The objective is to develop methodologies for the detection and observation of protein association in a single living cell using a time-resolved microluminescence spectrophotometer (= time-resolved microfluorescence and microphosphorescence spectrophotometer), and at the same time to establish time resolved microluminometry as a useful tool for biomedical research and medical diagnosis. The key idea is combination of the spotting capability of the microscope optics and time-resolved (real time) fluorescence or phosphorescence spectrophotometric measurements on the spot, i.e., we select a structure of our interest under the fluorescence microscope and obtain molecular information on that structure using spectroscopic means. We emphasize the time-resolved techniques because of the wealth of information and because the characteristic time (e.g. excited-state lifetime) is often the only quantitative parameter whose absolute value can be measured under the microscope. We already have almost all necessary equipment and we will concentrate our effort on methodological development for detection of membrane protein association in a single cell during the proposed project period. Our strategies for development and systematic evaluation of time-resolved microluminometric methods include [A] use of rhodopsin in reconstituted membranes and rod outer segment membranes as model samples, [B] comparison of results obtained by microspectroscopic methods with those obtained by usual (macroscopic) spectroscopic methods, [C] measurement of time-dependent depolarization of phosphorescence and delayed fluorescence for observation of rotational diffusion, which is very sensitive to the size of the diffusing unit (oligomers), [D] use of the time-resolved fluorescence energy transfer method for evaluation of (average) inter-protein distances, and [E] development of """"""""antibody probes"""""""" specifically tailored to rotational diffusion and fluorescence energy transfer measurements to observe association of a specific protein in a cell. The developed methods will be applied to our desmosome project: Desmosomal protein association in plasma membranes is observed at initial stages of calcium-induced synchronous formation of desmosomes in mouse epidermal cells in culture. We will detect formation of oligomeric complexes and colocalize at the molecular level two or more kinds of proteins in a cellular structure.
| Kusumi, A; Tsuji, A; Murata, M et al. (1991) Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells. Biochemistry 30:6517-27 |
| Tsuji, A; Kawasaki, K; Ohnishi, S et al. (1988) Regulation of band 3 mobilities in erythrocyte ghost membranes by protein association and cytoskeletal meshwork. Biochemistry 27:7447-52 |
| Merkle, H; Subczynski, W K; Kusumi, A (1987) Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes. Biochim Biophys Acta 897:238-48 |
| Kusumi, A; Winkelhake, J L (1986) Fc:Fc interactions revealed by spin-labeled IgG heterosaccharides in model immune complexes. Biochem Biophys Res Commun 137:237-43 |
