Related Experiment Video
Updated: Sep 6, 2025
Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line
Pin Ren1, Christian Tiede2, Sean W Fanning3
1Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois; Center for Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois.
Abstract:
Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.
Insights
Researchers developed a fluorescent Affimer protein to image estrogen receptor alpha (ERα) mutants in living breast cancer cells. This tool efficiently labels ERα, distinguishing between key mutants and aiding drug response studies.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Intracellular estrogen receptor alpha (ERα) mutations are linked to 70% of breast cancers.
- Imaging ERα mutants in living cells is crucial for understanding cancer progression and drug efficacy.
Purpose of the Study:
- To develop a novel imaging tool for ERα mutants in living cancer cells.
- To assess the ability of a small Affimer protein to label and differentiate ERα mutants.
- To investigate the nuclear localization and DNA targeting of Affimer-bound ERα.
Main Methods:
- Development of a small (13 kDa) fluorescently labeled Affimer protein via phage display.
- Introduction of the Affimer into living cancer cells using streptolysin O to create temporary membrane pores.
- Single-molecule fluorescence imaging to visualize Affimer-ERα interactions and nuclear translocation.
Main Results:
- The Affimer efficiently labels ERα, with preferential binding to the Y537S mutant.
- The Affimer distinguishes between L536S, Y537S, and D538G ERα mutants.
- Affimer-ERαY537S demonstrates efficient nuclear localization and navigation to target DNA.
- The Affimer differentiates the effects of selective estrogen receptor modulators on ERα.
Conclusions:
- A small Affimer protein can be efficiently delivered into living cells to image endogenous proteins with minimal perturbation.
- This Affimer serves as a valuable tool for studying ERα mutants in breast cancer research.
- The Affimer's ability to differentiate ERα mutants and drug effects opens avenues for personalized cancer therapy studies.
