title: Quantitative and robust assay to measure cell-cell contact assembly and maintenance.
abstract: Epithelial junction formation and maintenance are multistep processes that rely on the clustering of macromolecular complexes. These events are highly regulated by signalling pathways that involve Rho small GTPases. Usually, when analysing the contribution of different components of Rho-dependent pathways to cell-cell adhesion, the localisation of adhesion receptors at junctions is evaluated by immunofluorescence. However, we find that this method has limitations on the quantification (dynamic range), ability to detect partial phenotypes and to differentiate between the participation of a given regulatory protein in assembly and/or maintenance of cell-cell contacts.In this chapter, we describe a suitable method, the aggregation assay, in which we adapted a quantitative strategy to allow objective and reproducible detection of partial phenotypes. Importantly, this methodology estimates the ability of cells to form junctions and their resistance to mechanical shearing forces (stabilisation).
authors.affiliation: Faculty of Medicine, Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Nola
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Erasmus
authors.forename: Vania M M
authors.initials: VM
authors.lastname: Braga
mesh_terms: D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D002449:Cell Aggregation / Q000502:physiology; D002478:Cells, Cultured; D003584:Cytological Techniques; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D008853:Microscopy / Q000379:methods
chemical_list: D015820:Cadherins