Noriyuki Hatsugai

Publications (3)16.19 Total impact

• Article: Luminescent proteins for high-speed single-cell and whole-body imaging.

  Kenta Saito, Y-F Chang, Kazuki Horikawa, Noriyuki Hatsugai, Yuriko Higuchi, Mitsuru Hashida, Yu Yoshida, Tomoki Matsuda, Yoshiyuki Arai, Takeharu Nagai
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  ABSTRACT: The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca(2+), cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.
  Nature Communications 12/2012; 3:1262. · 7.40 Impact Factor
• Article: Changes in Cytosolic ATP Levels and Intracellular Morphology during Bacteria-Induced Hypersensitive Cell Death as Revealed by Real-Time Fluorescence Microscopy Imaging.

  Noriyuki Hatsugai, Vadim Perez Koldenkova, Hiromi Imamura, Hiroyuki Noji, Takeharu Nagai
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  ABSTRACT: Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.
  Plant and Cell Physiology 08/2012; 53(10):1768-75. · 4.70 Impact Factor
• Source

  Available from: Toru Matsu-ura

  Article: Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.

  Kenta Saito, Noriyuki Hatsugai, Kazuki Horikawa, Kentaro Kobayashi, Toru Matsu-Ura, Katsuhiko Mikoshiba, Takeharu Nagai
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  ABSTRACT: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We applied BRET to develop an autoluminescent Ca(2+) indicator, BRAC, which is composed of Ca(2+)-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca(2+) dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca(2+)-independent signal drifts due to change in cell shape, focus shift, etc. The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca(2+) imaging not only in single live cells but also in small living subjects.
  PLoS ONE 01/2010; 5(4):e9935. · 4.09 Impact Factor