Yoshitaro Nakano

Regulation of nuclear import by light‐induced activation of caged nuclear localization signal in living cells

A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine‐128 residue in SV40 T‐antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78±0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm2). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally.

Imaging of rotational Brownian motion of biomolecules in living cells using fluorescence depolarization microscopy

Two-dimensional fluorescence depolarization measurement was achieved by a fluorescence microscope equipped with polarizing devices and an image intensified CCD camera. Anisotropy images were acquired using living cells stained with a membrane specific binding dye. It was found that the bound dye is spatially distinguishable from the free dye which does not bind to the membrane of a cell, due to the difference in rotational Brownian motion. In addition, we succeeded in obtaining fluorescence depolarization images by means of time- resolved measurement and two-photon excitation measurement. Two-photon excitation images showed a superior signal-to-noise ratio compared to one-photon excitation images. Fluorescence depolarization imaging will therefore prove a powerful tool for studying molecular functions in cells.