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Assay Drug Dev Technol. 2016 Oct;14(8):478-488. Epub 2016 Sep 23.

A Phenotypic High-Content Screening Assay to Identify Regulators of Membrane Protein Localization.

Author information

1
1 Cell Cycle and Cancer Genetics Laboratory, Peter MacCallum Cancer Centre , Victoria, Australia .
2
2 The Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre , Victoria, Australia .
3
3 Sir Peter MacCallum Department of Oncology, University of Melbourne , Parkville, Australia .
4
4 Department of Pathology, University of Melbourne , Parkville, Australia .
5
5 Department of Biochemistry and Molecular Biology, University of Melbourne , Parkville, Australia .
6
6 La Trobe Institute for Molecular Science, Department of Biochemistry and Genetics, La Trobe University , Melbourne, Australia .

Abstract

Correct subcellular localization of proteins is a requirement for appropriate function. This is especially true in epithelial cells, which rely on the precise localization of a diverse array of epithelial polarity and cellular adhesion proteins. Loss of cell polarity and adhesion is a hallmark of cancer, and mislocalization of core polarity proteins, such as Scribble, is observed in a range of human epithelial tumors and is prognostic of poor survival. Despite this, little is known about how Scribble membrane localization is regulated. Here, we describe the development and application of a phenotypic high-content screening assay that is designed to specifically quantify membrane levels of Scribble to identify regulators of its membrane localization. A screening platform that is capable of resolving individual cells and quantifying membrane protein localization in confluent epithelial monolayers was developed by using the cytoplasm-to-cell-membrane bioapplication integrated with the Cellomics ArrayScan high-content imaging platform. Application of this method to a boutique human epithelial polarity and signaling small interfering RNA (siRNA) library resulted in highly robust coefficient-of-variance and Z' factor values. As proof of concept, we present two candidate genes whose depletion specifically reduces Scribble protein levels at the membrane. Data mining revealed that these proteins interact with components of the Scribble polarity complex, providing support for the utility of the screening approach. This method is broadly applicable to genome-wide and large-scale compound screening of membrane-bound proteins, and when coupled with pathway analysis the dataset becomes even more valuable and can provide predictive mechanistic insight.

KEYWORDS:

cell-based; imaging; membrane; microscopy; screening

PMID:
27661290
DOI:
10.1089/adt.2016.733
[Indexed for MEDLINE]

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