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Measurement of Mechanical Tension at cell-cell junctions using two-photon laser ablation   

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Original research article

A brief version of this protocol appeared in:
Developmental Cell
Apr 2016

Abstract

The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreux et 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 (Gomez et al., 2015); actin-binding proteins such as tropomyosins (Caldwell et al., 2014) and N-WASP (Wu et al., 2014); Myosin II (Priya et al., 2015) and coronin-1B (Michael et 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.

Keywords: Laser ablation, Tension, Cell-cell junction, Two-photon, Viscoelasticity, Epithelial cells

Background

Physical tension on junctions has been revealed by a variety of microscopy methods. These include laser ablation (Ratheesh et al., 2012; Smutny et al., 2015; Michael et al., 2016), optical tweezers (Bambardekar et al., 2015), FRET tension sensors (Grashoff et al., 2010; Borghi et al., 2012; Conway et al., 2013; Leerberg et al., 2014) and immunofluorescence for protein epitopes that are revealed under tension (Yonemura et al., 2010). Among these, laser ablation has become the most popular method, as it is easy to implement and provide a direct measurement of mechanical tension compared with other methods (e.g., FRET or immunofluorescence where the evidence for mechanical tension is more indirect). However, special considerations need to be taken to set up these experiments as well as its analysis, which are important for the correct interpretation of results. This protocol, provides the basic steps needed for the setup and optimization of laser ablation experiments in confluent monolayers of epithelial cells as well as a complete description of the image analysis procedure for measurements of initial recoil after ablation, which is an index of junctional tension.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Liang, X., Michael, M. and Gomez, G. A. (2016). Measurement of Mechanical Tension at cell-cell junctions using two-photon laser ablation. Bio-protocol 6(24): e2068. DOI: 10.21769/BioProtoc.2068.
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