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A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity

Cell polarity is an evolutionarily conserved process of asymmetric spatial organization within cells and is essential to tissue structure, signal transduction, cell migration, and cell division. The establishment and maintenance of polarity typically involves extensive protein-protein interactions t...

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Autor principal: Johnston, Christopher A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649242/
https://www.ncbi.nlm.nih.gov/pubmed/33195282
http://dx.doi.org/10.3389/fcell.2020.598492
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author Johnston, Christopher A.
author_facet Johnston, Christopher A.
author_sort Johnston, Christopher A.
collection PubMed
description Cell polarity is an evolutionarily conserved process of asymmetric spatial organization within cells and is essential to tissue structure, signal transduction, cell migration, and cell division. The establishment and maintenance of polarity typically involves extensive protein-protein interactions that can be made further intricate by cell cycle-dependent regulation. These aspects can make interpreting phenotypes within traditional in vivo genetic systems challenging due to pleiotropic effects in loss-of-function experiments. Minimal reconstitution methods offer investigators the advantage of stricter control of otherwise complex systems and allow for more direct assessment of the role of individual components to the process of interest. Here I provide a detailed protocol for a cell adhesion-based method of inducing cell polarity within non-polarized Drosophila S2 cells. This technique is simple, cost effective, moderate throughput, and amenable to RNAi-based loss-of-function studies. The ability to “plug-and-play” genes of interest allows investigators to easily assess the contribution of individual protein domains and post-translational modifications to their function. The system is ideally suited to test not only the requirement of individual components but also their sufficiency, and can provide important insight into the epistatic relationship among multiple components in a protein complex. Although designed for use within Drosophila cells, the general premise and protocol should be easily adapted to mammalian cell culture or other systems that may better suit the interests of potential users.
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spelling pubmed-76492422020-11-13 A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity Johnston, Christopher A. Front Cell Dev Biol Cell and Developmental Biology Cell polarity is an evolutionarily conserved process of asymmetric spatial organization within cells and is essential to tissue structure, signal transduction, cell migration, and cell division. The establishment and maintenance of polarity typically involves extensive protein-protein interactions that can be made further intricate by cell cycle-dependent regulation. These aspects can make interpreting phenotypes within traditional in vivo genetic systems challenging due to pleiotropic effects in loss-of-function experiments. Minimal reconstitution methods offer investigators the advantage of stricter control of otherwise complex systems and allow for more direct assessment of the role of individual components to the process of interest. Here I provide a detailed protocol for a cell adhesion-based method of inducing cell polarity within non-polarized Drosophila S2 cells. This technique is simple, cost effective, moderate throughput, and amenable to RNAi-based loss-of-function studies. The ability to “plug-and-play” genes of interest allows investigators to easily assess the contribution of individual protein domains and post-translational modifications to their function. The system is ideally suited to test not only the requirement of individual components but also their sufficiency, and can provide important insight into the epistatic relationship among multiple components in a protein complex. Although designed for use within Drosophila cells, the general premise and protocol should be easily adapted to mammalian cell culture or other systems that may better suit the interests of potential users. Frontiers Media S.A. 2020-10-26 /pmc/articles/PMC7649242/ /pubmed/33195282 http://dx.doi.org/10.3389/fcell.2020.598492 Text en Copyright © 2020 Johnston. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Johnston, Christopher A.
A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity
title A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity
title_full A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity
title_fullStr A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity
title_full_unstemmed A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity
title_short A Cell Adhesion-Based Reconstitution Method for Studying Cell Polarity
title_sort cell adhesion-based reconstitution method for studying cell polarity
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649242/
https://www.ncbi.nlm.nih.gov/pubmed/33195282
http://dx.doi.org/10.3389/fcell.2020.598492
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