirms the possibility of using conventional microscopy devices to gather both qualitative and quantitative data from immunofluorescent stainings. This method will be useful in determining the relative membranous and nuclear amounts of β-catenin in treated cells, and in screening for compounds that could help in preserving cellular adhesion properties following nephrotoxic insults.