Bioimaging of geographically adjacent proteins in a single cell by quantum dot-based fluorescent resonance energy
Won Jun Kang1, Mee Hyang Ko, Dong Soo Lee
1Department of Radiology, Division of Nuclear Medicine, College of Medicine, Yonsei University, Seoul, Korea.
Abstract:
Thousands of proteins are simultaneously involved in the maintenance of a single cancer cell. Fluorescent resonance energy transfer (FRET) is one of the most general techniques for imaging biologically interacting molecules in a cell. Here, we applied FRET to image the co-localization of two proteins that do not interact biologically (nucleolin and integrin α(v) β(3),) both of which are highly expressed in the plasma membrane of cancer cells. AS1411 aptamer, which targets nucleolin, was labeled by Cy3 (Cy3-AS1411) and arginine-glycine-aspartic acid (RGD) peptide, which targets integrin α(v) β(3) , was conjugated with quantum dot (525 nm, Qd) Qd arginine-glycine-aspartic acid (Qd-RGD). FRET activities between Cy3-AS1411 and Qd-RGD were measured in HeLa cells, a human cervical cancer cell line. FRET phenomena between Qd and Cy3 showed good compatibility according to proximity. The fluorescence signature using Qd-RGD and Cy3-AS1411 showed that nucleolin and integrin α(v) β(3) proteins were highly expressed in HeLa cells. Co-incubation of Qd-RGD and Cy3-AS1411 in a single HeLa cell demonstrated that the fluorescence overlay by FRET was quantitatively and geographically quite different from that of individual confocal images. These results suggest that Qd-based FRET analysis can provide information on geographical co-localization of proteins in naïve cells, which is very important for determining the molecular and cellular functions of genes involved in cancers and other clinical diseases.
Insights
This study used quantum dot-based fluorescence resonance energy transfer (FRET) to visualize non-interacting proteins, nucleolin and integrin α(v) β(3), in cancer cells. The findings reveal distinct protein co-localization patterns crucial for understanding cancer biology.
Area of Science:
- Biophysics
- Molecular Biology
- Cancer Research
Background:
- Cancer cells involve thousands of proteins in their maintenance.
- Fluorescent resonance energy transfer (FRET) is a key technique for imaging molecular interactions within cells.
Purpose of the Study:
- To apply FRET for imaging the co-localization of nucleolin and integrin α(v) β(3) in cancer cells.
- To assess the utility of quantum dot-based FRET for analyzing protein geographical distribution.
Main Methods:
- Utilized Cy3-labeled AS1411 aptamer targeting nucleolin and quantum dot-conjugated RGD peptide targeting integrin α(v) β(3).
- Performed FRET measurements in HeLa cervical cancer cells.
- Analyzed fluorescence overlay and geographical distribution using confocal imaging.
Main Results:
- Demonstrated FRET compatibility between quantum dots and Cy3, indicating proximity.
- Confirmed high expression of nucleolin and integrin α(v) β(3) in HeLa cells.
- Observed distinct quantitative and geographical fluorescence overlays via FRET compared to individual protein images.
Conclusions:
- Quantum dot-based FRET analysis provides valuable insights into protein geographical co-localization in cells.
- This technique is important for determining the molecular and cellular functions of genes in cancer and other diseases.
