Measurement of Mechanical Tension at cell-cell junctions using two-photon laser ablation
采用双光子激光烧蚀测定细胞间连接处的机械张力
发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2068 浏览次数: 10598
评审: Anonymous reviewer(s)
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The authors used this protocol in:
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 (激光烧蚀)
Tension (张力)
Cell-cell junction (细胞间连接)
Two-photon (双光子)
Viscoelasticity (粘弹性)
Epithelial cells (上皮细胞)
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.
Materials and Reagents
- MCF-7 or Caco-2 cells from ATCC®
Note: This protocol can be easily extended to any other endothelial or epithelial cell line with well defined cell-cell junctions like AML12 cells. - Plasmids (or lentivirus) to express a junctional marker like E-cadherin-GFP.
See Bio-protocol e937 by Priya and Gomez (2013) for lentivirus preparation for expression of mouse E-cadherin-GFP in cells knockdown for endogenous human E-cadherin. In this protocol, we describe the transfection of cells for overexpression of E-cadherin-GFP. - Purified plasmid DNA encoding E-cadherin-GFP (Addgene, catalog number: 67937 ) or any other junctional protein like ZO-1 (Addgene, catalog number: 30313 ), vinculin (Addgene, catalog number: 30312 ), MRLC (Addgene, catalog number: 35680 ), or the actin marker Utrophin (Addgene, catalog number: 26737 )
- Lipofectamine 3000 and P3000 reagent (Thermo Fisher Scientific, InvitrogenTM, catalog number: L3000015 )
- Dulbecco’s modified Eagle’s medium high glucose with stable L-glutamine (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11995-073 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
- PBS without Ca2+ and Mg2+ (Astral Scientific, catalog number: 09-8912-100 )
- Opti-MEM media (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
- Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
- CaCl2
- Imaging media (see Recipes)
Equipment
- Laser scanning confocal microscope, LSM 510 Meta Zeiss confocal microscope (Zeiss, Jena, Germany) equipped with:
An acoustic optical tunable filter (AOTF) for bleaching of selected areas
A heated chamber (37 °C) for live cell imaging
A tunable two-photon laser (700-1100 nm, > 2,000 mW power, Chameleon Laser, Coherent Inc.)
A 30 mW argon laser (458, 488 and 514 nm laser lines)
A 60x objective, 1.4 NA oil Plan Apochromat (Zeiss) immersion lens
Dichroic and emission filters for the use of the 488 nm laser lines and detection of GFP fluorescence - Glass bottom dishes, No. 1.5 Coverslip (35 mm diameter, MATTEK, catalog number: P35G-1.5-20-C or 29 mm diameter, Shengyou Biotechnology, catalog number: D29-10-1.5-N )
Software
- ImageJ software (https://imagej.nih.gov/ij/)
- Fiji software (http://imagej.net/Fiji)
- MTrackJ pluging (http://www.imagescience.org/meijering/software/mtrackj/manual/)
- Microsoft Excel (https://products.office.com/en-au/excel)
- GraphPad PRISM software (http://www.graphpad.com/scientific-software/prism/)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
细胞生物学 > 细胞成像 > 双光子显微镜
细胞生物学 > 细胞结构 > 细胞粘附
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