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A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation
- Source :
- Scientific Reports, Vol 11, Iss 1, Pp 1-16 (2021), Scientific Reports
- Publication Year :
- 2021
- Publisher :
- Nature Portfolio, 2021.
-
Abstract
- 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 KRASG12V 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 KRASG12V-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.
- Subjects :
- Proteome
Science
Lumen (anatomy)
Apicobasal polarity
Cellular imaging
Proteomics
Article
03 medical and health sciences
0302 clinical medicine
Spheroids, Cellular
Cell polarity
Humans
Cell Culture Techniques, Three Dimensional
030304 developmental biology
0303 health sciences
RALB
Multidisciplinary
Chemistry
Spheroid
Cell Polarity
Epithelial Cells
Phenotype
Cell biology
Intestines
030220 oncology & carcinogenesis
Lumen maintenance
Medicine
PTPN14
Subjects
Details
- Language :
- English
- ISSN :
- 20452322
- Volume :
- 11
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Scientific Reports
- Accession number :
- edsair.doi.dedup.....b82743bc82f3296b79fa23e9ee7cb1bd