2013 Fiscal Year Research-status Report
二量化、クラスタ構成、および膜受容体細胞内輸送に関する単一分子蛍光の研究
| Project/Area Number |
24750029
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| Research Institution | National Institute of Advanced Industrial Science and Technology |
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Principal Investigator |
BIJU V・Pillai 独立行政法人産業技術総合研究所, 健康工学研究部門, 主任研究員 (60392651)
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| Keywords | EGFR / receptor dimerization / receptor clustering / FRET / intracellular delivery |
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| Research Abstract |
Research plan in 2013 was the evaluation of the kinetics of formation and intracellular delivery of EGFR using fluorescence imaging and FRET analysis. This plan was implemented by first labeling EGF or biotinylated EGF with CdSe/ZnS quantum dots and anti-EGFR antibody with Cy5 dye. Successively, human lung epithelial adenocarcinoma cells (H1650) that over-express EGFR are cultured up to 70% confluence, washed and labeled with the Cy5-antibody conjugate. After removing the unbound antibody-dye conjugate by washing, EGFRs in cells are activated using quantum dot-labeled EGF. The cells are then washed and supplemented with cell culture medium and immediately observed using a fluorescence microscope and a fluorescence lifetime system. FRET from quantum dot to Cy5, which was studied using fluorescence lifetime measurements, enabled us for identifying the labeling process. The kinetics of clustering is evaluated by recording temporal changes in the fluorescence intensity of single quantum dots into dimer-like and successively cluster-like assemblies of EGF-EGFR. Fluorescence images of single and clusters of EFGR at different concentrations of QD-EGF conjugates and different time intervals are recorded and analyzed. A uniform distribution of single quantum dots observed initially changed into smaller and then larger clusters with time under experiment. Finally, the clusters are internalized and delivered inside the cytoplasm, which is identified from the perinuclear fluorescence.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Detection of FRET from labeled cells, fluorescence of single-molecule as well as clusters of EGFR, transformation of monomers into clusters, and intracellular delivery of clusters of EGFR are within the expected results. Activation or labeling of cells with quantum dot-labeled EGF and Cy5-labeled anti-EGFR antibody enabled us to efficiently detect fluorescence and FRET from labeled EGFR in living cells. Previous FRET studies and single-molecule fluorescence detections were helpful for recording and analyzing the kinetics of EGFR clustering in the current project. The present status of results will enable me to optimize FRET and single molecule studies and report the mechanism of dimerization, clustering and intracellular delivery of EGFR in live cells.
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| Strategy for Future Research Activity |
Research plan in this fiscal year is on the reconfirmation and quantification of the kinetics of dimerization of EGFR single-molecules and association of dimers into clusters, and evaluation of the number of EGFR molecules involved in the intracellular delivery, all in living H1650 cells. First, EGFR in H1650 cells will be activated using 0.5 nM to 2 nM EGF-quantum dot conjugate and the fluorescence images of single-molecules in single cells will be recorded at different time intervals. In another cell sample, cells will be first labeled with Cy5-labeled anti-EGFR antibody and then with quantum dot-labeled EGF. FRET efficiency is expected to increase during the clustering process due to a large number of Cy5-antibody-EGFR conjugates proximal to EGFR activated using EGF-quantum dot conjugate. Also, the number of EGFR required for clustering will be re-examined by the labeling of cells using quantum dot-labeled anti-EGFR antibody. The results about dimerization, clustering and intracellular delivery of EGFR will be summarized and published.
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Research Products
(3 results)
