We combine pulsed laser, supercontinuum radiation source and fast single-photon counting peripherals to obtain a multifunctional
micro/nano-scope. This provides us with better spatial and temporal resolution to observe fast dynamics.
Performing fluorescence correlation spectroscopy for fast dynamics (<μs) with sub-diffraction limit resolution to observe
the initial state of single-lipid dynamics in supported lipid bilayers and living cells is our goal. Lipid raft serves as a
platform for recruiting signaling components of effective signal transduction. However, the dynamics of sub-200nm
rapidly aggregated lipid rafts are still not elucidated in living cells. We here report our recent progress on the
construction of this multi-functional stage-scanning fluorescence micro/nanoscope for single-lipid dynamics study.
Zero-mode waveguides (ZMWs) are optical nanostructures to confine fluorescent excitation within sub-diffraction
volumes and are commonly used for single-molecule analysis. However, the conventional ZMWs with aluminum film on
fused silica have limitations on living cell studies. The same surfaces composed of hydroxyl group inside and outside
each ZMW restrict specific surface functionalization. The sharp-cylinder shaped and rough edge of each ZMW produces
a steric interference of molecular dynamics on cell membrane. In this study, selectively surface functionalization inside
and outside of each ZMW was achieved with tri-metal-layer film on fused silica. Moreover, bowl-shaped and smooth edge of each ZMW was manufactured in large area. The improvement of ZMWs provides a broad way for monitoring molecular dynamics in living cells.
Integrin receptors serve as both mechanical links and signal transduction mediators between the cell and its environment. Experimental evidence demonstrates that conformational changes and lateral clustering of the integrin proteins may affect their binding to ligands and regulate downstream cellular responses; however, experimental links between the structural and functional correlations of the ligand-receptor interactions are not yet elucidated. In the present report, we utilized optical tweezers to measure the dynamic binding between the snake venom rhodostomin, coated on a microparticle and functioned as a ligand, and the membrane receptor integrin alpha(IIb)beta(3) expressed on a Chinese Hamster Ovary (CHO) cell. A progressive increase of total binding affinity was found between the bead and CHO cell in the first 300 sec following optical tweezers-guided contact. Further analysis of the cumulative data revealed the presence of "unit binding force" presumably exerted by a single rhodostomin-integrin pair. Interestingly, two such units were found. Among the measurements of less total binding forces, presumably taken at the early stage of ligand-receptor interactions, a unit of 4.15 pN per molecule pair was derived. This unit force dropped to 2.54 pN per molecule pair toward the later stage of interactions when the total binding forces were relatively large. This stepped change of single molecule pair binding affinity was not found when mutant rhodostomin proteins were used as ligands (a single unit of 1.81 pN per pair was found). These results were interpreted along with the current knowledge about the conformational changes of integrins during the "molecule activation" process.
The conformational change of integrin αIIbβ3 plays an important role in clot formation. However, the correlation between the structure and the function of integrin αIIbβ3 in interacting with its ligand is still not clear. In this report, we focus on the dynamic variation of the binding between integrin αIIbβ3 and its ligand, rhodostomin by using a photonic force microscopy (PFM). The PFM is used to trap a rhodostomin-coated bead and, then, shift it to bind a surrounding CHO αIIbβ3 cell. Meanwhile, it tracks, with a resolution of 1MHz, the Brownian fluctuations of the trapped bead. Theoretically, the smaller the amplitude of the Brownian fluctuations, the stronger the stiffness of the binding force between the rhodostomin and the CHO αIIbβ3 cell. Experimentally, a significant decrease of the Brownian fluctuations was observed during the interval between the 360th seconds and the 400th seconds after the trapped rhodostomin-coated bead contacted an integrin-expressed CHO αIIbβ3 cell. This observation reveals that it takes the rhodostomin 360 seconds to seek the correct position to bind to the integrin αIIbβ3. After 400 seconds, the rhodostomin has bound rigidly with the integrin αIIbβ3. We presume that the integrin αIIbβ3 has reached its final stage of conformational change.
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