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細胞内微視領域での時間分解蛍光分光を目指した低温顕微鏡システムの展開

Fujita Yuki 東北大学

2022.03.25

概要

Oxygenic photosynthesis is performed by the two photoactive units, photosystem I (PSI) and photosystem II (PSII), that utilize light energy to generate the electron flow from water to NADPH. Photosynthetic organisms have developed mechanisms called state transitions to regulate the excitation balance between the two units, since the balance is constantly disturbed by fluctuation in light quality. The traditional state transitions model assumes shuttling of a lightharvesting complex called LHCII between the two PSs. In addition, some previous studies have suggested the appearance of the isolated LHCII bound to neither PSs, in parallel with the shuttling of LHCII. This isolated or free LHCII down-regulates the energy flow to PSs. These functions have a significant effect on the efficiency of photosynthesis, which is a potential key to improve a crop production. However, there has been no direct observation of the intracellular rearrangements and the excitation energy transfer kinetics of LHCII upon state transitions. Recently, the establishment of cryo-electron microscopy and cryo-electron tomography methods has facilitated our understandings of the molecular structures and intracellular distributions of the photosynthetic proteins. On the other hand, changes in protein-protein interactions in response to the external stimulation, such as the state transitions, are reproduced only under the physiological environment within the chloroplast. My doctoral research has focused on the development of novel cryogenic optical microscope systems that is the first in the world to provide fluorescence spectrum and fluorescence lifetime simultaneously at every pixel position on an image of a cryotreated specimen. This was achieved by combining the cryo-microscope system with either the time-correlated single photon counting setup or the streak camera. Here, I demonstrate that this system is an innovative imaging method to realize more comprehensive investigation on the dynamic changes in excitation energy transfer kinetics based on the rearrangement of protein supercomplexes in living cells. I have succeeded for the first time in the world in spatially resolving the intracellular PSII-rich and PSI-rich regions by operating the developed system for imaging of a unicellular green alga Chlamydomonas reinhardtii at 80 K. A time-resolved fluorescence spectrum measurement within the identified local regions have revealed the change in the excitation energy transfer kinetics from LHCII to PSs upon the state transitions. Moreover, the simultaneous acquisitions of the spectrum and fluorescence decay kinetics enabled to specify the presence of the highly quenched and red-shifted (to 690-695 nm) LHCII which colocalized in the PSI-rich region upon the state2 induction. The observed red shift of the peak wavelength suggested the formation of LHCII aggregates. In addition, I have challenged to develop a STED microscope system that operates at low temperatures to more clearly resolve segregations of intracellular PSII-rich and PSI-rich regions. Although I have confirmed the advantages of the lowtemperature environment for efficient stimulated emission, I could not succeed in improving the spatial resolution probably due to the problem in the quality of the donut-shaped STED beam at the focal plane. In parallel with the development of the cryogenic optical microscope system, I also began collaborative research on the development of a novel model sample with thylakoid membrane arranged on the substrate for easy microscopic observation. I devoted my doctoral research to establish a novel in-vivo platform to understand the photophysical and biochemical processes in photosynthesis and to inspire the design of genetically modified crops for improvement of crop production.

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