title: Measurement of Mechanical Tension at Cell-cell Junctions Using Two-photon Laser Ablation.
abstract: The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreuxet al., 2012). When coupled to E-cadherin cell-cell adhesion, cortical actomyosin generates junctional tension that influences many aspects of tissue function, organization and morphogenesis (Lecuit and Yap, 2015). Uncovering the molecular mechanisms underlying the generation of junctional tension requires tools for measuring it in live cells with a high spatio-temporal resolution. For this, we have set up a technique of laser ablation, in which we use the high power output of a two-photon laser to physically cut the actin cortex at the sites of cell-cell adhesion labeled with E-cadherin-GFP. Tension, thus is visualized as the outwards recoil of the vertices that define a junction after this was ablated/cut. Analysis of recoil versus time allows extracting parameters related to the amount of contractile force that is applied to the junction before ablation (initial recoil) and the ratio between elasticity of the junction and viscosity of the media (cytoplasm) in which the junctional cortex is immersed. Using this approach we have discovered how Src protein-tyrosine kinase (Gomezet al., 2015); actin-binding proteins such as tropomyosins (Caldwellet al., 2014) and N-WASP (Wuet al., 2014); Myosin II (Priyaet al., 2015) and coronin-1B (Michaelet al., 2016) contribute to the molecular apparatus responsible for generating tension at the cell-cell junctions. This protocol describes the experimental procedure for setting up laser ablation experiments and how to optimize ablation and acquisition conditions for optimal measurements of junctional tension. It also provides a full description, step by step, of the post-acquisition analysis required to evaluate changes in contractile force as well as cell elasticity and/or cytoplasm viscosity.
authors.affiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia.
authors.forename: Xuan
authors.initials: X
authors.lastname: Liang
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London, UK.
authors.forename: Magdalene
authors.initials: M
authors.lastname: Michael
authors.affiliation: Divisions of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Australia.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Gomez
keywords: Cell-cell junction; Epithelial cells; Laser ablation; Tension; Two-photon; Viscoelasticity