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A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation
Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspens...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610992/ https://www.ncbi.nlm.nih.gov/pubmed/34815476 http://dx.doi.org/10.1038/s41598-021-02178-2 |
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author | Wang, Li-Ting Proulx, Marie-Ève Kim, Anne D. Lelarge, Virginie McCaffrey, Luke |
author_facet | Wang, Li-Ting Proulx, Marie-Ève Kim, Anne D. Lelarge, Virginie McCaffrey, Luke |
author_sort | Wang, Li-Ting |
collection | PubMed |
description | Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRAS(G12V) exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRAS(G12V)-expressing cells. This represents an applicable approach to explore protein–protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells. |
format | Online Article Text |
id | pubmed-8610992 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-86109922021-11-24 A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation Wang, Li-Ting Proulx, Marie-Ève Kim, Anne D. Lelarge, Virginie McCaffrey, Luke Sci Rep Article Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRAS(G12V) exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRAS(G12V)-expressing cells. This represents an applicable approach to explore protein–protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells. Nature Publishing Group UK 2021-11-23 /pmc/articles/PMC8610992/ /pubmed/34815476 http://dx.doi.org/10.1038/s41598-021-02178-2 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Wang, Li-Ting Proulx, Marie-Ève Kim, Anne D. Lelarge, Virginie McCaffrey, Luke A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
title | A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
title_full | A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
title_fullStr | A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
title_full_unstemmed | A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
title_short | A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
title_sort | proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610992/ https://www.ncbi.nlm.nih.gov/pubmed/34815476 http://dx.doi.org/10.1038/s41598-021-02178-2 |
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