Ichiro Sase

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.

Focusing on an individual.

Every element of life, at all levels of hierarchy down to a single molecule, is a functional entity that by itself performs a delicate task (s). Understanding of the mechanism of biological machinery therefore requires a close observation of the performance of individual cells, organelles, or molecules. Three examples are presented, in which single-cell or single-molecule observation under an optical microscope was crucial in the analysis: ionic regulation of the acrosome reaction in starfish sperm revealed by multi-view microscopy, elucidation of elementary steps of actomyosin motors using optical tweezers, and detection of molecular rotation via single fluorophore imaging.