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High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy
Published on: July 25, 2014
Engineering of cell-surface receptors for analysis of receptor internalization and detection of receptor-specific
Chang-Hee Lee1, Sookil Park1, Sanggil Kim2
1Department of Chemistry, Yonsei University Seoul 03722 Republic of Korea injae@yonsei.ac.kr.
Abstract:
The epidermal growth factor receptor (EGFR) is a cell-surface glycoprotein that is involved mainly in cell proliferation. Overexpression of this receptor is intimately related to the development of a broad spectrum of tumors. In addition, glycans linked to the EGFR are known to affect its EGF-induced activation. Because of the pathophysiological significance of the EGFR, we prepared a fluorescently labeled EGFR (EGFR128-AZDye 488) on the cell surface by employing the genetic code expansion technique and bioorthogonal chemistry. EGFR128-AZDye 488 was initially utilized to investigate time-dependent endocytosis of the EGFR in live cells. The results showed that an EGFR inhibitor and antibody suppress endocytosis of the EGFR promoted by the EGF, and that lectins recognizing glycans of the EGFR do not enhance EGFR internalization into cells. Observations made in studies of the effects of appended glycans on the entry of the EGFR into cells indicate that a de-sialylated or de-fucosylated EGFR is internalized into cells more efficiently than a wild-type EGFR. Furthermore, by using the FRET-based imaging method of cells which contain an EGFR linked to AZDye 488 (a FRET donor) and cellular glycans labeled with rhodamine (a FRET acceptor), sialic acid residues attached to the EGFR were specifically detected on the live cell surface. Taken together, the results suggest that a fluorescently labeled EGFR will be a valuable tool in studies aimed at gaining an understanding of cellular functions of the EGFR.
Insights
Researchers developed fluorescently labeled epidermal growth factor receptor (EGFR) to study its cell surface dynamics. This tool revealed how EGFR endocytosis is regulated by inhibitors and glycan modifications, aiding cancer research.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Oncology
Background:
- Epidermal growth factor receptor (EGFR) is a key regulator of cell proliferation, and its overexpression is linked to various cancers.
- Glycans attached to EGFR influence its activation and cellular behavior.
- Understanding EGFR dynamics is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To develop a method for fluorescently labeling EGFR on the cell surface for live-cell imaging.
- To investigate the mechanisms of EGFR endocytosis and the role of glycans in this process.
- To establish EGFR as a tool for studying cellular functions.
Main Methods:
- Genetic code expansion and bioorthogonal chemistry were used to create fluorescently labeled EGFR (EGFR128-AZDye 488).
- Time-dependent endocytosis assays were performed on live cells.
- Inhibitors, antibodies, and lectins were used to probe EGFR internalization.
- Förster Resonance Energy Transfer (FRET)-based imaging was employed to detect specific glycan residues.
Main Results:
- EGFR inhibitors and antibodies were found to suppress EGF-induced EGFR endocytosis.
- Lectins targeting EGFR glycans did not enhance EGFR internalization.
- De-sialylated or de-fucosylated EGFR showed more efficient internalization compared to wild-type EGFR.
- Sialic acid residues on EGFR were successfully detected on live cell surfaces using FRET imaging.
Conclusions:
- Fluorescently labeled EGFR is a valuable tool for studying EGFR dynamics and cellular functions in real-time.
- Glycan composition, particularly sialic acid and fucose residues, significantly impacts EGFR internalization.
- This approach provides new insights into EGFR regulation relevant to cancer biology.
