Quantitative Analysis of Melanocyte Migration in vitro Based on Automated Cell Tracking under Phase Contrast Microscopy Considering the Combined Influence of Cell Division and Cell-Matrix Interactions

V. Letort¹, S. Fouliard¹, G. Letort¹, I. Adanja², M. Kumasaka^3,4, S. Gallagher^3,4, O. Debeir², L. Larue^3,4 and F. Xavier^1*

¹ Laboratory of Applied Mathematics, Ecole Centrale Paris, F-92295 Chatenay-Malabry, France
² Laboratory of Image Synthesis and Analysis (LISA), Faculty of Applied Sciences, Universite Libre de Bruxelles, 1050 Brussels, Belgium
³ Institut Curie, Centre Recherche, Developmental Genetics of Melanocytes, F-91405 Orsay, France
⁴ CNRS UMR146, F-91405 Orsay, France

^* Corresponding author. E-mail: francoise.xavier@ecp.fr

Abstract

The aim of this study was to describe and analyze the regulation and spatio-temporal dynamics of melanocyte migration in vitro and its coupling to cell division and interaction with the matrix. The melanocyte lineage is particularly interesting because it is involved in both embryonic development and oncogenesis/metastasis (melanoma). Biological experiments were performed on two melanocyte cell lines established from wild-type and β-catenin-transgenic mice (bcat*). The multi-functional β-catenin molecule is known to be able to regulate the transcription of various genes involved in cell proliferation and migration, particularly in the melanocyte lineage. We also investigated fibronectin, an extra-cellular matrix protein that binds integrins, thereby providing adhesion points for cells and encouraging migration. As the migration of individual cells were followed, automated methods were required for processing the large amount of data generated by the time-lapse video-microscopy. A model-based approach for automated cell tracking was evaluated on a sample by comparison with manual tracking. This method was found reliable in studying overall cell behaviour. Its application to all the observed sequences provided insight into the factors affecting melanocyte migration in vitro: melanocytes of mutated form of β-catenin showed higher division rates and no contact inhibition of growth was induced by the resulting increase in cell density. However, cell density limited the amplitude of cell displacements, although their motility was less affected. The high fibronectin concentration bound to substratum promoted cell migration and motility, the effect being more intense for wild-type cells than for cells with β-catenin over-expression. During the division process, cell migration speed increased rapidly then decreased slowly. Analyses of such data is expected to lead both to biological answers and to a framework for a better modeling processes in the future.