압타머와 양자점 복합체를 이용한 단일 플라즈마 막 단백질의 동역학 연구

Abstract

Ligand-receptor interactions on the plasma membrane initiate cellular events. The interactions in the cellular processes may be heterogeneous and dynamic. Such a heterogeneity and dynamics can be revealed through the real time observation at the single-molecule level. Total internal reflection fluorescence (TIRF) microscope allows us to track individual membrane proteins with a high spatio-temporal resolution by reducing autofluorescence from cell. I used aptamer, a modified single stranded DNA, to label a membrane protein in live cells with Quantum dot (Qdot) that is a semiconductor but widely used as a fluorophore for bio-imaging and bio-sensors and that is highly resistant to photo-bleaching. Aptamer can specifically recognize a protein with a high affinity and specificity and is superior to antibody: less interruption of a ligand binding due to a smaller size (< 30 kDa), reproducible production with a DNA synthesizer, and chemically easy modification. However the use of aptamer gave rise to a nonspecific binding to the PLL coated glass of the cell chamber. The utilization of a bare glass and Dextran dramatically suppressed the nonspecific binding of aptamer. The aptamer-Quantum dot complex was successfully applied for following individual insulin receptors in real time. I could not observe a significant change of the Brownian motion and the diffusion coefficient of the insulin receptors after adding insulin as a ligand. However cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin decreases the diffusion coefficient of insulin receptors. It might be thought that the depletion of cholesterol increases the stiffness of the membrane by changing liquid ordered phase (cholesterol-sphingolipid complexes) to quasi-solid phase (sphingolipids).