Researcher Profile and Settings

Affiliation

• Faculty of Advanced Life Science　Functional Life Sciences　Functional Cell Science

Job Title

• Lecturer

Degree

• Ph.D.（Kyoto University）

J-Global ID

• 200901073020874800

Research Interests

• Chaperone RNA   proteostasis   FRAP   FRET   Fluorescence correlation spectroscopy   Polyglutamine disease   Amyotrophic lateral sclerosis   Proteostasis   Fluorescence imaging   Neurodegenerative disease   Molecular chaperone   Protein folding   Protein aggregation   

Research Areas

• Life sciences / Functional biochemistry
• Life sciences / Biophysics
• Life sciences / Molecular biology
• Life sciences / Cell biology

Educational Organization

•  Bachelor's degree program　School of Science
•  Master's degree program　Graduate School of Life Science
•  Doctoral (PhD) degree program　Graduate School of Life Science

Academic & Professional Experience

• 2023/08 - Today RIKEN Center for Biosystems Dynamics Research Visiting Researcher
• 2023/08 - Today Hokkaido University Faculty of Advanced Life Science Senior Lecturer (Tenure and PI)
• 2022/10 - Today PRIME, AMED
• 2019/10 - Today Hokkaido University Research and Education Center for Brain Science
• 2019/04 - 2023/07 Hokkaido University Faculty of Advanced Life Science Senior Lecturer (Tenure-track)
• 2017 - 2020/03 JSPS Scientist for Joint International Research
• 2010/04 - 2019/03 Hokkaido University Faculty of Advanced Life Science Assistant Professor
• 2018/08 - 2018/11 Northwestern University Prof. Richard Morimoto Laboratory Visiting Scholar
• 2018/05 - 2018/06 KTH Royal Institute of Technology Department of Applied Physics JSPS Scientist for Joint International Research
• 2017/10 - 2017/12 KTH Royal Institute for Technology Dept. Applied Physics JSPS Scientist for Joint International Research
• 2008/04 - 2010/03 Faculty of Advanced Life Science, Hokkaido University JSPS Research fellow PD
• 2006/04 - 2008/03 Kyoto University, Institute for Frontier Medical Sciences JSPS Research fellow DC2

Education

•        - 2008  Kyoto University  Graduate School of Science  Division of Biological Sciences
•        - 2008  Kyoto University  Graduate School, Division of Natural Science
•        - 2005  Kyoto University  Graduate School of Science  Division of Biological Sciences
•        - 2003  Kyoto University  Faculty of Engineering  School of Industrial Chemistry
•        - 2003  Kyoto University  Faculty of Engineering

Association Memberships

• JAPAN SOCIETY FOR CELL BIOLOGY   THE BIOPHYSICAL SOCIETY OF JAPAN   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   The RNA Society of Japan   

Research Activities

Published Papers

• Spike protein of the SARS-CoV-2 omicron variant interacts with actin

  Ai Fujimoto, Haruki Kawai, Rintaro Kawamura, Akira Kitamura

  bioRxiv (preprint) 2024/05/17 [Not refereed]
  
• Stress Granule Dysfunction via Chromophore-Associated Light Inactivation

  Takumi Koizumi, Ai Fujimoto, Haruka Kawaguchi, Tsumugi Kurosaki, Akira Kitamura

  ACS Omega 2024/05/14 [Refereed][Not invited]
  
• 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  2023/10/01 [Refereed]
   

  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 2023/07/22 [Refereed][Not invited]
   

  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.
• Light-Induced Condensates Show Accumulation-Prone and Less Dynamic Properties in the Nucleus Compared to the Cytoplasm

  Yuta Hamada, Akira Kitamura

  Spectroscopy Journal 2023/07/10 [Refereed][Not invited]
  
• 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 2023/03/21 [Refereed][Not invited]
  
• 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  2023/03/14 [Refereed][Not invited]
  
• Short repeat RNA reduces cytotoxicity by preventing the aggregation of TDP-43 and its 25 kDa carboxy-terminal fragment

  Ai Fujimoto, Masataka Kinjo, Akira Kitamura

  bioRxiv (preprint) 2022/07/04 [Not refereed][Not invited]
  
• 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

  bioRxiv (preprint) 2022/05/23
• Physico- and chemical biology using nanomanipulation and micromanipulation technologies

  Akira Kitamura, Ryo Iizuka

  Biophysics and Physicobiology 2022 [Refereed][Invited]
  
• 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 2021/11/12 [Refereed][Invited]
   

  Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is initiated by the interaction between a receptor protein, angiotensin-converting enzyme type 2 (ACE2) on the cell surface, and the viral spike (S) protein. This interaction is similar to the mechanism in SARS-CoV, a close relative of SARS-CoV-2, which was identified in 2003. Drugs and antibodies that inhibit the interaction between ACE2 and S proteins could be key therapeutic methods for preventing viral infection and replication in COVID-19. Here, we demonstrate the interaction between human ACE2 and a fragment of the S protein (S1 subunit) derived from SARS-CoV-2 and SARS-CoV using two-color fluorescence cross-correlation spectroscopy (FCCS), which can detect the interaction of fluorescently labeled proteins. The S1 subunit of SARS-CoV-2 interacted in solution with soluble ACE2, which lacks a transmembrane region, more strongly than that of SARS-CoV. Furthermore, one-to-one stoichiometry of the two proteins during the interaction was indicated. Thus, we propose that this FCCS-based interaction detection system can be used to analyze the interaction strengths of various mutants of the S1 subunit that have evolved during the worldwide pandemic, and also offers the opportunity to screen and evaluate the performance of drugs and antibodies that inhibit the interaction.
• 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 0006-291X 2021/06 [Refereed][Not invited]
  
• Pinhole Closure Improves Spatial Resolution in Confocal Scanning Microscopy

  Akira Kitamura

  Methods in Molecular Biology 385 - 389 1064-3745 2021/05 [Not refereed][Invited]
  
• Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

  Akira Kitamura, Ai Fujimoto, Masataka Kinjo

  Journal of Visualized Experiments (170) e62576  2021/04/25 [Refereed][Invited]
   

  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 2021/04/20 [Refereed]
   

  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 1010-6030 2021/03 [Refereed][Not invited]
   

  © 2020 Elsevier B.V. Herein, we evaluated the intersystem crossing quantum yield (ΦISC) of a silicon phthalocyanine derivatized from IRDye700DX (IR700) which is used as a photosensitizer for photoimmunotherapy (PIT), using fluorescence correlation spectroscopy (FCS). The calculated ΦISC was 0.019 ± 0.002. The FCS measurement was validated by experiment in the presence of potassium iodide, which can change the kinetics of the relaxation process in the excited state.
• 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 0743-7463 2021/01/12 [Refereed][Not invited]
  
• 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) 2331-8325 2020 [Refereed][Invited]
  
• 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 2019/03/04 [Refereed][Not invited]
   

  Mysterin, also known as RNF213, is an intracellular protein that forms large toroidal oligomers. Mysterin was originally identified in genetic studies of moyamoya disease (MMD), a rare cerebrovascular disorder of unknown etiology. While mysterin is known to exert ubiquitin ligase and putative mechanical ATPase activities with a RING finger domain and two adjacent AAA+ modules, its biological role is poorly understood. Here, we report that mysterin is targeted to lipid droplets (LDs), ubiquitous organelles specialized for neutral lipid storage, and markedly increases their abundance in cells. This effect was exerted primarily through specific elimination of adipose triglyceride lipase (ATGL) from LDs. The ubiquitin ligase and ATPase activities of mysterin were both important for its proper LD targeting. Notably, MMD-related mutations in the ubiquitin ligase domain of mysterin significantly impaired its fat-stabilizing activity. Our findings identify a unique new regulator of cytoplasmic LDs and suggest a potential link between the pathogenesis of MMD and fat metabolism.
• 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 0006-291X 2019/02 [Refereed][Not invited]
   

  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 0003-2700 2019/02 [Refereed][Not invited]
   

  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 2018/09/01 [Refereed][Not invited]
  
• 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 2018/08 [Refereed][Not invited]
   

  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 2018/07 [Refereed][Not invited]
   

  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.
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• 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  1422-0067 2018/04/01 [Refereed][Invited]
   

  Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N& B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases.
• 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 1090-2104 2018/02/26 [Refereed][Not invited]
   

  Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis.
• 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  1422-0067 2018/02/06 [Refereed][Not invited]
   

  The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football-and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.
• TDP-43 depletion: mechanism of neuronal cell death in ALS.

  Akira Kitamura

  Future Neurology 13 (3) 2018 [Refereed][Invited]
  
• 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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• U6 snRNA expression prevents toxicity in TDP-43-knockdown cells

  Masao Yahara, Akira Kitamura, Masataka Kinjo

  PLOS ONE 12 (11) e0187813  1932-6203 2017/11 [Refereed][Not invited]
   

  Depletion of amyotrophic lateral sclerosis (ALS)-associated transactivation response (TAR) RNA/DNA-binding protein 43 kDa (TDP-43) alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs) as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.
• 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 1356-9597 2017/06 [Refereed][Not invited]
   

  The mechanism and cause of motor neuronal cell death in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder, are unknown; gain of function of oligomers and aggregation of misfolded proteins, including carboxyl-terminal fragments (CTFs) of TAR RNA/DNA-binding protein 43 kDa (TDP-43), have been proposed as important causative factors in the onset of ALS. We recently reported that a nuclear localization signal (NLS)-tagged 25-kDa CTF of TDP-43 (TDP25) could decrease the cell-death proportion compared with that promoted by TDP25. Here, we show oligomeric states of NLS-TDP25 and its detailed localization property using super-resolution fluorescence microscopy, FRET, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy analysis. NLS-TDP25 efficiently formed a nucleolar cap structure via RNA binding in the presence of actinomycin D, but TDP25 did not. Although cytoplasmic inclusion bodies including TDP25 had a disordered and immobile structure, NLS-TDP25 in the nucleolus was ordered and dynamic. In the diffuse state, TDP25 formed fewer oligomers and interacted with the molecular chaperone, HSP70; however, NLS-TDP25 formed oligomers. These results suggested that NLS-tagged TDP25 can change its structure to use ordered oligomeric but nontoxic state. Moreover, the structure of ordered oligomers as well as nuclear sequestration may be important in mediating cytotoxicity in ALS pathology.
• 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  2045-2322 2016/03 [Refereed][Not invited]
   

  The intracellular accumulation of amyloid-beta (A beta) oligomers critically contributes to disease progression in Alzheimer's disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of A beta oligomers in vivo is key for drug discovery research, however, it has been challenging because A beta aggregation inhibits the fluorescence from fusion proteins. Here, we developed A beta(1-42)-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of A beta-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between A beta and GFP, we generated two fusion proteins with "a long-linker" and "a short-linker", and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with A beta-GFP plasmids and A beta-GFP transgenic C. elegans. We found that A beta-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel A beta-GFP fusion proteins could be utilized for studying the physiological functions of A beta oligomers in living cells and animals, and for drug screening by analyzing A beta toxicity.
• 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  2045-2322 2016/03 [Refereed][Not invited]
   

  Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Frster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.
• 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 2211-5463 2016/02 [Refereed][Not invited]
   

  Fibroin modulator-binding protein 1 (FMBP-1) is a silkworm transcription factor that has a unique DNA-binding domain called the one score and three amino acid peptide repeat (STPR). Here we used fluorescence correlation spectroscopy (FCS) to analyze the diffusion properties of an enhanced green fluorescent protein-tagged FMBP-1 protein (EGFP-FMBP-1) expressed in posterior silk gland (PSG) cells of Bombyx mori at the same developmental stage as natural FMBP-1 expression. EGFP-FMBP-1 clearly localized to cell nuclei. From the FCS analyses, we identified an immobile DNA-bound component and three discernible diffusion components. We also used FCS to observe the movements of wild-type and mutant EGFP-FMBP-1 proteins in HeLa cells, a simpler experimental system. Based on previous in vitro observation, we also introduced a single amino acid substitution in order to suppress stable FMBP-1-DNA binding; specifically, we replaced the ninth Arg in the third repeat within the STPR domain with Ala. This mutation completely disrupted the slowest diffusion component as well as the immobile component. The diffusion properties of other FMBP-1 mutants (e.g. mutants with N-terminal or C-terminal truncations) were also analyzed. Based on our observations, we suggest that the four identifiable movements might correspond to four distinct FMBP-1 states: (a) diffusion of free protein, (b) and (c) two types of transient interactions between FMBP-1 and chromosomal DNA, and (d) stable binding of FMBP-1 to chromosomal DNA.
• 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  2045-2322 2016/01 [Refereed][Not invited]
   

  A hallmark of amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, is formation of inclusion bodies (IBs) from misfolded proteins in neuronal cells. TAR RNA/DNA-binding protein 43 kDa (TDP43) is an ALS-causative protein forming IBs in ALS patients. The relation between localization of the IBs and neurotoxicity remains largely unknown. We characterized aggregation of fluorescently tagged TDP43 and its carboxyl-terminal fragments (CTFs) by analytical fluorescence imaging techniques. Quantitative time-lapse analysis in individual live cells showed that fluorescent-protein-tagged TDP43 was cleaved and a 35 kDa TDP43 CTF (TDP35) formed ubiquitin (Ub)-negative cytoplasmic IBs. Although TDP35 formed mildly toxic Ub-negative IBs in the cytoplasm, TDP25, another type of a TDP43 CTF, efficiently formed sufficiently toxic Ub-positive IBs. One-or two-color fluorescence correlation spectroscopy (FCS/FCCS) revealed that coaggregation of TDP25 with TDP43 was initiated by depletion of the RNA that binds to TDP25. Moreover, nuclear localization tagging TDP25 reduced the rate of neuronal cell death. These observations point to the need to elucidate the novel sequestration mechanism and details of the toxicity of the misfolded and aggregation-prone TDP43 CTFs (as well as the RNA binding and nuclear retention) in order to identify possible preventive interventions against ALS.
• 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 0006-291X 2015/07 [Refereed][Not invited]
   

  Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLSSV40) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLSSV40 in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLSSV40 formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLSSV40 likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLSSV40 can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. (C) 2015 Elsevier Inc. All rights reserved.
• 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 1422-0067 2015/03 [Refereed][Invited]
   

  Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Forster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.
• 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 2014/06 [Refereed][Invited]
  
• 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  2045-2322 2014/03 [Refereed][Not invited]
   

  Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.
• 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 1356-9597 2014/03 [Refereed][Not invited]
   

  A hallmark of protein conformational disease, exemplified by neurodegenerative disorders, is the expression of misfolded and aggregated proteins. The relationship between protein aggregation and cellular toxicity is complex, and various models of experimental pathophysiology have often yielded conflicting or controversial results. In this study, we examined the biophysical properties of amyotrophic lateral sclerosis (ALS)-linked mutations of Cu/Zn superoxide dismutase 1 (SOD1) expressed in human tissue culture cells. Fluorescence correlation spectroscopy (FCS) and Forster resonance energy transfer (FRET) analyses revealed that changes in proteasome activity affected both the expression of FCS- and FRET-detected oligomers and cellular toxicity. Under normal conditions, highly aggregation-prone mutant SOD1 exhibited very little toxicity. However, when the activity of the proteasome was transiently inhibited, only upon recovery did we observe the appearance of ordered soluble oligomers, which were closely correlated with cellular toxicity. These results shed light on the importance of balance in proteostasis and suggest that transient shifts of activity in the cellular machinery can alter the course of protein conformational transitions and dysregulate modulation of proteasome activity. In neurodegenerative disorders including ALS, such changes may be a risk factor for pathogenesis.
• 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 0006-8993 2014/01 [Refereed][Not invited]
   

  Protein aggregation is observed in various neurodegeneration diseases, including Parkinson's disease (PD). Alpha-synuclein, a causative gene product of familial PD, is a major component of large aggregates (inclusion bodies) in PD. Prefoldin, a molecular chaperone comprised of six subunits, PFD1 similar to 6, prevents misfolding of newly synthesized nascent polypeptides and also prevents aggregation of protein such as a' pathogenic form of Huntingtin, a causative gene product of Huntington disease. In this study, we first found that aggregation of TagRFP-tagged wild-type alpha-synuclein and its pathogenic mutants, but not that of GFP-tagged alpha-synuclein, occurred in transfected Neuro-2a cells. The fluorescence of GFP is weakened under the condition of pH 4.5-5.0, and TagRFP is a stable red fluorescence protein under an acidic condition. Aggregated TagRFP-wild-type alpha-synuclein and its pathogenic mutants in Neuro-2a cells were ubiquitinated and were colocalized with the prefoldin complex in the lysosome under this condition. Furthermore, knockdown of PFD2 and PFD5 disrupted prefoldin formation in alpha-synuclein-expressing cells, resulting in accumulation of aggregates of wild-type and pathogenic alpha-synuclein and in induction of cell death. The levels of aggregation and cell death in pathogenic alpha-synuclein-transfected cells tended to be higher than those in wild-type alpha-synuclein-transfected cells. These results suggest that prefoldin works as a protective factor in aggregated alpha-synucleininduced cell death. (C) 2013 Elsevier B.V. All rights reserved.
• 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 0006-291X 2013/12 [Refereed][Not invited]
   

  Prions are self-propagating amyloids. Yeast prion [PSI+] is a protein-based heritable element, in which amyloid aggregates of the Sup35 protein are transmitted to daughter cells. Hsp104, an ATP-dependent disaggregase, and other chaperones are essential to maintain [PSI+] Although previous reports have demonstrated the physical interactions of Hsp104 and Sup35 amyloids, the mechanism how Hsp104 interacts with Sup35 amyloids remains to be elucidated. Here we investigated the interaction between Hsp104 and Sup35 in the lysates of [PSI+] cells using fluorescence cross-correlation spectroscopy (FCCS), which can analyze the codiffusion events of different fluorophores. FCCS analysis showed a strong interaction between Hsp104 and Sup35 in [PSI+] lysates, but not in [psi(-)] lysates, suggesting that Hsp104 recognizes the amyloid aggregates of Sup35. Although the interaction was retained in ATP-depleted [PSI+] lysates, addition of ATP or guanidine hydrochloride, which is an inhibitor of Hspl 04, to [PSI+] lysates weakened the interaction. (C) 2013 Elsevier Inc. All rights reserved.
• 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 0021-9258 2013/07 [Refereed][Not invited]
   

  Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than similar to 40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.
• 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 1356-9597 2013/02 [Refereed][Not invited]
   

  Nonmuscle myosin II forms a folded conformation (10S form) in the inactivated state; however, the physiological importance of the 10S form is still unclear. To investigate the role of 10S form, we generated a chimeric mutant of nonmuscle myosin IIB (IIB-SK1 center dot 2), in which S1462-R1490 and L1551-E1577 were replaced with the corresponding portions of skeletal muscle myosin heavy chain. The IIB-SK1 center dot 2 mutant did not fold into a 10S form under physiological condition in vitro. IIB-SK1 center dot 2 was less dynamic by stabilizing the filamentous form and accumulated in the posterior region of migrating cells. IIB-SK1 center dot 2 functioned properly in cytokinesis but altered migratory properties; the rate and directional persistence were increased by IIB-SK1 center dot 2 expression. Surprisingly, endogenous nonmuscle myosin IIA was excluded from the posterior region of migrating cells expressing IIB-SK1 center dot 2, which may underlie the change of the cellular migratory properties. These results suggest that the 10S form is necessary for maintaining nonmuscle myosin II in an unassembled state and for recruitment of nonmuscle myosin II to a specific region of the cell.
• Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor, IRE1 beta

  Daisuke Oikawa, Akira Kitamura, Masataka Kinjo, Takao Iwawaki

  PLOS ONE 12 7 (12) e51290  1932-6203 2012/12 [Refereed][Not invited]
   

  IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1 alpha and IRE1 beta, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1 beta senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1 beta clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1 beta complex is robustly increased upon ER stress. Moreover, unlike IRE1 alpha, the luminal domain of IRE1 beta showed antiaggregation activity in vitro, and IRE1 beta was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1 beta, while IRE1 alpha was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1 alpha, the luminal event mediated by IRE1 beta involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.
• 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 0021-9525 2012/07 [Refereed][Not invited]
   

  During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.
• Analyzing the aggregation of polyglutamine-expansion proteins and its modulation by molecular chaperones

  Hiroshi Kubota, Akira Kitamura, Kazuhiro Nagata

  METHODS 53 (3) 267 - 274 1046-2023 2011/03 [Refereed][Not invited]
   

  Polyglutamine (polyQ)-expansion proteins cause protein aggregation in the cytosol and nucleus of neuronal cells, leading to neurodegenerative diseases. For example, expansion of the polyQ tract (>40 repeats) in huntingtin (htt) proteins leads to Huntington disease, while polyQ-expanded ataxins cause several types of ataxias. PolyQ-rich inclusions are found in neuronal cells of patients, suggesting that polyQ disease is caused by protein misfolding. However, the mechanisms by which polyQ-expansion proteins exert neuronal toxicity are largely unknown. Here, we review experimental procedures to analyze the roles of molecular chaperones in preventing polyQ aggregation and toxicity as well as to measure the characteristics and dynamics of polyQ aggregation, particularly focusing on cellular models and dynamic imaging of fluorescently-labeled polyQ-expansion proteins and their modulation by chaperones. (C) 2010 Elsevier Inc. All rights reserved.
• Amyloid oligomers: dynamics and toxicity in the cytosol and nucleus

  Akira Kitamura, Hiroshi Kubota

  FEBS JOURNAL 277 (6) 1369 - 1379 1742-464X 2010/03 [Refereed][Not invited]
   

  The accumulation of misfolded proteins in the cytosol and nucleus of neuronal cells leads to neurodegenerative disorders. Polyglutamine diseases are caused by polyglutamine-expanded proteins, whereas mutations in superoxide dismutase 1 lead to amyotrophic lateral sclerosis. These structurally unstable mutant species perturb essential interactions between normal proteins and tend to aggregate because of the presence of exposed hydrophobic surfaces. Accumulating evidence suggests that soluble species, including misfolded monomers and oligomers, are more toxic than large insoluble aggregates or inclusions. Spectroscopic analysis, including fluorescence recovery after photobleaching and fluorescence loss in photobleaching, in living cells revealed that protein aggregates of misfolded proteins are dynamic structures that interact with other proteins, such as molecular chaperones. Fluorescence correlation spectroscopy analysis detected soluble oligomers/aggregates of misfolded proteins in cell extracts. Fluorescence resonance energy transfer analysis supported the notion that soluble oligomers/aggregates are formed before the formation of inclusions in vivo. Here, we reviewed the characteristics of oligomers and aggregates of misfolded proteins, with a particular focus on those revealed by spectroscopic analysis, and discussed how these oligomers may be toxic to cells.
• 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  0168-0102 2010 [Refereed][Not invited]
  
• 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 1059-1524 2009/06 [Refereed][Not invited]
   

  Type I collagen is a major component of the extracellular matrix, and mutations in the collagen gene cause several matrix-associated diseases. These mutant procollagens are misfolded and often aggregated in the endoplasmic reticulum (ER). Although the misfolded procollagens are potentially toxic to the cell, little is known about how they are eliminated from the ER. Here, we show that procollagen that can initially trimerize but then aggregates in the ER are eliminated by an autophagy-lysosome pathway, but not by the ER-associated degradation (ERAD) pathway. Inhibition of autophagy by specific inhibitors or RNAi-mediated knockdown of an autophagy-related gene significantly stimulated accumulation of aggregated procollagen trimers in the ER, and activation of autophagy with rapamycin resulted in reduced amount of aggregates. In contrast, a mutant procollagen which has a compromised ability to form trimers was degraded by ERAD. Moreover, we found that autophagy plays an essential role in protecting cells against the toxicity of the ERAD-inefficient procollagen aggregates. The autophagic elimination of aggregated procollagen occurs independently of the ERAD system. These results indicate that autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that are not able to be removed by ERAD.
• Effects of MM-1 and molecular chaperones an formation of polyglutamine aggregation

  Tashiro Erika, Muto Hideki, Zako Tamotsu, Miyazawa Makoto, Kitaura Hirotake, Kitamura Akira, Kubota Hiroshi, Maeda Mizuo, Iguchi-Ariga Sanae M. M, Kinjo Masataka, Ariga Hiroyoshi

  NEUROSCIENCE RESEARCH 65 S248  0168-0102 2009 [Refereed][Not invited]
  
• 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 1059-1524 2008/03 [Refereed][Not invited]
   

  McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP) 70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.
• MKKSは中心体輸送タンパク質であり、その病原性変異体はE3酵素CHIPの品質管理を介して分解される(MKKS is a centrosome-shuttling protein and its disease-causing mutants are degraded via quality control E3 enzyme CHIP)

  平山 尚志郎, 山崎 祐自, 北村 朗, 小田 裕香子, 木村 宏, 大川 克也, 森戸 大介, 久保田 広志, 永田 和宏

  日本発生生物学会・日本細胞生物学会合同大会要旨集 40回・59回 78 - 78 2007/05
• 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 1465-7392 2006/10 [Not refereed][Not invited]
   

  Polyglutamine (polyQ)-expansion proteins cause neurodegenerative disorders including Huntington's disease, Kennedy's disease and various ataxias. The cytotoxicity of these proteins is associated with the formation of aggregates or other conformationally toxic species. Here, we show that the cytosolic chaperonin CCT (also known as TRiC) can alter the course of aggregation and cytotoxicity of huntingtin (Htt) - polyQ proteins in mammalian cells. Disruption of the CCT complex by RNA-imediated knockdown enhanced Htt - polyQ aggregate formation and cellular toxicity. Analysis of the aggregation states of the Htt - polyQ proteins by fluorescence correlation spectroscopy revealed that CCT depletion results in the appearance of soluble Htt - polyQ aggregates. Similarly, overexpression of all eight subunits of CCT suppressed Htt aggregation and neuronal cell death. These results indicate that CCT has an essential role in protecting against the cytotoxicity of polyQ proteins by affecting the course of aggregation.
• 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 1059-1524 2006/05 [Refereed][Not invited]
   

  Heat-shock protein of 47 kDa (Hsp47) is a molecular chaperone that recognizes collagen triple helices in the endoplasmic reticulum (ER). Hsp47-knockout mouse embryos are deficient in the maturation of collagen types I and IV, and collagen triple helices formed in the absence of Hsp47 show increased susceptibility to protease digestion. We show here that the fibrils of type I collagen produced by Hsp47(-/-) cells are abnormally thin and frequently branched. Type I collagen was highly accumulated in the ER of Hsp47(-/-) cells, and its secretion rate was much slower than that of Hsp47(+/+) cells, leading to accumulation of the insoluble aggregate of type I collagen within the cells. Transient expression of Hsp47 in the Hsp47(-/-) cells restored normal extracellular fibril formation and intracellular localization of type I collagen. Intriguingly, type I collagen with unprocessed N-terminal propeptide (N-propeptide) was secreted from Hsp47(-/-) cells and accumulated in the extracellular matrix. These results indicate that Hsp47 is required for correct folding and prevention of aggregation of type I collagen in the ER and that this function is indispensable for efficient secretion, processing, and fibril formation of collagen.

Books etc

• 新・生細胞蛍光イメージング

  北村 朗 (Joint work第21章，実習8-2)
  共立出版 2015/11

Conference Activities & Talks

• Molecular cell biology through induction, kinetic analysis, and inactivation of biomolecules using ultimate light technology  [Not invited]

  Akira Kitamura

  MBSJ2023  2023/12
• Detective tools of RNA structural transition using fluorescence blinking  [Invited]

  Akira Kitamura

  45th MBSJ annual meeting  2022/11
• Diffusion coefficient measurement using fluorescence recovery after photobleaching ―a homework during summer vacation with refreshment―  [Invited]

  Akira Kitamura

  60th BSJ annual meeting  2022/09
• Basics and applications of chromophore-assisted light inactivation (CALI)  [Invited]

  Akira Kitamura

  60th BSJ annual meeting  2022/09
• Intracellular structure and function of chaperone RNA  [Invited]

  Akira Kitamura

  International Workshop on Fluorescence Methods and Applications 2022  2022/09
• The N-terminal rule in the aggregate formation of C-terminal fragments of TDP-43  [Not invited]

  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  2021/07
• The domain role for condensation of carboxyl-terminal fragments of TDP-43  [Not invited]

  Akira Kitamura

  FASEB SRC 2021 "The Protein Aggregation Conference: Function, Dysfunction, and Disease"
• Cold Spring Harbor Laboratory 2020 Meeting "Protein homeostasis in Health & Disease"  [Not invited]

  Akira Kitamura

  Cold Spring Harbor Laboratory 2020 Meeting "Protein homeostasis in Health & Disease"  2020/11
• 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  [Invited]

  北村朗

  サントリー生有研シンポジウム  2019/12
• Aggregation process of carboxyl terminal fragments of TDP-43  [Invited]

  Akira Kitamura

  QBP seminar, KTH Royal Institute of Technology  2019/11
• Aggregation process of carboxyl terminal fragments of TDP-43  [Not invited]

  Akira Kitamura

  Asian Pacific Prion Symposium 2019  2019/10
• RNA prevents ALS-linked protein aggregation  [Invited]

  Akira Kitamura

  QBP-seminar, KTH Royal Institute of Technology  2018/11
• 蛍光ゆらぎを利用した生体分子構造変化の解析  [Invited]

  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  2018/07
• Basics and application of fluorescence correlation spectroscopy  [Invited]

  Akira Kitamura

  5th AIST International Imaging Workshop  2018/01
• FCS and FCCS in living cells  [Invited]

  Akira Kitamura

  Course Functional Fluorescence Microscopy Imaging (fFMI) in Biomedical Research  2017/12  Stockholm, Sweden
• Basics and application of fluorescence correlation spectroscopy  [Invited]

  Akira Kitamura

  4th AIST International Imaging Workshop  2017/01  AIST, Tsukuba
• Frontiers of neurodegenerative disorder research using FRAP and FCS  [Invited]

  Akira Kitamura

  36th MBSJ annual meeting  2016/12
• FCS and FCCS in living cells  [Invited]

  Akira Kitamura

  Course Functional Fluorescence Microscopy Imaging (fFMI) in Biomedical Research  2016/11  Karolinska Institutet, Stockholm, Sweden
• Elucidation for aggregation mechanism of ALS-linked TDP43 using fluorescence imaging techniques  [Invited]

  Akira Kitamura

  7th ALS forum  2016/07  Tokyo, Japan
• Basics and application of fluorescence correlation spectroscopy  [Invited]

  Akira Kitamura

  3rd AIST International Imaging Workshop  2016/01
• 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  [Not invited]

  北村 朗

  第55回日本植物生理学会年会  2014/03
• 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
  
• 神経変性疾患に おける細胞内タンパク質恒常性  [Invited]

  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  2013/11
• 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－  [Not invited]

  北村 朗

  第五回「光塾」  2013/09
• 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？―  [Not invited]

  佐々木章, 北村 朗

  第四回「光塾」  2012/08
• 生命科学画像解析における数学の基礎  [Not invited]

  北村 朗

  第三回「光塾」  2011/10
• 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－  [Not invited]

  北村 朗

  第二回「光塾」  2010/12
• Molecular chaperone prefoldin inhibits polyglutamine aggregation and cytotoxicity  [Not invited]

  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
  
• 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－  [Not invited]

  北村 朗

  第一回「光塾」  2009/08
• 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
  
• 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
  

MISC

• 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  2023/08  [Not refereed][Invited]
  
• 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  2023/03  [Not refereed][Invited]
  
• 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  2022/07  [Not refereed][Invited]
  
• Realizing RNA roles during protein aggregation associated with neurodegenerative diseases

  Akira Kitamura  The CELL  53-  (9)  578  -581  2021/08  [Not refereed][Invited]
  
• Protein aggregations and their gene therapy in neurodegenerative disorders

  Akira Kitamura, Ai Fujimoto  Bio Clinica  36-  (5)  453  -456  2021/07/12  [Not refereed][Invited]
  
• Session 2SHP report—decoding intracellular architecture using visualizing device development and mathematical modeling

  Akira Kitamura, Kazuya Kabayama  Biophysical Reviews  12-  (2)  281  -282  2020/04  [Not refereed][Not invited]
  
• 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  [Not refereed][Invited]
  
• New Trends in Fluorescence Correlation Spectroscopy, Development and Application

  YAMAMOTO Johtaro, KITAMURA Akira, KINJO Masataka  Seibutsu Butsuri  59-  (3)  125  -131  2019  [Not refereed][Not invited]
   

  <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>
• 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  2012/03/05  [Not refereed][Not invited]
  
• 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  2010/09  [Not refereed][Not invited]
  
• 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  2010/05/15  [Not refereed][Not invited]
  
• 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  2010/03  [Not refereed][Not invited]
   

  Proteins synthesized in living cells often fail to properly fold, and misfolded proteins are potentially toxic to cells due to the existence of exposed hydrophobic surfaces. For example, misfolded proteins tend to aggregate each other and capture other normal proteins into aggregates. Genetic mutations often cause protein misfolding, and these mutations induce neuronal cell death in neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, polyglutamine disease and amyotrophic lateral sclerosis). However, how misfolded proteins exert cytotoxicity is poorly understood. In living cells, there are two strategies to prevent the toxicity of misfolded proteins: one is prevention of aggregation by molecular chaperones and the other is degradation by proteases. These two systems coordinately monitor quality of proteins and decide the fate of proteins (e.g., reuse after refolding or destruction by protease digestion). We have used aggregationprone proteins tagged with green fluorescent proteins and derivatives to analyze the dynamics of cytotoxic misfolded proteins in living cells. We discuss how fluorescently labeled proteins are useful to understand the dynamics of misfolded proteins, particularly for those causing neurodegenerative diseases. We also discuss kinetics of interaction between misfolded proteins and binding proteins like molecular chaperones.
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Refereed][Not invited]
  
• 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  2005/06  [Not refereed][Not invited]
  
• 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  2005/06  [Not refereed][Not invited]
  
• 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  2005/06  [Refereed][Not invited]
  
• 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  2004/05  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  
• 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  [Not refereed][Not invited]
  

Industrial Property Rights

• 特願PCT/JP2022/041001: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  2022年/11/02

  北村朗, 藤本愛  国立大学法人 北海道大学
• 特開2017-214339:ヘモシアニン会合体を用いた包摂体の製造方法  2017/12/07

  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

Awards & Honors

• 2022/09 The biophysical Society of Japan 2022 Biophysics and Physicobiology (BPPB) Awards
   

  受賞者: Akira Kitamura

Research Grants & Projects

• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2029/03 

  Author : 北村 朗
• 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

  AMED：Advanced Research & Development Programs for Medical Innovation (AMED-PRIME) "Understanding proteostasis and discovering innovative medical applications"

  Date (from‐to) : 2022/10 -2026/03 

  Author : Akira Kitamura
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/04 -2025/03 

  Author : 金城 政孝, 北村 朗
• 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

  Hoansha：研究助成

  Date (from‐to) : 2021/04 -2025/03
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/06 -2024/03 

  Author : 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。
• 非公開

  AMED：Translational Research Network Program (Seeds A)

  Date (from‐to) : 2023/04 -2024/03
• 非公開

  AMED：Translational Research Network Program (Seeds H)

  Date (from‐to) : 2022/09 -2024/03 

  Author : Akira Kitamura
• 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

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2022/04 -2024/03 

  Author : 北村 朗
• 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

  HAGIWARA Foundation：2021 2nd Grant Program

  Date (from‐to) : 2022/04 -2023/03 

  Author : Akira Kitamura
• 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

  NAKATANI Foundation：Grant Program

  Date (from‐to) : 2022/04 -2023/03 

  Author : Akira Kitamura
• 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

  NAKATANI Foundation：新型コロナウイルス感染症対策 助成プログラム

  Date (from‐to) : 2020/07 -2021/06
• 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

  JSPS：Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2018/04 -2021/03 

  Author : Akira Kitamura
• 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

  Canon fundation：Research fund

  Date (from‐to) : 2018/04 -2020/03 

  Author : Akira Kitamura
• Development of polarization-dependent fluorescence correlation spectroscopy for initial process analysis of protein aggregation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2019/03 

  Author : 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.
• Elucidation for the regulation of motility and lifespan by proteostasis(Fostering Joint International Research)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017 -2019 

  Author : 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.
• 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

  一般社団法人 日本ALS協会：ALS基金

  Date (from‐to) : 2016/06 -2017/03 

  Author : 北村 朗
• 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

  三井住友信託銀行公益信託：「生命の彩」ALS研究助成基金

  Date (from‐to) : 2016/05 -2017/03 

  Author : 北村 朗
• Establishment of unified theory of cellular inclusion bodies including misfolded protein

  JSPS：KAKENHI

  Date (from‐to) : 2014/04 -2017/03 

  Author : 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.
• 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

  JST：Development of systems and technology for advanced measurement and analysis

  Date (from‐to) : 2013/10 -2017/03 

  Author : Yasushi Hiraoka
• Analysis of neuronal cell death by ALS-associated misfolded protein aggregation with modulation of cellular RNA homeostasis

  JSPS：KAKENHI

  Date (from‐to) : 2011/04 -2015/03 

  Author : 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.
• 多点時空間相関解析法による細胞内分子複合体研究

  MEXT：KAKENHI

  Date (from‐to) : 2009/05 -2014/03 

  Author : 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。
• Regulation of cellular functions by highly evolved two chaperonins

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011 -2013 

  Author : 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.
• Study of membrane binding protein complex using total internal reflection fluorescence correlation spectroscopy

  MEXT：KAKENHI

  Date (from‐to) : 2007 -2011 

  Author : 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.
• 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

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2008 -2010 

  Author : 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.
• 細胞質シャペロニンの機能解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2006 -2007 

  Author : 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.

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• 2024/02 - Today   Frontiers in Biophysics   Review editor
• 2024/01 - Today   Biophysics and Physicobiology (BPPB)   Editorial Board Members
• 2023 - Today   Spectroscopy Journal   Editorial Board Members