Sep. 2022, The biophysical Society of Japan, 2022 Biophysics and Physicobiology (BPPB) Awards　　　　　　　　　　　　　　　
Akira Kitamura

Advances in Conventional and Extended Fluorescence Correlation Spectroscopy for the Analysis of Biological Clusters and Aggregates
Akira Kitamura
Spectroscopy Journal, 3, 4, 31, 05 Nov. 2025, [Peer-reviewed], [Invited], [Lead author, Last author, Corresponding author]
English, Scientific journal, 46048265

Optimizing Automated Detection for Cytoplasmic TDP25 Aggregates in Fluorescence Imaging
Sumire Sogawa, Kotetsu Sasaki, Akira Kitamura
Spectroscopy Journal, 3, 2, 18, 19 May 2025, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, 46048265

Enhancement of Sensitivity in Aggregation-Based Whole-Cell Arsenite Sensor Utilizing Arsenic Metabolism Regulation
Shiryu Abe, Rina Ayuba, Kyohei Ouchi, Yu-ki Tanaka, Ai Fujimoto, Akira Kitamura, Yasumitsu Ogra, Yuki Kimura, Daisuke Umeno, Shigeko Kawai-Noma
ACS Omega, 15 Apr. 2025, [Peer-reviewed]
English, Scientific journal

Malabaricone C isolated from edible plants as a potential inhibitor of SARS-CoV-2 infection
Mutmainah, Yuta Murai, Ai Fujimoto, Rintaro Kawamura, Akira Kitamura, Sajeer Koolath, Seigo Usuki, Michihito Sasaki, Yasuko Orba, Yasuyuki Igarashi, Hirofumi Sawa, Akihiko Sato, Kenji Monde
Scientific Reports, 15, 1, Springer Science and Business Media LLC, 12 Mar. 2025, [Peer-reviewed]
English, Scientific journal

Short Repeat Ribonucleic Acid Reduces Cytotoxicity by Preventing the Aggregation of TDP-43 and Its 25 KDa Carboxy-Terminal Fragment
Ai Fujimoto, Masataka Kinjo, Akira Kitamura
JACS Au, 28 Oct. 2024, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, 46048265

Interaction of Receptor-Binding Domain of the SARS-CoV-2 Omicron Variant with hACE2 and Actin
Ai Fujimoto, Haruki Kawai, Rintaro Kawamura, Akira Kitamura
Cells, 13, 16, 1318, 07 Aug. 2024, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, 46048265

Hetero-oligomerization of TDP-43 carboxy-terminal fragments with cellular proteins contributes to proteotoxicity
Akira Kitamura, Ai Fujimoto, Rei Kawashima, Yidan Lyu, Kotetsu Sasaki, Yuta Hamada, Kanami Moriya, Ayumi Kurata, Kazuho Takahashi, Reneé Brielmann, Laura C. Bott, Richard I. Morimoto, Masataka Kinjo
Communications Biology, 7, 1, 743, Springer Science and Business Media LLC, 20 Jun. 2024, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, Abstract

Carboxy terminal fragments (CTFs) of TDP-43 contain an intrinsically disordered region (IDR) and form cytoplasmic condensates containing amyloid fibrils. Such condensates are toxic and associated with pathogenicity in amyotrophic lateral sclerosis. However, the molecular details of how the domain of TDP-43 CTFs leads to condensation and cytotoxicity remain elusive. Here, we show that truncated RNA/DNA-recognition motif (RRM) at the N-terminus of TDP-43 CTFs leads to the structural transition of the IDR, whereas the IDR itself of TDP-43 CTFs is difficult to assemble even if they are proximate intermolecularly. Hetero-oligomers of TDP-43 CTFs that have recruited other proteins are more toxic than homo-oligomers, implicating loss-of-function of the endogenous proteins by such oligomers is associated with cytotoxicity. Furthermore, such toxicity of TDP-43 CTFs was cell-nonautonomously affected in the nematodes. Therefore, misfolding and oligomeric characteristics of the truncated RRM at the N-terminus of TDP-43 CTFs define their condensation properties and toxicity., 46048265

Stress Granule Dysfunction via Chromophore-Associated Light Inactivation
Takumi Koizumi, Ai Fujimoto, Haruka Kawaguchi, Tsumugi Kurosaki, Akira Kitamura
ACS Omega, 14 May 2024, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, 40045923

Axonal transport of Frizzled5 by Alcadein α-containing vesicles is associated with kinesin-1.
Yuzuha Shiraki, Monet Mitsuma, Ritsuko Takada, Saori Hata, Akira Kitamura, Shinji Takada, Masataka Kinjo, Hidenori Taru, Ulrike C Müller, Tohru Yamamoto, Yuriko Sobu, Toshiharu Suzuki
Molecular biology of the cell, 34, 11, ar110, 01 Oct. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, Alcadein α (Alcα) and amyloid-β protein precursor (APP) are cargo receptors that associate vesicles with kinesin-1. These vesicles, which contain either Alcα or APP, transport various proteins/cargo molecules into axon nerve terminals. Here, we analyzed immune-isolated Alcα- and APP-containing vesicles of adult mouse brains with LC-MS/MS and identified proteins present in vesicles that contained either Alcα or APP. Among these proteins, Frizzled-5 (Fzd5), a Wnt receptor, was detected mainly in Alcα vesicles. Although colocalization ratios of Fzd5 with Alcα are low in the neurites of differentiating neurons by a low expression of Fzd5 in embryonic brains, the suppression of Alcα expression decreased the localization of Fzd5 in neurites of primary cultured neurons. Furthermore, Fzd5-EGFP expressed in primary cultured neurons was preferentially transported in axons with the transport velocities of Alcα vesicles. In synaptosomal fractions of adult-mice brains that express higher levels of Fzd5, the amount of Fzd5 and the phosphorylation level of calcium/calmodulin-dependent protein kinase-II were reduced in the Alcα-deficient mice. These results suggest that reduced transport of Fzd5 by Alcα-containing vesicles associated with kinesin-1 in axon terminals may impair the response to Wnt ligands in the noncanonical Ca2+-dependent signal transduction pathway at nerve terminals of mature neurons.

Increased intracellular crowding during hyperosmotic stress.
Akira Kitamura, Sho Oasa, Haruka Kawaguchi, Misato Osaka, Vladana Vukojević, Masataka Kinjo
Scientific reports, 13, 1, 11834, 11834, 22 Jul. 2023, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced green fluorescent protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster resonance energy transfer sensor (GimRET). This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. Our approach can therefore be used to reveal in live cells short-lived transient states through which MMC builds over time, which could not be observed when measuring changes in other physical properties that occur at slower time scales., 40045923

Light-Induced Condensates Show Accumulation-Prone and Less Dynamic Properties in the Nucleus Compared to the Cytoplasm
Yuta Hamada, Akira Kitamura
Spectroscopy Journal, 10 Jul. 2023, [Peer-reviewed], [Last author, Corresponding author]
English, Scientific journal, 40045923

Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of exogeneous RNA G-quadruplexes in live cells
Akira Kitamura, Johan Tornmalm, Baris Demirbay, Joachim Piguet, Masataka Kinjo, Jerker Widengren
Nucleic Acids Research, 21 Mar. 2023, [Peer-reviewed], [Lead author]
English, Scientific journal, 40045923

Intracellular Conformation of Amyotrophic Lateral Sclerosis-Causative TDP-43
Akira Kitamura, Sachiko Yuno, Rintaro Kawamura, Masataka Kinjo
International Journal of Molecular Sciences, 24, 6, 5513, 14 Mar. 2023, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal, 40045923

Physico- and chemical biology using nanomanipulation and micromanipulation technologies
Akira Kitamura, Ryo Iizuka
Biophysics and Physicobiology, Biophysical Society of Japan, 2022, [Peer-reviewed], [Invited], [Lead author, Corresponding author]
Symposium, 40045923

Interaction between Spike Protein of SARS-CoV-2 and Human Virus Receptor ACE2 Using Two-Color Fluorescence Cross-Correlation Spectroscopy
Ai Fujimoto, Yidan Lyu, Masataka Kinjo, Akira Kitamura
Applied Sciences, 11, 22, 10697, 10697, MDPI AG, 12 Nov. 2021, [Peer-reviewed], [Invited], [Last author, Corresponding author]
English, Scientific journal, 33376192

Conformational stabilization of optineurin by the dynamic interaction of linear polyubiquitin
Akira Kitamura, Rika Numazawa, Masataka Kinjo
Biochemical and Biophysical Research Communications, 559, 203, 209, Elsevier BV, Jun. 2021, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Pinhole Closure Improves Spatial Resolution in Confocal Scanning Microscopy
Akira Kitamura
Methods in Molecular Biology, 385, 389, Springer US, May 2021, [Invited], [Lead author, Last author, Corresponding author]
English, In book

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
Akira Kitamura, Ai Fujimoto, Masataka Kinjo
Journal of Visualized Experiments, 170, e62576, MyJove Corporation, 25 Apr. 2021, [Peer-reviewed], [Invited], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

Molecular basis of functional exchangeability between ezrin and other actin-membrane associated proteins during cytokinesis.
Guang Yang, Shota Hiruma, Akira Kitamura, Masataka Kinjo, Mithilesh Mishra, Ryota Uehara
Experimental cell research, 403, 2, 112600, 112600, 20 Apr. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis.

Analysis of the triplet-state kinetics of a photosensitizer for photoimmunotherapy by fluorescence correlation spectroscopy
Hideo Takakura, Yuto Goto, Akira Kitamura, Toshitada Yoshihara, Seiji Tobita, Masataka Kinjo, Mikako Ogawa
Journal of Photochemistry and Photobiology A: Chemistry, 408, 113094, 113094, Elsevier BV, Mar. 2021, [Peer-reviewed]
English, Scientific journal

Membrane Surface Modulates Slow Diffusion in Small Crowded Droplets
Kanae Harusawa, Chiho Watanabe, Yuta Kobori, Kazuho Tomita, Akira Kitamura, Masataka Kinjo, Miho Yanagisawa
Langmuir, 37, 1, 437, 444, American Chemical Society (ACS), 12 Jan. 2021, [Peer-reviewed]
English, Scientific journal

Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images
Akira Kitamura, Masakata Kinjo
BIO-PROTOCOL, 10, 10, Bio-Protocol, LLC, 2020, [Peer-reviewed], [Invited], [Lead author, Corresponding author]
English, Scientific journal

The AAA+ ATPase/ubiquitin ligase mysterin stabilizes cytoplasmic lipid droplets.
Munechika Sugihara, Daisuke Morito, Shiori Ainuki, Yoshinobu Hirano, Kazutoyo Ogino, Akira Kitamura, Hiromi Hirata, Kazuhiro Nagata
The Journal of cell biology, 218, 3, 949, 960, 04 Mar. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal

Encapsulation of biomacromolecules by soaking and co-crystallization into porous protein crystals of hemocyanin.
Tsubasa Hashimoto, Yuxin Ye, Asuka Matsuno, Yuki Ohnishi, Akira Kitamura, Masataka Kinjo, SatoshiAbe, Takafumi Ueno, MinYao, Tomohisa Ogawa, Takashi Matsui, Yoshikazu Tanaka
Biochemical and Biophysical Research Communications, 509, 2, 577, 584, Feb. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110 Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250 kDa, i.e., 27 kDa green fluorescence protein, 105 kDa allophycocyanin, 220 kDa C-phycocyanin, and 250 kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440 kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.

Characterization of Intracellular Crowding Environments with Topology-Based DNA Quadruplex Sensors.
Takahashi S, Yamamoto J, Kitamura A, Kinjo M, Sugimoto N
Analytical chemistry, 91, 4, 2586, 2590, Feb. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Molecular crowding creates a unique environment in cells and imposes physical constraints such as the excluded volume effect, water activity, and dielectric constant that can affect the structure and function of biomolecules. It is therefore important to develop a method for quantifying the effects of molecular crowding in cells. In this study, we developed a Förster resonance energy transfer (FRET) probe based on a guanine-quadruplex (G4) DNA motif that shows distinct FRET signals in response to crowding conditions in the presence of salt and poly(ethylene glycol). FRET efficiencies varied in different solutions, reflecting the dependence of G4 stability and topology on salt concentration and water activity. In living cells, FRET signals in the nucleus were higher than those in the cytosol; the signals in membraneless nuclear compartments (i.e., nucleolus) were especially high, suggesting that a decrease in water activity is important for the crowding effect in the nucleus. Thus, the use of DNA sensors with variable structures can elucidate the local effects of molecular crowding in cells.

Determination of cytoplasmic optineurin foci sizes using image correlation spectroscopy
Akira Kitamura, Hiroki Shimizu, Masataka Kinjo
The Journal of Biochemistry, Oxford University Press ({OUP}), 01 Sep. 2018, [Peer-reviewed]
English, Scientific journal

The cytoplasmic region of the amyloid β-protein precursor (APP) is necessary and sufficient for the enhanced fast velocity of APP transport by kinesin-1.
Maoko Tsukamoto, Kyoko Chiba, Yuriko Sobu, Yuzuha Shiraki, Yuka Okumura, Saori Hata, Akira Kitamura, Tadashi Nakaya, Seiichi Uchida, Masataka Kinjo, Hidenori Taru, Toshiharu Suzuki
FEBS letters, 592, 16, 2716, 2724, Aug. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Amyloid β-protein precursor (APP) is transported mainly by kinesin-1 and at a higher velocity than other kinesin-1 cargos, such as Alcadein α (Alcα); this is denoted by the enhanced fast velocity (EFV). Interaction of the APP cytoplasmic region with kinesin-1, which is essential for EFV transport, is mediated by JNK-interacting protein 1 (JIP1). To determine the roles of interactions between the APP luminal region and cargo components, we monitored transport of chimeric cargo receptors, Alcα (luminal)-APP (cytoplasmic) and APP (luminal)-Alcα (cytoplasmic). Alcα-APP is transported at the EFV, whereas APP-Alcα is transported at the same velocity as wild-type Alcα. Thus, the cytoplasmic region of APP is necessary and sufficient for the EFV of APP transport by kinesin-1.

Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy.
Akira Kitamura, Ai Shibasaki, Kayo Takeda, Ryoji Suno, Masataka Kinjo
Biochemistry and biophysics reports, 14, 58, 63, Jul. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Normal function and abnormal aggregation of transactivation response (TAR) DNA/RNA-binding protein 43 kDa (TDP-43) are directly associated with the lethal genetic diseases: cystic fibrosis, amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The binding of TDP-43 to single-stranded DNA (ssDNA) or RNA is involved in transcriptional repression, regulation of RNA splicing, and RNA stabilization. Equilibrium dissociation constants (Kd) of TDP-43 and ssDNA or RNA have been determined using various methods; however, methods that can measure Kd with high sensitivity in a short time using a small amount of TDP-43 in solution would be advantageous. Here, in order to determine the Kd of TDP-43 and fluorescence-labeled ssDNA as well as the binding stoichiometry, we use fluorescence correlation spectroscopy (FCS), which detects the slowed diffusion of molecular interactions in solution with single-molecule sensitivity, in addition to electrophoretic mobility shift assay (EMSA). Using tandem affinity chromatography of TDP-43 dually tagged with glutathione-S-transferase and poly-histidine tags, highly purified protein was obtained. FCS successfully detected specific interaction between purified TDP-43 and TG ssDNA repeats, with a Kd in the nanomolar range. The Kd of the TDP-43 mutant was not different from the wild type, although mutant oligomers, which did not bind ssDNA, were observed. Analysis of the fluorescence brightness per dimerized TDP-43/ssDNA complex was used to evaluate their binding stoichiometry. The results suggest that an assay combining FCS and EMSA can precisely analyze ssDNA recognition mechanisms, and that FCS may be applied for the rapid and quantitative determination of the interaction strength between TDP-43 and ssDNA or RNA. These methods will aid in the elucidation of the substrate recognition mechanism of ALS- and FTLD-associated variants of TDP-43.

State-of-the-art fluorescence fluctuation-based spectroscopic techniques for the study of protein aggregation
Akira Kitamura, Masataka Kinjo
International Journal of Molecular Sciences, 19, 4, 964, MDPI AG, 01 Apr. 2018, [Peer-reviewed], [Invited]
English, Scientific journal

Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy
Akira Kitamura, Yoshihito Ishida, Hiroshi Kubota, Chan-Gi Pack, Takayuki Homma, Shinya Ito, Kazutaka Araki, Masataka Kinjo, Kazuhiro Nagata
Biochemical and Biophysical Research Communications, 497, 1, 279, 284, Elsevier B.V., 26 Feb. 2018, [Peer-reviewed]
English, Scientific journal

Physicochemical properties of the mammalian molecular chaperone HSP60
Ryuichi Ishida, Tomoya Okamoto, Fumihiro Motojima, Hiroshi Kubota, Hiroki Takahashi, Masako Tanabe, Toshihiko Oka, Akira Kitamura, Masataka Kinjo, Masasuke Yoshida, Michiro Otaka, Ewa Grave, Hideaki Itoh
International Journal of Molecular Sciences, 19, 2, 489, MDPI AG, 06 Feb. 2018, [Peer-reviewed]
English, Scientific journal

TDP-43 depletion: mechanism of neuronal cell death in ALS.
Akira Kitamura
Future Neurology, 13, 3, 2018, [Peer-reviewed], [Invited]
English, Scientific journal

Molecular chaperone HSP70 prevents formation of inclusion bodies of the 25-kDa C-terminal fragment of TDP-43 by preventing aggregate accumulation.
Akira Kitamura, Nodoka Iwasaki, Masataka Kinjo
Cell Stress and Chaperones, 2018, [Peer-reviewed]
English, Scientific journal

Determination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy
Akira Kitamura, Masataka Kinjo
Biophysics and Physicobiology, 15, 1, 7, 2018, [Peer-reviewed]
English, Scientific journal

U6 snRNA expression prevents toxicity in TDP-43-knockdown cells
Masao Yahara, Akira Kitamura, Masataka Kinjo
PLOS ONE, 12, 11, e0187813, Nov. 2017, [Peer-reviewed]
English, Scientific journal

Different aggregation states of a nuclear localization signal-tagged 25-kDa C-terminal fragment of TAR RNA/DNA-binding protein 43kDa
Akira Kitamura, Sachiko Yuno, Hideki Muto, Masataka Kinjo
GENES TO CELLS, 22, 6, 521, 534, Jun. 2017, [Peer-reviewed]
English, Scientific journal

Development of new fusion proteins for visualizing amyloid-beta oligomers in vivo
Tomoyo Ochiishi, Motomichi Doi, Kazuhiko Yamasaki, Keiko Hirose, Akira Kitamura, Takao Urabe, Nobutaka Hattori, Masataka Kinjo, Tatsuhiko Ebihara, Hideki Shimura
SCIENTIFIC REPORTS, 6, 22712, Mar. 2016, [Peer-reviewed]
English, Scientific journal

Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it
Takamitsu J. Morikawa, Hideaki Fujita, Akira Kitamura, Takashi Horio, Johtaro Yamamoto, Masataka Kinjo, Akira Sasaki, Hiroaki Machiyama, Keiko Yoshizawa, Taro Ichimura, Katsumi Imada, Takeharu Nagai, Tomonobu M. Watanabe
SCIENTIFIC REPORTS, 6, 22342, Mar. 2016, [Peer-reviewed]
English, Scientific journal

In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor
Motosuke Tsutsumi, Hideki Muto, Shohei Myoba, Mai Kimoto, Akira Kitamura, Masakatsu Kamiya, Takashi Kikukawa, Shigeharu Takiya, Makoto Demura, Keiichi Kawano, Masataka Kinjo, Tomoyasu Aizawa
FEBS OPEN BIO, 6, 2, 106, 125, Feb. 2016, [Peer-reviewed]
English, Scientific journal

Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity
Akira Kitamura, Yusaku Nakayama, Ai Shibasaki, Ayami Taki, Sachiko Yuno, Kayo Takeda, Masao Yahara, Naoki Tanabe, Masataka Kinjo
SCIENTIFIC REPORTS, 6, 19230, Jan. 2016, [Peer-reviewed]
English, Scientific journal

Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding
Akira Kitamura, Yusaku Nakayama, Masataka Kinjo
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 463, 3, 401, 406, Jul. 2015, [Peer-reviewed]
English, Scientific journal

Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques
Akira Kitamura, Kazuhiro Nagata, Masataka Kinjo
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 16, 3, 6076, 6092, Mar. 2015, [Peer-reviewed], [Invited]
English, Scientific journal

蛍光相関分光法とFRETを用いた細胞内タンパク質品質管理機構の解析
北村 朗
バイオイメージング, 23, 1, 12, 17, Jun. 2014, [Peer-reviewed], [Invited], [Lead author, Last author, Corresponding author]
Japanese, Scientific journal

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA plus ATPase, which dynamically changes its oligomeric state
Daisuke Morito, Kouki Nishikawa, Jun Hoseki, Akira Kitamura, Yuri Kotani, Kazumi Kiso, Masataka Kinjo, Yoshinori Fujiyoshi, Kazuhiro Nagata
SCIENTIFIC REPORTS, 4, 4442, Mar. 2014, [Peer-reviewed]
English, Scientific journal

Dysregulation of the proteasome increases the toxicity of ALS-linked mutant SOD1
Akira Kitamura, Noriko Inada, Hiroshi Kubota, Gen Matsumoto, Masataka Kinjo, Richard I. Morimoto, Kazuhiro Nagata
GENES TO CELLS, 19, 3, 209, 224, Mar. 2014, [Peer-reviewed]
English, Scientific journal

Prefoldin prevents aggregation of alpha-synuclein
Mariko Takano, Erika Tashiro, Akira Kitamura, Hiroshi Maita, Sanae M. M. Iguchi-Ariga, Masataka Kinjo, Hiroyoshi Ariga
BRAIN RESEARCH, 1542, 186, 194, Jan. 2014, [Peer-reviewed]
English, Scientific journal

The interaction of Hsp104 with yeast prion Sup35 as analyzed by fluorescence cross-correlation spectroscopy
Shohei Ohta, Shigeko Kawai-Noma, Akira Kitamura, Chan-Gi Pack, Masataka Kinjo, Hideki Taguchi
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 442, 1-2, 28, 32, Dec. 2013, [Peer-reviewed]
English, Scientific journal

Prefoldin Protects Neuronal Cells from Polyglutamine Toxicity by Preventing Aggregation Formation
Erika Tashiro, Tamotsu Zako, Hideki Muto, Yoshinori Itoo, Karin Soergjerd, Naofumi Terada, Akira Abe, Makoto Miyazawa, Akira Kitamura, Hirotake Kitaura, Hiroshi Kubota, Mizuo Maeda, Takashi Momoi, Sanae M. M. Iguchi-Ariga, Masataka Kinjo, Hiroyoshi Ariga
JOURNAL OF BIOLOGICAL CHEMISTRY, 288, 27, 19958, 19972, Jul. 2013, [Peer-reviewed]
English, Scientific journal

Nonmuscle myosin II folds into a 10S form via two portions of tail for dynamic subcellular localization
Takayuki Kiboku, Tsuyoshi Katoh, Akio Nakamura, Akira Kitamura, Masataka Kinjo, Yota Murakami, Masayuki Takahashi
GENES TO CELLS, 18, 2, 90, 109, Feb. 2013, [Peer-reviewed]
English, Scientific journal

Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor, IRE1 beta
Daisuke Oikawa, Akira Kitamura, Masataka Kinjo, Takao Iwawaki
PLOS ONE, 7, 12, e51290, Dec. 2012, [Peer-reviewed]
English, Scientific journal

Atg9 vesicles are an important membrane source during early steps of autophagosome formation
Hayashi Yamamoto, Soichiro Kakuta, Tomonobu M. Watanabe, Akira Kitamura, Takayuki Sekito, Chika Kondo-Kakuta, Rie Ichikawa, Masataka Kinjo, Yoshinori Ohsumi
JOURNAL OF CELL BIOLOGY, 198, 2, 219, 233, Jul. 2012, [Peer-reviewed]
English, Scientific journal

Analyzing the aggregation of polyglutamine-expansion proteins and its modulation by molecular chaperones
Hiroshi Kubota, Akira Kitamura, Kazuhiro Nagata
METHODS, 53, 3, 267, 274, Mar. 2011, [Peer-reviewed]
English

Amyloid oligomers: dynamics and toxicity in the cytosol and nucleus
Akira Kitamura, Hiroshi Kubota
FEBS JOURNAL, 277, 6, 1369, 1379, Mar. 2010, [Peer-reviewed]
English

Molecular chaperone prefoldin inhibits polyglutamine aggregation and cytotoxicity
Tashiro Erika, Muto Hideki, Zako Tamotsu, Miyazawa Makoto, Kitaura Hirotake, Kitamura Akira, Kubota Hiroshi, Maeda Mizuo, Kinjo Masataka, Ariga Hiroyoshi
NEUROSCIENCE RESEARCH, 68, E310, 2010, [Peer-reviewed]

Autophagic Elimination of Misfolded Procollagen Aggregates in the Endoplasmic Reticulum as a Means of Cell Protection
Yoshihito Ishida, Akitsugu Yamamoto, Akira Kitamura, Shireen R. Lamande, Tamotsu Yoshimori, John F. Bateman, Hiroshi Kubota, Kazuhiro Nagata
MOLECULAR BIOLOGY OF THE CELL, 20, 11, 2744, 2754, Jun. 2009, [Peer-reviewed]
English, Scientific journal

MKKS is a centrosome-shuttling protein degraded by disease-causing mutations via CHIP-mediated ubiquitination
Shoshiro Hirayama, Yuji Yamazaki, Akira Kitamura, Yukako Oda, Daisuke Morito, Katsuya Okawa, Hiroshi Kimura, Douglas M. Cyr, Hiroshi Kubota, Kazuhiro Nagata
MOLECULAR BIOLOGY OF THE CELL, 19, 3, 899, 911, Mar. 2008, [Peer-reviewed]
English, Scientific journal

Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state
Akira Kitamura, Hiroshi Kubota, Chan-Gi Pack, Gen Matsumoto, Shoshiro Hirayama, Yasuo Takahashi, Hiroshi Kimura, Masataka Kinjo, Richard I. Morimoto, Kazuhiro Nagata
Nature Cell Biology, 8, 10, 1163, U224, Oct. 2006, [Lead author]
English, Scientific journal

Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis
Y Ishida, H Kubota, A Yamamoto, A Kitamura, HP Bachinger, K Nagata
MOLECULAR BIOLOGY OF THE CELL, 17, 5, 2346, 2355, May 2006, [Peer-reviewed]
English, Scientific journal

蛍光明滅速度変化でRNAの立体構造を識別する　　　　　　　　　　　　　　　
北村 朗, 月刊 光アライアンス, 34, 8, 8, 11, Aug. 2023, [Invited], [Lead author, Last author, Corresponding author]
Japanese, Introduction commerce magazine

TDP-43タンパク質凝集形成過程におけるシャペロンRNAの役割　　　　　　　　　　　　　　　
北村朗, 藤本愛, Bio Clinica, 38, 3, 56, 59, Mar. 2023, [Invited], [Lead author, Corresponding author]
Japanese, Introduction commerce magazine

ALSとFTD関連TDP-43タンパク質凝集形成過程におけるRNAの役割　　　　　　　　　　　　　　　
北村朗, 藤本愛, 月刊「細胞」, 54, 8, 474, 477, Jul. 2022, [Invited], [Lead author, Corresponding author]
Japanese, Introduction commerce magazine

Realizing RNA roles during protein aggregation associated with neurodegenerative diseases　　　　　　　　　　　　　　　
Akira Kitamura, The CELL, 53, 9, 578, 581, Aug. 2021, [Invited], [Lead author, Last author, Corresponding author]
Japanese, Introduction scientific journal

Protein aggregations and their gene therapy in neurodegenerative disorders
Akira Kitamura, Ai Fujimoto, Bio Clinica, 36, 5, 453, 456, 12 Jul. 2021, [Invited], [Lead author, Corresponding author], [Domestic magazines]
Japanese

Session 2SHP report—decoding intracellular architecture using visualizing device development and mathematical modeling
Akira Kitamura, Kazuya Kabayama, Biophysical Reviews, 12, 2, 281, 282, Apr. 2020, [Lead author, Corresponding author]
Springer Science and Business Media LLC, English

ALSにおけるタンパク質凝集体と遺伝子治療　　　　　　　　　　　　　　　
北村朗, メディカル・サイエンス・ダイジェスト (MSD), 46, 12, 763, 765, 2020, [Invited], [Lead author, Last author, Corresponding author]
Japanese, Introduction commerce magazine

New Trends in Fluorescence Correlation Spectroscopy, Development and Application
YAMAMOTO Johtaro, KITAMURA Akira, KINJO Masataka, Seibutsu Butsuri, 59, 3, 125, 131, 2019
<p>Fluorescence correlation spectroscopy (FCS) is a potential tool to measure the dynamics of fluorescent molecules, either in solution or in a cell. FCS can quantify the diffusion coefficient, the target molecule concentration, and brightness of a single molecule. These parameters allow estimations of the molecular size, the molecular shape, and the affinities of molecular interactions. Moreover, varieties of FCS methods have been developed on the concept of the fluorescence lifetime, triplet state, decrease of fluorescent intensity and toward two-dimensional imaging, in order to reveal the molecular interaction in live cell and molecular crowding.</p>, The Biophysical Society of Japan General Incorporated Association, Japanese

蛍光相関分光法を用いた精製リコンビナントTDP43タンパク質のDNA結合活性解析
柴崎愛, 北村朗, 寿野良二, 金城政孝, 日本分子生物学会年会プログラム・要旨集(Web), 37th, 2014

分子シャペロンPrefoldinは変異ハンチンチンのオリゴマーおよび凝集体形成を阻害する
田代絵梨佳, 武藤秀樹, 座古保, 宮澤誠, 北浦廣剛, 北村朗, 久保田広志, 前田瑞夫, 金城政孝, 有賀寛芳, 日本薬学会年会要旨集, 132nd, 3, 139, 05 Mar. 2012
Japanese

分子シャペロンPrefoldinは変異ハンチンチンのオリゴマーおよび凝集体形成を阻害して細胞死を減少させる
田代絵梨佳, 武藤秀樹, 座古保, 宮澤誠, 北浦廣剛, 北村朗, 久保田広志, 前田瑞夫, 金城政孝, 有賀寛芳, 次世代を担う若手ファーマ・バイオフォーラム講演要旨集, 9th, 40, Sep. 2010
Japanese

ポリグルタミンタンパク質凝集における分子シャペロンプレフォルディンの役割
伊藤禎宣, 伊藤禎宣, 座古保, ソルヤード カリン, 寺田尚史, 田代絵梨佳, 北村朗, 久保田広志, 武藤秀樹, 金城政孝, 有賀寛芳, 前田瑞夫, 日本蛋白質科学会年会プログラム・要旨集, 10th, 91, 15 May 2010
Japanese

Fluorescently labeled proteins as a tool for analyzing the dynamics of protein quality control in living cells
Hiroshi Kubota, Akira Kitamura, Hideaki Itoh, Masataka Kinjo, Kazuhiro Nagata, International Journal of the Society of Material Engineering for Resources, 17, 1, 1, 4, Mar. 2010
English, Book review

分子シャペロンPrefoldinは変異ハンチンチンのオリゴマーおよび凝集体形成を阻害して細胞死を減少させる
田代絵梨佳, 武藤秀樹, 座古保, 宮澤誠, 北浦廣剛, 北村朗, 久保田広志, 前田瑞夫, 金城政孝, 有賀寛芳, 生化学, ROMBUNNO.1T4-6, 2010
Japanese

蛍光相関分光で見る生体系の情報伝達(6)原核生物における蛍光相関分光法測定-原核細胞内タンパク質品質管理機構の解明に向けて-
北村朗, 北村朗, 寿野良二, 寿野良二, 秋山芳展, 吉田腎右, 金城政孝, 蛍光相関分光で見る生体系の情報伝達6 理研シンポジウム 平成21年, 2009

MM‐1およびPrefoldinがポリグルタミン凝集に与える影響
田代絵梨佳, 武藤秀樹, 座古誠, 宮澤誠, 北浦廣剛, 北村朗, 久保田広志, 前田瑞夫, 金城正孝, 有賀芳寛, 日本分子生物学会年会講演要旨集, 32nd, Vol.1, 206, 2009
Japanese

マックジック－カウフマン病タンパク質は細胞質と中心体を素早く行き来し，その疾患原因変異体タンパク質は品質管理E3 リガーゼCHIP により素早く分解される　　　　　　　　　　　　　　　
Shoshiro Hirayama, Yuji Yamazaki, Akira Kitamura, Yukako Oda, Daisuke Morito, Katsuya Okawa, Hiroshi Kimura, Douglas M. Cyr, Hiroshi Kubota, Kazuhiro Nagata, 第40 回日本発生生物学会・第59 回日本細胞生物学会合同大会，福岡市，2007.5.28－30, 2007, [Peer-reviewed]
Japanese

McKusick-Kaufman syndromeタンパク質の疾患原因変異体はE3リガーゼCHIPを介してユビキチン-プロテアソーム系で素早く分解される
平山尚志郎, 山崎祐自, 北村朗, 小田祐香子, 森戸大介, 大川克也, 木村宏, 久保田広志, 永田和宏, 臨床ストレス応答学会大会抄録集, 1st, 2006

Analysis of the collagen-binding sites by mutation of the ER molecular chaperone HSP47
Yasuhiro Matsuoka, Hiroshi Kubota, Akira Kitamura, Kazuhiro Nagata, CELL STRUCTURE AND FUNCTION, 30, 48, 48, Jun. 2005
English, Summary international conference

Aggregation of actin and tubulin by RNAi-mediated knockdown of cytosolic chaperonin CCT in human cells
Akira Kitamura, Hiroshi Kubota, Kazuhiro Nagata, CELL STRUCTURE AND FUNCTION, 30, 45, 45, Jun. 2005
English, Summary international conference

Mckusick-Kaufman syndrome gene product is a chaperonin-like molecule abundant in the centrosome and degraded upon disease-causing mutations
Hiroshi Kubota, Yuji Yamazaki, Akira Kitamura, Yukako Oda, Shoshiro Hirayama, Kazuhiro Nagata, CELL STRUCTURE AND FUNCTION, 30, 45, 45, Jun. 2005, [Peer-reviewed]
English, Summary international conference

Mutational analysis of the ER molecular chaperone HSP47 for identifying the collagen-binding sites
Yasuhiro Matsuoka, Hiroshi Kubota, Akira Kitamura, Kazuhiro Nagata, CELL STRUCTURE AND FUNCTION, 29, 96, 96, May 2004
English, Summary international conference

Mckusick-Kaufman syndromeタンパク質：中心体に豊富な新規シャペロニン　　　　　　　　　　　　　　　
久保田広志, 山崎裕自, 吉田導雄, 安永卓生, 北村朗, 小田裕香子, 永田和宏, 第77回日本生化学会大会, 2004

シャペロニン様Mckusick-Kaufman syndromeタンパク質の病因変異体はプロテアソーム依存的に急速に分解される　　　　　　　　　　　　　　　
久保田広志, 山崎裕自, 吉田尊雌, 安永卓生, 北村朗, 小田裕香子, 永田和宏, 第27回日本分子生物学会大会, 2004

中心体に豊富なシャペロニン様タンパク質MKKSPの病因変具体は急速な細胞内分解を受ける　　　　　　　　　　　　　　　
久保田広志, 山崎裕自, 吉田尊雄, 安永卓生, 北村朗, 小田裕香子, 永田和宏, 第9回臨床ストレス蛋白質研究会, 2004

新・生細胞蛍光イメージング　　　　　　　　　　　　　　　
北村 朗, 第21章，実習8-2
共立出版, Nov. 2015, [Joint work]

None
Akira Kitamura
The 44th Hoansha Research Grant Meeting, 04 Jul. 2025, Japanese, Invited oral presentation
46048265, [Invited]

The world of protein aggregates from using fluorescence correlation spectroscopy and its advanced methods
Akira Kitamura
The 81st Annual Meeting of The Japanese Society of Microscopy, 10 Jun. 2025, Japanese, Invited oral presentation
46048265, [Invited]

The Future of Fluorescence Correlation Spectroscopy in Chemical Biology
Akira Kitamura
The 19th Annual Meeting of Japanese Society for Chemical Biology, ZEISS Luncheon Seminar, 04 Jun. 2025, Japanese, Invited oral presentation
46048265, [Invited]

これまでの蛍光相関分光法とこれからの蛍光相関分光法
北村 朗
第47回 日本分子生物学会年会, 28 Nov. 2024, Japanese, Invited oral presentation
46048265, [Invited]

先端相関分光と光イメージングで捉える 細胞内クラスター動態とその機能
北村 朗
第76回 日本細胞生物学会大会, 18 Jul. 2024, Japanese, Invited oral presentation
46048265, [Invited]

Molecular cell biology through induction, kinetic analysis, and inactivation of biomolecules using ultimate light technology
Akira Kitamura
MBSJ2023, 07 Dec. 2023, Japanese, Nominated symposium
40045816

Detective tools of RNA structural transition using fluorescence blinking
Akira Kitamura
45th MBSJ annual meeting, 30 Nov. 2022, Japanese, Invited oral presentation
40045923, [Invited]

Diffusion coefficient measurement using fluorescence recovery after photobleaching ―a homework during summer vacation with refreshment―
Akira Kitamura
60th BSJ annual meeting, 28 Sep. 2022, English, Public discourse
40045816, [Invited]

Basics and applications of chromophore-assisted light inactivation (CALI)
Akira Kitamura
60th BSJ annual meeting, 28 Sep. 2022, English, Invited oral presentation
40045816, [Invited]

Intracellular structure and function of chaperone RNA
Akira Kitamura
International Workshop on Fluorescence Methods and Applications 2022, 15 Sep. 2022, English, Invited oral presentation
15 Sep. 2022 - 16 Sep. 2022, 11610265, [Invited]

The N-terminal rule in the aggregate formation of C-terminal fragments of TDP-43　　　　　　　　　　　　　　　
北村朗, 呂依丹, 藤本愛, 川島怜維, 森谷香南, 高橋一帆, 金城正孝
72th JSCB annual meeting, 02 Jul. 2021, Japanese, Poster presentation
29 Jun. 2021 - 02 Jul. 2021

The domain role for condensation of carboxyl-terminal fragments of TDP-43　　　　　　　　　　　　　　　
Akira Kitamura
FASEB SRC 2021 "The Protein Aggregation Conference: Function, Dysfunction, and Disease", English, Poster presentation
23 Jun. 2021 - 26 Jun. 2021

Cold Spring Harbor Laboratory 2020 Meeting "Protein homeostasis in Health & Disease"　　　　　　　　　　　　　　　
Akira Kitamura
Cold Spring Harbor Laboratory 2020 Meeting "Protein homeostasis in Health & Disease", 11 Nov. 2020, English, Poster presentation
11 Nov. 2020 - 14 Nov. 2020

蛍光相関分光法とその応用法を用いたALS関連細胞内凝集体形成機構の解析
北村朗
サントリー生有研シンポジウム, 16 Dec. 2019, Japanese, Public discourse
12176697, [Invited]

Aggregation process of carboxyl terminal fragments of TDP-43
Akira Kitamura
QBP seminar, KTH Royal Institute of Technology, 28 Nov. 2019, English, Public discourse
11610265, [Invited]

Aggregation process of carboxyl terminal fragments of TDP-43
Akira Kitamura
Asian Pacific Prion Symposium 2019, 04 Oct. 2019, English, Oral presentation
03 Oct. 2019 - 04 Oct. 2019, 12176697

RNA prevents ALS-linked protein aggregation
Akira Kitamura
QBP-seminar, KTH Royal Institute of Technology, 15 Nov. 2018, English, Public discourse
11610265, [Invited]

蛍光ゆらぎを利用した生体分子構造変化の解析　　　　　　　　　　　　　　　
北村 朗
第55回 日本生化学会北海道支部例会，日本生物物理学会北海道支部合同シンポジウム, 13 Jul. 2018, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

Basics and application of fluorescence correlation spectroscopy　　　　　　　　　　　　　　　
Akira Kitamura
5th AIST International Imaging Workshop, 24 Jan. 2018, English, Public discourse
[Invited], [International presentation]

FCS and FCCS in living cells　　　　　　　　　　　　　　　
Akira Kitamura
Course Functional Fluorescence Microscopy Imaging (fFMI) in Biomedical Research, 03 Dec. 2017, English, Public discourse
Stockholm, Sweden, [Invited], [International presentation]

Basics and application of fluorescence correlation spectroscopy　　　　　　　　　　　　　　　
Akira Kitamura
4th AIST International Imaging Workshop, 21 Jan. 2017, English, Invited oral presentation
AIST, Tsukuba, [Invited]

Frontiers of neurodegenerative disorder research using FRAP and FCS　　　　　　　　　　　　　　　
Akira Kitamura
36th MBSJ annual meeting, 02 Dec. 2016, Japanese, Public discourse
[Invited], [Domestic Conference]

FCS and FCCS in living cells　　　　　　　　　　　　　　　
Akira Kitamura
Course Functional Fluorescence Microscopy Imaging (fFMI) in Biomedical Research, 14 Nov. 2016, English, Invited oral presentation
Karolinska Institutet, Stockholm, Sweden, [Invited]

Elucidation for aggregation mechanism of ALS-linked TDP43 using fluorescence imaging techniques　　　　　　　　　　　　　　　
Akira Kitamura
7th ALS forum, 30 Jul. 2016, Japanese, Invited oral presentation
Tokyo, Japan, [Invited]

Basics and application of fluorescence correlation spectroscopy　　　　　　　　　　　　　　　
Akira Kitamura
3rd AIST International Imaging Workshop, 21 Jan. 2016, English, Invited oral presentation
[Invited]

蛍光相関分光法とFRETを用いた細胞内タンパク質品質管理機構の解析　　　　　　　　　　　　　　　
北村 朗
第55回日本植物生理学会年会, 20 Mar. 2014, Japanese, Nominated symposium
[Domestic Conference]

1P299 Analysis of structural difference in ALS-linked mutant of TDP43 by fluorescence imaging in living cells(27. Bioimaging,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Yuno Sachiko, Kitamura Akira, Shibasaki Ai, Kinjo Masataka
Seibutsu Butsuri, 2014, The Biophysical Society of Japan General Incorporated Association, English
2014 - 2014

神経変性疾患に おける細胞内タンパク質恒常性　　　　　　　　　　　　　　　
北村 朗
「医と食の融合によるビジネス創出を目指して」 北大Ｒ＆ＢＰセミナー, 21 Nov. 2013, Japanese, Public discourse
[Invited], [Domestic Conference]

生細胞内FRET可視化法－フリーソフトウェアImageJを用いた画像演算実習－　　　　　　　　　　　　　　　
北村 朗
第五回「光塾」, 07 Sep. 2013, Public discourse

画像相関分光解析実習―点と点の相関性から何がわかるのか？―　　　　　　　　　　　　　　　
佐々木章, 北村 朗
第四回「光塾」, 25 Aug. 2012, Public discourse

生命科学画像解析における数学の基礎　　　　　　　　　　　　　　　
北村 朗
第三回「光塾」, 22 Oct. 2011, Public discourse

細胞内分子運動の解析－基礎から応用まで熱風のアシスト－　　　　　　　　　　　　　　　
北村 朗
第二回「光塾」, 11 Dec. 2010, Public discourse

Molecular chaperone prefoldin inhibits polyglutamine aggregation and cytotoxicity
Erika Tashiro, Hideki Muto, Tamotsu Zako, Makoto Miyazawa, Hirotake Kitaura, Akira Kitamura, Hiroshi Kubota, Mizuo Maeda, Masataka Kinjo, Hiroyoshi Ariga
NEUROSCIENCE RESEARCH, 2010, ELSEVIER IRELAND LTD, English
2010 - 2010

画像解析プラティカ －ImageJ tipsからフリーウェアを用いた数値解析処理まで－　　　　　　　　　　　　　　　
北村 朗
第一回「光塾」, 15 Aug. 2009, Public discourse

3P-027 Aggregation and disaggregation analysis of mutant SOD1 using fluorescence correlation spectroscopy and FLIM-FRET(The 46th Annual Meeting of the Biophysical Society of Japan)
Kitamura Akira, Inada Noriko, Matsumoto Gen, Morimoto Richard I., Kubota Hiroshi, Nagata Kazuhiro, Kinjo Masataka
Seibutsu Butsuri, 2008, The Biophysical Society of Japan General Incorporated Association, English
2008 - 2008

2P113 Fluorescence correlation spectroscopy detects polyglutamine small oligomers increased by knock-down of cytosolic chaperonin CCT.(31. Protein folding and misfolding (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)
Kitamura Akira, Kubota Hiroshi, Pack Chan-Gi, Matsumoto Gen, Hirayama Shoshiro, Takahashi Yasuo, Kumura Hiroshi, Kinjo Masataka, Morimoto Richard I., Nagata Kazuhiro
Seibutsu Butsuri, 2006, The Biophysical Society of Japan General Incorporated Association, English
2006 - 2006

Functional Cellular Sciences: Cellular and Molecular Science　　　　　　　　　　　　　　　
Hokkaido University, Graduate School of Life Science
2024 - Present

Cell Structural Science II　　　　　　　　　　　　　　　
Hokkaido University, School of Science
2023 - Present

生命融合科学概論　　　　　　　　　　　　　　　
北海道大学大学院生命科学院
2023 - Present

Life science special lecture III (Advanced Light Microscope in Life Science Research) as the Hokkaido Summer Institute　　　　　　　　　　　　　　　
Hokkaido University, Graduate School of Life Science
2016 - Present

Functional Fluorescence Microscopy Imaging (fFMI) in Biomedical Research　　　　　　　　　　　　　　　
Kalorinska Institutet
2016 - Present

Laboratory Work on Bio-macromolecular Science II　　　　　　　　　　　　　　　
Hokkaido University, School of Science
2013 - Present

Fundamental Laboratory Work on Bio-macromolecular Science　　　　　　　　　　　　　　　
Hokkaido University, School of Science
2012 - Present

脳科学研究の展開（先端脳機能イメージング）　　　　　　　　　　　　　　　
北海道大学脳科学教育研究センター
2011 - Present

Reaction Kinetics for Life Science　　　　　　　　　　　　　　　
School of Science, Hokkaido University
2018 - 2023

Experimental Biological Science　　　　　　　　　　　　　　　
Hokkaido University, School of Science
2012 - 2023

Cell Structural Science III　　　　　　　　　　　　　　　
Hokkaido University
2013 - 2022

AIST International Imaging Workshop　　　　　　　　　　　　　　　
Association for Iron & Steel Technology, Japan
2016 - 2019

Functional Cellular Sciences　　　　　　　　　　　　　　　
Hokkaido University, Graduate School of Life Science
2013 - 2017

生物物理学　　　　　　　　　　　　　　　
秋田大学工学資源学部
2012 - 2015

生命融合科学概論　　　　　　　　　　　　　　　
北海道大学大学院生命科学院
2010 - 2010

バイオロジカルクラスター解析のための極限時空間相関分光法の確立
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2029
北村 朗
日本学術振興会, 学術変革領域研究(A), 北海道大学, 24H02286

Not applicable
The 5th Research Grant (2024)
Apr. 2025 - Mar. 2026
Hagiwara Foundation of Japan, Principal investigator

Not applicable
39th (2025 FY) Research Grant
Apr. 2025 - Mar. 2026
Suharakinennzaidann Co., Ltd., Principal investigator

細胞内プロテオスタシスを維持するシャペロンRNAの作動機序解明
Advanced Research & Development Programs for Medical Innovation (AMED-PRIME) "Understanding proteostasis and discovering innovative medical applications"
Oct. 2022 - Mar. 2026
Akira Kitamura
AMED, Hokkaido University, Principal investigator

タンパク質2量体安定化機構へ与える細胞内微環境の影響解明
科学研究費助成事業
01 Apr. 2022 - 31 Mar. 2025
金城 政孝, 北村 朗
細胞情報伝達には多くのタンパク質が関与している。その中でも2量体化して細胞質から核へ移行し，情報を伝達する仕組みは，情報伝達因子や転写因子に多く存在している。しかし「2量体化」と「移行」の間には相反する要因がある。それは2量体化することで構造的には安定化すると考えられているが，2量体化することで分子量が大きくなれば，移行するためには多くのエネルギーが必要であり，また輸送時間がかかることから，情報伝達では不利になる。このようにタンパク質が2量体化をする理由については不明なことが多く，その理由は明確にされていない。


特に核内受容体の一つであるグルココルチコイドレセプター (GR)は，細胞情報伝達分子として糖代謝のみならず，免疫制御・抗炎症作用から精神的な情緒まで広く関与している。GRのシグナル伝達の最終段階は2量体化しDNAに結合する転写活性にあるが，申請者らはその前段階のホモならびにヘテロ2量体形成量が核内受容体の多様な機能を支えると考えた。
本研究ではGRの2量体化量とそれに与える細胞内微環境の影響を同時に解明するために分子量サイズの変化に敏感で回転拡散測定可能な偏光蛍光相互相関分光装置（Pol-FCCS）の新規構築を続ける。特にPol-FCCSから得られるパラメーターのうち，分子数を決定することのため，Alexa系蛍光色素と，Fluorescein系蛍光色素を用いて，共焦点領域の大きさの決定を行った。
また，細胞測定に向けて，細胞内タンパク質の発現パターンを調べる必要性から科研費予算を利用して外注してNeuro2a並びに293細胞のtoral RNA解析を行った。
日本学術振興会, 基盤研究(B), 北海道大学, 23K23842

タンパク質2量体安定化機構へ与える細胞内微環境の影響解明
科学研究費助成事業
Apr. 2022 - Mar. 2025
金城 政孝, 北村 朗
日本学術振興会, 基盤研究(B), 北海道大学, Coinvestigator, 22H02578

神経変性疾患の核酸医薬創薬に向けたシャペロン RNA の実証
研究助成
Apr. 2021 - Mar. 2025
Hoansha, 北海道大学, Principal investigator

細胞由来微粒子迅速測定のための蛍光と散乱光測定の融合
科学研究費助成事業
30 Jun. 2022 - 31 Mar. 2024
金城 政孝, 北村 朗
細胞内の液-液相分離による分子集合体やタンパク質凝縮体、また細胞外に放出される細胞外小胞など、様々な形態や大きさを有する新規の構造体が細胞情報伝達や疾患など生体の重要な機能に関連することが認識されている。本研究では、そのような多彩な構造と幅広いサイズ分布を持つ分子集合体を一括して『細胞由来微粒子』として捉え、それらを対象とした散乱光による網羅的検出と蛍光による特異的検出を統合した新規定量的検出法の確立を目指している。
生体分子を対象とした蛍光と散乱光測定を融合した同時計測方法はこれまでになく、シグナル強度だけを見ると、散乱強度は極端に強く、蛍光は微弱であり、単純に比較することは困難である。申請者等は溶液中の分子や微粒子の特徴の一つである「揺れ動く」性質に着目した。蛍光・散乱測定に共通する時系列シグナル変化を基礎とする「自己相関解析」と、さらに蛍光と散乱光シグナル間の「相互相関解析」の同時利用で蛍光と散乱測定の二つの情報の統合と高度利用法を目指している。
具体的測定手段は申請者等が構築した不等分割光ファイバー型蛍光相関分光装置の散乱同時測定への展開と高度化である。本研究では特に細胞外微粒子の中でもガン診断の指標等として注目されている抗原提示エクソソームの定量的検出・同定法の確立を通してその性能を実証し、新規細胞由来微粒子検出法としての可能性を実証する
不当分割光ファイバーを利用した2色蛍光相互相関分光装置（FCrossCS）は、通常の励起光ダイクロイックミラー（第１次DM）を必要としないことから、これまで測定毎に必要とされた光軸・ピンホール調整等のキャリブレーションは一切不要となり、小型で長期安定な蛍光測定装置として利用でき、光軸調整不要なことから広い拡張性を有する。その拡張性の一つに、検出側の蛍光分光ダイクロイックミラー（２次）をハーフミラーに交換することで蛍光と散乱の同時測定を行なった。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 22K19886

非公開
Translational Research Network Program (Seeds A)
Apr. 2023 - Mar. 2024
AMED, Hokkaido University, Principal investigator

非公開
Translational Research Network Program (Seeds H)
Sep. 2022 - Mar. 2024
Akira Kitamura
AMED, Hokkaido University, Principal investigator, JP22ym0126814

蛍光動的消光測定による生細胞内RNA立体構造の情報物理解析
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
Apr. 2022 - Mar. 2024
北村 朗
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, Principal investigator, 22H04826

次世代画像相関分光法の確立による液滴内分子運動解析
2021 2nd Grant Program
Apr. 2022 - Mar. 2023
Akira Kitamura
HAGIWARA Foundation, Hokkaido University, Principal investigator

二量体特異的発色団補助光不活化法の確立とそれを用いた光破壊による細胞内機能解析
Grant Program
Apr. 2022 - Mar. 2023
Akira Kitamura
NAKATANI Foundation, Hokkaido University, Principal investigator

単一分子感度蛍光測定法を用いた新型コロナウイルス感染阻害剤スクリーニング法の確立
新型コロナウイルス感染症対策 助成プログラム
Jul. 2020 - Jun. 2021
NAKATANI Foundation, 北海道大学, Principal investigator

神経変性疾患に関連したタンパク質凝集形成を防ぐシャペロンRNAの実証
Grant-in-Aid for Scientific Research (C)
Apr. 2018 - Mar. 2021
Akira Kitamura
JSPS, Principal investigator, Competitive research funding

光ファイバー型蛍光相関分光システムの研究開発と生物応用　　　　　　　　　　　　　　　
Research fund
Apr. 2018 - Mar. 2020
Akira Kitamura
Canon fundation, Principal investigator, Competitive research funding

Development of polarization-dependent fluorescence correlation spectroscopy for initial process analysis of protein aggregation
Grants-in-Aid for Scientific Research
Apr. 2016 - Mar. 2019
Yamamoto Johtaro, Kitamura Akira
In this work, we developed a polarization-dependent fluorescence correlation spectroscopy (Pol-FCS), which analyzes translational and rotational diffusion of molecules such as proteins. Size measurement of molecular aggregation using Pol-FCS inside living cells was successfully demonstrated. In addition, we found the Pol-FCS can also estimate orientation of molecules (ordered/disordered) and degree of macromolecular crowding. Thus, the Pol-FCS was a more powerful tool than we expected. In future, we expect the Pol-FCS will be widely used in biology such as the researches about aggregation prone proteins.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Coinvestigator, 16K07312

Elucidation for the regulation of motility and lifespan by proteostasis(Fostering Joint International Research)
Grants-in-Aid for Scientific Research
2017 - 2019
Kitamura Akira
The accumulation of misfolded proteins formed upon disruption of intracellular proteinstasis often results in the formation of aggregates in the cell. Such protein aggregates are thought to be toxic and cause death of neurons. However, the mechanism of reducing the toxicity of the aggregates formed in the cells at the individual level has not been elucidated, and its application to the treatment of specific neurodegenerative diseases has not been possible. In this study, we established a nematode strain expressing TDP25, a causative protein of amyotrophic lateral sclerosis (ALS), and analyzed its motility and lifespan. We further established a novel fluorescence spectroscopy method and a system to read out the structure of RNAs that bind to aggregate proteins in living cells. We also established a method to read out the status of intracellular macromolecular crowding, which is thought to be involved in aggregation formation, using the fluorescence lifetime of GFP.
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research), Hokkaido University, 16KK0156

TDP43に潜在する非古典的核内輸送シグナル配列の同定と， 細胞質における神経細胞毒性低減機構の解明　　　　　　　　　　　　　　　
ALS基金
Jun. 2016 - Mar. 2017
北村 朗
一般社団法人 日本ALS協会, Principal investigator, Competitive research funding

ALSのRNA治療に向けたTDP43のプリオン様構造転移および凝集形成を抑制するRNA分子の同定　　　　　　　　　　　　　　　
「生命の彩」ALS研究助成基金
May 2016 - Mar. 2017
北村 朗
三井住友信託銀行公益信託, Principal investigator, Competitive research funding

Establishment of unified theory of cellular inclusion bodies including misfolded protein
KAKENHI
Apr. 2014 - Mar. 2017
Akira Kitamura
Accumulation of misfolded proteins due to inappropriate stabilization results in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). In this study, we clarify structural differences of carboxyl terminal fragments of ALS-causative TDP43 (TDP35 or TDP25) by using fluorescence resonance energy transfer (FRET) or related procedure. We elucidate that nuclear localization signal sequence (NLS)-tagged TDP25 can reduce cytotoxicity through the structural change.
JSPS, Grant-in-Aid for Scientific Research (C), Hokkaido University, Principal investigator, Competitive research funding, 26440090

細胞内化学反応解析のための超高速光計測システムの開発　　　　　　　　　　　　　　　
Development of systems and technology for advanced measurement and analysis
Oct. 2013 - Mar. 2017
Yasushi Hiraoka
JST, Competitive research funding

Analysis of neuronal cell death by ALS-associated misfolded protein aggregation with modulation of cellular RNA homeostasis
KAKENHI
Apr. 2011 - Mar. 2015
Akira Kitamura
TAR-RNA/DNA binding protein 43 kDa is a disease gene product associated with amyotrophic lateral sclerosis (ALS), a motor neuron disease. In this research, fluorescence correlation spectroscopy (FCS) measurement in cell lysate revealed that a 25 kDa carboxyl-terminal fragment of TDP43 (TDP25) formed aggregation after dissociation of RNA. Next, TDP43 was sequestrated into cytoplasmic inclusion bodies of TDP25 in living cells. Neuronal cells expressing TDP25 showed higher cell death efficiency than that of TDP43. These results suggest that TDP25 may be an aggregation-causative and toxic seed, and RNA may play an important role for inhibition of aggregation-formation of TDP25.
JSPS, Grant-in-Aid for Young Scientists (B), Hokkaido University, Principal investigator, Competitive research funding, 23770215

多点時空間相関解析法による細胞内分子複合体研究
KAKENHI
May 2009 - Mar. 2014
Masataka Kinjo
蛍光相関分光法（Fluorescence Correlation Spectroscopy，FCS）はダイナミックな分子間相互作用を単一分子レベルで解析する手法でるが、その一方で、励起光源にレーザー光が必要であり、その焦点領域である、特定の１点でしか測定出来ない。このことは生細胞が様々な区画に分かれていて、さらにその区画の中も不均一であるため、細胞生物学的応用の際の問題となっている。その解決のために本年も引き続き多点同時測定FCSの構築と生細胞測定への応用を目指ざしている。
そこでこれまで多点測定のためのまず、多点の焦点領域を作る必要がある。そのために空間光変調素子を利用したホログラフィ技術により複数照射の励起光パターンを作りだすことを可能とした。次に検出側としてマルチ光ファイバーと各端面に光電子増倍管を接続した多点検出装置を用いて，実際に生細胞内の多点におけるタンパク質の拡散運動を検出する装置を作成した。
次に本年は細胞応用測定に重点を置き、細胞質ならび細胞核を区別して7点同時測定を行った。
生細胞内GFPを対象としてガラス面からZ軸方向（上方向）に0.5ミクロンステップの空間分解能があることを証明し、次に細胞核膜を境にして同時7点測定FCS測定が可能であることを実証した。次に機能性タンパク質としてリガンド刺激により、細胞質から核へ移行するグルココルチコイドレセプター（GR）のGFP融合タンパク質を対象とした測定を行い、15秒おきのFCS多点同時測定に成功した。さらに本年は精度を上げるために細胞運動の抑制方法を種々検討し、ノコダゾール処理と、緩衝液を調整することで、長期の観測を可能とした。
安定測定が可能となったので、さらに時間分解を上げることが可能となったので、単一タンパク質分子の動態について、検出と解析を試みている。
MEXT, 基盤研究(S), 北海道大学, Competitive research funding, 21221006

Regulation of cellular functions by highly evolved two chaperonins
Grants-in-Aid for Scientific Research
2011 - 2013
KUBOTA HIROSHI, NAGATA Kazuhiro, KINJO Masataka, KITAMURA Akira
Chaperonins are a group of molecular chaperone that assists in protein folding in the cell. In this project, we analyzed two eukaryotic chaperonins: cytosolic chaperonin CCT and mitochondrial chaperonin HSP60. In the study of CCT, we found that CCT possesses GTPase activity in addition to the ATPase activity. In the study of HSP60, we found that football-like complex plays an important role in the function of HSP60 after formation of the football-like complex from single-ring complex via double-ring complex. These results contain important findings to understand chaperonin functions.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Akita University, 23570220

Study of membrane binding protein complex using total internal reflection fluorescence correlation spectroscopy
KAKENHI
2007 - 2011
Masataka Kinjo
The functional molecules move around in the cell and interact with other molecules, and then support higher function of the cell. Moreover, even aggregate prone protein acts as aggregate and disaggregate under specific condition, so that each element of these aggregate protein is exchanged with free its molecules in solution. FCS (fluorescence correlation spectroscopy) detects the fluorescence fluctuations, the diffusion coefficient, molecular concentration and molecular interactions of molecules. We developed a multipoint FCS system which was based on an objective-type total internal reflection-FCS (TIR-FCS) in order to analysis molecular interaction in living cell and system of protein community.
MEXT, Grant-in-Aid for Scientific Research on Priority Areas, Hokkaido University, Competitive research funding, 19058001

神経変性疾患を引き起こすタンパク質凝集体形成と神経細胞死機構の時空間的解析
科学研究費助成事業
2008 - 2010
北村 朗
神経変性疾患に共通してみられる現象として,神経細胞の機能低下とそれに続く細胞死がある.このような神経細胞死を引き起こす原因として,細胞内外に蓄積したタンパク質凝集体があげられる.筋萎縮性側索硬化症(Amyotrophic Lateral Screlosis ; ALS)では,原因遺伝子の約20%をSuperoxide dismutase 1(SOD1)が占めており,このSOD1タンパク質に変異が入ると凝集体を形成することが知られている.これらの変異タンパク質の凝集体が,細胞内のどのような場所でどのような構造にあるとき,細胞毒性を持つのか.そして細胞死に至るのかといった問題点を解明するため,細胞内の分解機構の一つであるプロテアソーム経路の阻害剤を用いたところ,変異型SOD1は細胞内で凝集体を形成した.さらに,プロテアソームの活性を回復させたところ,凝集体は脱凝集を介して消失した.これらの脱凝集過程は,プロテアソームの活性に大きく依存しており,細胞内における別の分解経路として知られているオートファジー・リソソーム経路は必須ではないことを見出した.また,蛍光相関分光法技術を用いることで,変異型SOD1タンパク質は生細胞のサイトゾルで複合体を形成していることが判明した.蛍光相関分光法の解析により,この複合体は変異型SOD1タンパク質のみを含むものではなく,他の細胞内因子が含まれていることが示唆された.さらに,免疫共沈降法・マススペクトル解析により,分子シャペロンHsc70などが含まれていることが明らかとなった.このことから,細胞内において凝集体の毒性を抑制する際に,分子シャペロンが関与していることが示唆された.
日本学術振興会, 特別研究員奨励費, 北海道大学, 08J04474

細胞質シャペロニンの機能解析
科学研究費助成事業
2006 - 2007
北村 朗
本年度は,従来より研究を進めていたポリグルタミンタンパク質のように変異によって凝集体を形成し,神経細胞死を招くと考えられている変異型SOD1タンパク質の凝集体形成について主に解析を行った.SOD1タンパク質は,難病指定された神経変性疾患の一つである家族性筋萎縮性側索硬化症(fALS)の原因遺伝子である.最初に,プロテアソーム阻害剤であるMG-132処理により,変異型SOD1では,核近傍に,ユビキチン化されたタンパク質や分子シャペロンなどが集積したアグレソーム様の凝集体構造を形成していることを明らかにした.さらに,MG-132処理によって形成された凝集体構造は,プロテアソームの活性回復に伴い,消失することを見出した.この消失過程を詳細に解析した結果,一度形成された凝集体を解きほぐす脱凝集機構が関与していることが示唆された.また,凝集過程よりも脱凝集過程における細胞死の頻度が高いことから,脱凝集過程において発生する分子の構造もしくは多量体化状態が毒性を発揮する可能性があると考え,蛍光相関分光法による分子サイズの解析より,脱凝集過程において,可溶性のオリゴマーはすみやかに消失することから,毒性を発揮するのは,大きな多量体ではなく,低分子で構造の転移したものではないかという仮説提唱に至った.これらの結果は,昨年度公開した研究成果のようなポリグルタミンタンパク質で得られた知見とは異なり,SOD1の場合は,多量体化したオリゴマーが毒性を発揮するのではなく,低分子であっても毒性を発揮しうる状態があるのではないかと考えられる.
続いて,細胞質シャペロニンCCTと弱い相同性を持つタンパク質であるMKKSPについても解析を行った.MKKSPは中心体に豊富に局在していることが,これまでの解析から明らかにされていたが,今年度の研究では,MKKSPが中心体と細胞質の間をすみやかに移動しているが,少ない割合の成分は中心体に滞留し,何かしらの機能を発現していることが示唆された.
日本学術振興会, 特別研究員奨励費, 京都大学, 06J03037

ＢＤＮＦ分泌促進剤及び緑内障を治療又は予防するための薬剤
Patent right, 北村朗, 藤本愛, 河村綸太郎
特願2025-104771, 20 Jun. 2025
40045923

ＴＤＰ４３、ＴＤＰ３５、またはＴＤＰ２５の凝集を特徴とする神経変性疾患の治療剤、進行抑制剤、および予防剤　　　　　　　　　　　　　　　
Patent right, 北村朗, 藤本愛, 国立大学法人 北海道大学
特願PCT/JP2022/041001, 02 Nov. 2022

ヘモシアニン会合体を用いた包摂体の製造方法　　　　　　　　　　　　　　　
Patent right, 田中 良和, 松野 明日香, 北村 朗, 金城 政孝, 姚 閔, 上野 隆史, 安部 聡
特願2016-110309, 01 Jun. 2016
特開2017-214339, 07 Dec. 2017