A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation. 2021

Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, H3A 1A3, Canada.

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.

UI MeSH Term Description Entries
D007422 Intestines The section of the alimentary canal from the STOMACH to the ANAL CANAL. It includes the LARGE INTESTINE and SMALL INTESTINE. Intestine
D004847 Epithelial Cells Cells that line the inner and outer surfaces of the body by forming cellular layers (EPITHELIUM) or masses. Epithelial cells lining the SKIN; the MOUTH; the NOSE; and the ANAL CANAL derive from ectoderm; those lining the RESPIRATORY SYSTEM and the DIGESTIVE SYSTEM derive from endoderm; others (CARDIOVASCULAR SYSTEM and LYMPHATIC SYSTEM) derive from mesoderm. Epithelial cells can be classified mainly by cell shape and function into squamous, glandular and transitional epithelial cells. Adenomatous Epithelial Cells,Columnar Glandular Epithelial Cells,Cuboidal Glandular Epithelial Cells,Glandular Epithelial Cells,Squamous Cells,Squamous Epithelial Cells,Transitional Epithelial Cells,Adenomatous Epithelial Cell,Cell, Adenomatous Epithelial,Cell, Epithelial,Cell, Glandular Epithelial,Cell, Squamous,Cell, Squamous Epithelial,Cell, Transitional Epithelial,Cells, Adenomatous Epithelial,Cells, Epithelial,Cells, Glandular Epithelial,Cells, Squamous,Cells, Squamous Epithelial,Cells, Transitional Epithelial,Epithelial Cell,Epithelial Cell, Adenomatous,Epithelial Cell, Glandular,Epithelial Cell, Squamous,Epithelial Cell, Transitional,Epithelial Cells, Adenomatous,Epithelial Cells, Glandular,Epithelial Cells, Squamous,Epithelial Cells, Transitional,Glandular Epithelial Cell,Squamous Cell,Squamous Epithelial Cell,Transitional Epithelial Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000087603 Cell Culture Techniques, Three Dimensional Methods for growing cells in a way that they form three-dimensional shapes, such as on a three-dimensionally shaped substrate or as free-falling aggregates in stirred liquids. 3-D Cell Culture,3-Dimensional Cell Culture,3D Cell Culture,Scaffold 3D Cell Culture,Scaffold Cell Culture,Scaffold Cell Culture Techniques,Three-Dimensional Cell Culture,3 D Cell Culture,3 Dimensional Cell Culture,3-D Cell Cultures,3-Dimensional Cell Cultures,3D Cell Cultures,Cell Culture, 3-D,Cell Culture, 3-Dimensional,Cell Culture, 3D,Cell Culture, Scaffold,Cell Culture, Three-Dimensional,Cell Cultures, 3-D,Cell Cultures, 3-Dimensional,Cell Cultures, 3D,Cell Cultures, Scaffold,Cell Cultures, Three-Dimensional,Culture, 3-D Cell,Cultures, 3-D Cell,Cultures, 3D Cell,Scaffold Cell Cultures,Three Dimensional Cell Culture,Three-Dimensional Cell Cultures
D016764 Cell Polarity Orientation of intracellular structures especially with respect to the apical and basolateral domains of the plasma membrane. Polarized cells must direct proteins from the Golgi apparatus to the appropriate domain since tight junctions prevent proteins from diffusing between the two domains. Cell Polarities,Polarities, Cell,Polarity, Cell
D018874 Spheroids, Cellular Spherical, heterogeneous aggregates of proliferating, quiescent, and necrotic cells in culture that retain three-dimensional architecture and tissue-specific functions. The ability to form spheroids is a characteristic trait of CULTURED TUMOR CELLS derived from solid TUMORS. Cells from normal tissues can also form spheroids. They represent an in-vitro model for studies of the biology of both normal and malignant cells. (From Bjerkvig, Spheroid Culture in Cancer Research, 1992, p4) Multicellular Spheroids,Cellular Spheroid,Cellular Spheroids,Multicellular Spheroid,Spheroid, Cellular,Spheroid, Multicellular,Spheroids, Multicellular
D020543 Proteome The protein complement of an organism coded for by its genome. Proteomes

Related Publications

Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
February 2010, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery,
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
January 2013, Frontiers in bioscience (Elite edition),
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
January 2015, Acta medica (Hradec Kralove),
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
November 1999, The Prostate,
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
December 2020, ACS chemical biology,
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
September 2010, Genes & development,
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
February 2017, Molecular and cellular biology,
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
September 2010, Stem cells (Dayton, Ohio),
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
January 2017, Nature,
Li-Ting Wang, and Marie-Ève Proulx, and Anne D Kim, and Virginie Lelarge, and Luke McCaffrey
April 2018, Journal of neuroscience methods,
Copied contents to your clipboard!