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Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy p...

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Autores principales: Vedula, Pavan, Cruz, Lissette A., Gutierrez, Natasha, Davis, Justin, Ayee, Brian, Abramczyk, Rachel, Rodriguez, Alexis J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928050/
https://www.ncbi.nlm.nih.gov/pubmed/27357130
http://dx.doi.org/10.1038/srep28822
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author Vedula, Pavan
Cruz, Lissette A.
Gutierrez, Natasha
Davis, Justin
Ayee, Brian
Abramczyk, Rachel
Rodriguez, Alexis J.
author_facet Vedula, Pavan
Cruz, Lissette A.
Gutierrez, Natasha
Davis, Justin
Ayee, Brian
Abramczyk, Rachel
Rodriguez, Alexis J.
author_sort Vedula, Pavan
collection PubMed
description Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.
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spelling pubmed-49280502016-07-01 Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis Vedula, Pavan Cruz, Lissette A. Gutierrez, Natasha Davis, Justin Ayee, Brian Abramczyk, Rachel Rodriguez, Alexis J. Sci Rep Article Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation. Nature Publishing Group 2016-06-30 /pmc/articles/PMC4928050/ /pubmed/27357130 http://dx.doi.org/10.1038/srep28822 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Vedula, Pavan
Cruz, Lissette A.
Gutierrez, Natasha
Davis, Justin
Ayee, Brian
Abramczyk, Rachel
Rodriguez, Alexis J.
Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
title Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
title_full Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
title_fullStr Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
title_full_unstemmed Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
title_short Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
title_sort quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928050/
https://www.ncbi.nlm.nih.gov/pubmed/27357130
http://dx.doi.org/10.1038/srep28822
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