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High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells
Published on: January 7, 2019
Plasma membrane assays and three-compartment image cytometry for high content screening
Natalie L Prigozhina1, Ling Zhong, Edward A Hunter
1Vala Sciences, Inc., San Diego, CA 92121, USA.
Assay and Drug Development Technologies
|March 16, 2007
Summary
A new algorithm precisely quantifies plasma membrane proteins, revealing changes in cell division and adhesion. This image cytometry method accurately analyzes protein localization in cultured cells and skin cancer tissues.
Area of Science:
- Cell Biology
- Biotechnology
- Image Analysis
Background:
- High-throughput image cytometry relies on accurate cell region identification.
- Plasma membrane proteins regulate critical cellular functions like division, migration, and adhesion.
- Understanding protein localization changes is crucial for disease research, including cancer.
Purpose of the Study:
- To develop and validate a novel algorithm for high-throughput image cytometry focused on plasma membrane analysis.
- To quantify changes in specific plasma membrane-associated proteins (PKC alpha, cadherins) involved in cell regulation.
- To apply the algorithm to cultured cells and tissue sections from cancer models.
Main Methods:
- Development of a novel image cytometry algorithm to identify plasma membrane regions and associated proteins.
- Validation using phorbol 12-myristate 13-acetate (PMA) to induce protein kinase C (PKC) alpha translocation in HeLa cells.
- Assay of cadherin protein localization changes in response to PMA, serum-free medium, and EGTA in various cell lines.
- Application of the algorithm to analyze cadherin expression and DNA content in skin cancer tissue sections from transgenic and chemically induced models.
Main Results:
- The algorithm successfully quantified PMA-induced plasma membrane localization of PKCalpha (Z' = 0.88).
- PMA treatment significantly increased plasma membrane localization of N-cadherin, E-cadherin, and VE-cadherin (P < .01).
- VE-cadherin levels showed significant differences between serum-free medium and EGTA treatments (Z' = 0.52).
- Transgenic skin cancer model analysis revealed significantly lower cadherin expression (34%) in tumor cells compared to normal tissue, with increased DNA content.
- Chemically induced tumors did not show increased DNA content in tumor cells.
Conclusions:
- The novel plasma membrane image cytometry algorithm enables statistically significant quantification of protein localization.
- The algorithm is effective for analyzing both cultured cells and complex tissue sections.
- Findings suggest a link between PKCalpha activity and cadherin localization, with implications for cell adhesion and migration.
- The algorithm's application to cancer models highlights differential cadherin expression and DNA content changes in tumor development.
