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...

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Bibliographic Details
Published in:Scientific reports 2021-11, Vol.11 (1), p.22807-22807, Article 22807
Main Authors: Wang, Li-Ting, Proulx, Marie-Ève, Kim, Anne D., Lelarge, Virginie, McCaffrey, Luke
Format: Article
Language:English
Subjects:
631/80/2373
631/80/85/2361
Cell culture
Cell Culture Techniques, Three Dimensional - methods
Cell Polarity
Cysts
Epithelial Cells - cytology
Epithelial Cells - metabolism
Humanities and Social Sciences
Humans
Intestines - cytology
Intestines - metabolism
multidisciplinary
Phenotypes
Polarity
Protein interaction
Proteins
Proteome - analysis
Proteome - metabolism
Proteomics
Science
Science (multidisciplinary)
Spheroids, Cellular - cytology
Spheroids, Cellular - metabolism
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-021-02178-2