Faculty Profiles - NARITA, Akihiro

Papers - NARITA, Akihiro

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Cryo-EM structure of the Agrobacterium tumefaciens T4SS-associated T-pilus reveals stoichiometric protein-phospholipid assembly Reviewed International coauthorship

Stefan Kreida, Akihiro Narita, Matthew D Johnson, Elitza I Tocheva, Anath Das, Debnath Ghosal, Grant J Jensen

Structure 2023.2

Structures and mechanisms of actin ATP hydrolysis Reviewed

Yusuke Kanematsu, Akihiro Narita, Toshiro Oda, Ryotaro Koike, Motonori Ota, Yu Takano, Kei Moritsugu, Ikuko Fujiwara, Kotaro Tanaka, Hideyuki Komatsu, Takayuki Nagae, Nobuhisa Watanabe, Mitsusada Iwasa, Yuichiro Maéda, Shuichi Takeda

Proceedings of the National Academy of Sciences Vol. 119 ( 43 ) page： e2122641119 2022.10

ADF/cofilin regulation from a structural viewpoint Invited Reviewed

Narita Akihiro

JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY Vol. 41 ( 1 ) page： 141 - 151 2020.3

Helical rotation of the diaphanous-related formin mDia1 generates actin filaments resistant to cofilin Reviewed

Mizuno Hiroaki, Tanaka Kotaro, Yamashiro Sawako, Narita Akihiro, Watanabe Naoki

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vol. 115 ( 22 ) page： E5000-E5007 2018.5

Structural basis for cofilin binding and actin filament disassembly Reviewed

Tanaka Kotaro, Takeda Shuichi, Mitsuoka Kaoru, Oda Toshiro, Kimura-Sakiyama Chieko, Maeda Yuichiro, Narita Akihiro

NATURE COMMUNICATIONS Vol. 9 2018.5

Direct observation of the actin filament by tip-scan atomic force microscopy. Reviewed

Microscopy (Oxford, England) Vol. 65 ( 4 ) page： 370-7 2016.8

Direct determination of actin polarity in the cell Reviewed International coauthorship

Narita, A. Mueller, J. Urban, E. Vinzenz, M. Small, J. V. Maeda, Y.

Journal of molecular biology Vol. 419 ( 5 ) page： 359-68 2012.6

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton.

Actin branching in the initiation and maintenance of lamellipodia Reviewed International coauthorship

Vinzenz, M. Nemethova, M. Schur, F. Mueller, J. Narita, A. Urban, E. Winkler, C. Schmeiser, C. Koestler, S. A. Rottner, K. Resch, G. P. Maeda, Y. Small, J. V.

Journal of cell science Vol. 125 ( 11 ) page： 2775-85 2012.6

Language：English Publishing type：Research paper (scientific journal)

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 mum. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.9 nm resolution, which was comparable with that derived for the in vitro actin-Arp2/3 complex. Lamellipodium initiation was monitored in an intracellular wound-healing model and was found to involve branching from the sides of actin filaments oriented parallel to the plasmalemma. Many filament plus ends, presumably capped, terminated behind the lamellipodium tip and localized on the dorsal and ventral surfaces of the actin network. These findings reveal how branching events initiate and maintain a network of actin filaments of variable length, and provide the first structural model of the branch junction in vivo. A possible role of filament capping in generating the lamellipodium leaflet is discussed and a mathematical model of protrusion is also presented.

Minimum requirements for the actin-like treadmilling motor system Invited Reviewed

Akihiro Narita

Bioarchitecture Vol. 1 ( 5 ) page： 205-208 2011.9

Authorship：Lead author,　Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Actin is one of the most abundant proteins in eukaryote cells, which forms a double stranded filament. The actin filament is not only a main component of the cytoskeleton, but also acts as a motor protein which moves toward one specific end, the barbed end, driven by polymerization at the barbed end and depolymerization at the other end, the pointed end, without any associated proteins. This motor activity is referred to as "treadmilling" and it represents the simplest motor system known, consisting of only one 42 kDa protein, actin. Here we report the minimum requirements of the actin-like motor system elucidated by computer simulations: (1) Nucleotide binding and ATPase activity in the filament; (2) Polarity in the rates of polymerization and depolymerization between the two ends; and (3) The dependence of the subunit-subunit interactions on the bound nucleotide. These requirements are simple and this knowledge should facilitate the development of artificial molecular motor systems in the future.

Structural basis for the slow dynamics of the actin filament pointed end. Reviewed

Narita, A., Oda, T. & Maeda, Y.

The EMBO Journal Vol. 30 page： 1230-7 2011.3

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

The actin filament has clear polarity where one end, the pointed end, has a much slower polymerization and depolymerization rate than the other end, the barbed end. This intrinsic difference of the ends significantly affects all actin dynamics in the cell, which has central roles in a wide spectrum of cellular functions. The detailed mechanism underlying this difference has remained elusive, because high-resolution structures of the filament ends have not been available. Here, we present the structure of the actin filament pointed end obtained using a single particle analysis of cryo-electron micrographs. We determined that the terminal pointed end subunit is tilted towards the penultimate subunit, allowing specific and extra loop-to-loop inter-strand contacts between the two end subunits, which is not possible in other parts of the filament. These specific contacts prevent the end subunit from dissociating. For elongation, the loop-to-loop contacts also inhibit the incorporation of another actin monomer at the pointed end. These observations are likely to account for the less dynamic pointed end.

DOI： doi:10.1038/emboj.2011.48

Human Spire Interacts with the Barbed End of the Actin Filament Reviewed

Ito, T., Narita, A., Hirayama, T., Taki, M., Iyoshi, S., Yamamoto, Y., Maeda, Y. & Oda, T.

Journal of Molecular Biology Vol. 408 page： 26-39 2011.3

Language：English Publishing type：Research paper (scientific journal)

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell.

DOI： doi:10.1016/j.jmb.2010.12.045

Electron Microscopic Visualization of the Filament Binding Mode of Actin-Binding Proteins Reviewed

Ito, T, Hirayama, T., Taki, M., Iyoshi, S., Dai, S., Takeda, S., Sakiyama, C. K., Oda, T., Yamamoto, Y., Maeda, Y. & Narita, A.

Journal of Molecular Biology Vol. 408 page： 18-25 2011

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.

DOI： 10.1016/j.jmb.2011.01.054

The nature of the globular- to fibrous-actin transition. Reviewed

Oda T, Iwasa M, Aihara T, Maéda Y, Narita A.

Nature Vol. 457 page： 441-5 2009.1

Language：English Publishing type：Research paper (scientific journal)

Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

Molecular structure of the ParM polymer and the mechanism leading to its nucleotide-driven dynamic instability. Reviewed

Popp D, Narita A, Oda T, Fujisawa T, Matsuo H, Nitanai Y, Iwasa M, Maeda K, Onishi H, Maéda Y.

EMBO J. Vol. 27 ( 3 ) page： 570-9 2008.2

Language：English Publishing type：Research paper (scientific journal)

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the right-handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra- and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.

Three-dimensional structure of cytoplasmic dynein bound to microtubules. Reviewed

Mizuno N, Narita A, Kon T, Sutoh K, Kikkawa M.

Proc Natl Acad Sci U S A. Vol. 104 ( 52 ) page： 20832-7 2007.12

Language：English Publishing type：Research paper (scientific journal)

Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located approximately 280 A away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke.

Molecular determination by electron microscopy of the dynein-microtubule complex structure. Reviewed

Narita A, Mizuno N, Kikkawa M, Maéda Y.

J. Mol. Biol. Vol. 372 ( 5 ) page： 1320-36 2007.10

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Dynein is a minus-end-directed microtubule (MT) motor that is responsible for the wide range of MT-based motility in eukaryotic cells. Detailed mechanism of the dynein chemomechanical conversion is still unknown, partly because the structure of dynein is not studied at high resolution. To address this problem and reconstruct the dynein-MT complex at higher resolution, we have developed new procedures based on single particle analysis. To accurately determine the orientation of the dynein-MT complex, we introduced a "dynein track model" to restrict the possible dynein positions on the images. We tested our procedures by reconstructing structures from simulated dynein-MT complex images. Starting from the simulated noisy images generated using three different models of the dynein-MT complex, we have successfully recovered the original three-dimensional (3-D) structure. We also showed that our procedure is robust against fluctuation of the dynein molecules and can determine the structure even when the dynein position fluctuates to a certain extent. Convergence of the final 3-D structure can be tested with a "two-dimensional (2-D) agreement value," which we introduced to see whether the final structure is a result of overfit from fluctuating dynein or not. When the procedures did not work well due to the fluctuation, we could recognize the failure by this 2-D agreement value. Finally, the actual structure of the dynein-MT complex was determined from actual cryoelectron micrographs of Dictyostelium cytoplasmic dynein-MT complex. This method has revealed the detailed 3-D structures of the dynein-MT complex and will shed light on the motor mechanism of the dynein molecule.

Molecular determination by electron microscopy of the actin filament end structure. Reviewed

Narita A, Maéda Y.

J. Mol. Biol. Vol. 365 ( 2 ) page： 480-501 2007.1

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

In eukaryotic cells, actin filaments play various crucial roles by altering their spatial and temporal distributions in the cell. The distribution of actin filaments is regulated by the binding of end-binding proteins, including capping protein (CapZ in muscle), the Arp2/3 complex, gelsolin, formin and tropomodulin, to the end of the actin filament. In order to determine the nature of these regulations, structural elucidations of actin filament-end-binding protein complexes are crucially important. Here, we have developed new procedures on the basis of single-particle analysis to determine the structure of the end of actin filaments from electron micrographs. In these procedures, the polarity of the actin filament image, as well as the azimuth orientation and the axial position of each actin protomer within a short stretch near the filament end, were determined accurately. This improved both the stability and accuracy of the structural determination dramatically. We tested our procedures by reconstructing structures from simulated filament images, which were obtained from 24 model structures for the actin-CapZ complex. These model structures were generated by random docking of the atomic structure of CapZ to the barbed end of an atomic model of the actin filament. Of the 24 model structures, 23 were recovered correctly by the present procedures. We found that our analysis was robust against local aberrations of the helical twist near the end of the actin filament. Finally, the procedures were applied successfully to determine the structure of the actin-CapZ complex from real cryo-electron micrographs of the complex. This is the first method for elucidating the detailed 3D structures at the end of the actin filament.

Structural basis of actin filament capping at the barbed-end: a cryo-electron microscopy study. Reviewed

Narita A, Takeda S, Yamashita A, Maéda Y.

EMBO J. Vol. 25 ( 23 ) page： 5626-33 2006.11

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

The intracellular distribution and migration of many protein complexes and organelles is regulated by the dynamics of the actin filament. Many actin filament end-binding proteins play crucial roles in actin dynamics, since polymerization and depolymerization of actin protomers occur only at the filament ends. We present here an EM structure of the complex of the actin filament and hetero-dimeric capping protein (CP) bound to the barbed-end at 23 A resolution, by applying a newly developed methods of image analysis to cryo-electron micrographs. This structure was fitted by the crystal structure of CP and the proposed actin filament structure, allowing us to construct a model that depicts two major binding regions between CP and the barbed-end. This binding scheme accounted for the results of newly performed and previously published mutation experiments, and led us to propose a two-step binding model. This is the first determination of an actin filament end structure.

Ca(2+)-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy. Reviewed

Narita A, Yasunaga T, Ishikawa T, Mayanagi K, Wakabayashi T.

J. Mol. Biol. Vol. 308 ( 2 ) page： 241-261 2001.4

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.

Microscopic and structural observations of actin filament capping and severing by Cytochalasin D

Takahiro Mitani, Shuichi Takeda, Toshiro Oda, Akihiro Narita, Yuichiro Maeda, Hajime Honda, Ikuko Fujiwara

bioRxiv 2025.1

Dimerization GAS2 mediates microtubule and F-actin crosslinking Reviewed International coauthorship Open Access

Jiancheng An, Tsuyoshi Imasaki, Akihiro Narita, Shinsuke Niwa, Ryohei Sasaki, Tsukasa Makino, Ryo Nitta, and Masahide Kikkawa

EMBO J 2025

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

Kazuki Kasai, Kayo Imamura, Masatoshi Uno, Naotaka Sekiyama, Tomoko Miyata, Fumiaki Makino, Ryusei Yamada, Yoshiki Takahashi, Noriyuki Kodera, Keiichi Namba, Hidenori Ohnishi, Akihiro Narita, Hiroki Konno, Hidehito Tochio

bioRxiv 2024.9

Bacterial genome encoded ParMs International coauthorship

Samson Ali, Adrian Koh, David Popp, Kotaro Tanaka, Yoshihito Kitaoku, Naoyuki Miyazaki, Kenji Iwasaki, Kaoru Mitsuoka, Robert C. Robinson, Akihiro Narita

bioRxiv 2024.9

Influenza A virus exploits the motility of membrane cytoskeletal actomyosin filaments for its genome packaging in the host cell International coauthorship

I-Hsuan Wang, Jiro Usukura, Yasuyuki Miyake, Eiji Usukura, Akihiro Narita, Yohei Yamauchi, Yoshihiro Kawaoka

bioRxiv 2024.9

Mechanical Stress Decreases the Amplitude of Twisting and Bending Fluctuations of Actin Filaments

Okura, K; Matsumoto, T; Narita, A; Tatsumi, H

JOURNAL OF MOLECULAR BIOLOGY Vol. 435 ( 22 ) page： 168295 2023.11

Mutagenic analysis of actin reveals the mechanism of His161 flipping that triggers ATP hydrolysis

Iwasa, M; Takeda, S; Narita, A; Maéda, Y; Oda, T

FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Vol. 11 page： 1105460 2023.3

ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming Reviewed

Daichi Takahashi, Ikuko Fujiwara, Yuya Sasajima, Akihiro Narita, Katsumi Imada and Makoto Miyata

Open Biology 2022.10

ATP-dependent polymerization dynamics of bacterial actin proteins involved in <i>Spiroplasma</i> swimming

Takahashi, D; Fujiwara, I; Sasajima, Y; Narita, A; Imada, K; Miyata, M

OPEN BIOLOGY Vol. 12 ( 10 ) page： 220083 2022.10

Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution Reviewed International coauthorship

Caner Akıl, Samson Ali, Linh T Tran, Jérémie Gaillard, Wenfei Li, Kenichi Hayashida, Mika Hirose, Takayuki Kato, Atsunori Oshima, Kosuke Fujishima, Laurent Blanchoin, Akihiro Narita, Robert C Robinson

Science Advances Vol. 8 ( 12 ) page： eabm2225 2022.3

Structural analysis of filamentous complexes by cryo-electron microscopy Invited Reviewed

Akihiro Narita

Translational and Regulatory Sciences Vol. 4 ( 3 ) page： 68 - 75 2022

A cryo-TSEM with temperature cycling capability allows deep sublimation of ice to uncover fine structures in thick cells. Reviewed

Usukura J, Narita A, Matsumoto T, Usukura E, Sunaoshi T, Watanabe S, Tamba Y, Nagakubo Y, Mizuo T, Azuma J et al

Scientific reports Vol. 11 page： 21406 2021.11

Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization

Tan, YQ; Ali, S; Xue, B; Teo, WZ; Ling, LH; Go, MK; Lv, H; Robinson, RC; Narita, A; Yew, WS

BIOMACROMOLECULES Vol. 22 ( 10 ) page： 4095 - 4109 2021.10

Structure of a Minimal alpha-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization. Reviewed International coauthorship

Tan YQ, Ali S, Xue B, Teo WZ, Ling LH, Go MK, Lv H, Robinson RC, Narita A, Yew WS

Biomacromolecules Vol. 22 ( 10 ) page： 4095 - 4109 2021.8

Structural insights into the regulation of actin capping protein by twinfilin C-terminal tail Reviewed

Shuichi Takeda, Ryotaro Koike, IkukoFujiwara, AkihiroNarita, Makoto Miyata, MotonoriOta, Yuichiro Maéda

Journal of Molecular Biology Vol. 433 ( 9 ) 2021.2

Crystal structure of human V-1 in the apo form Reviewed

Takeda, S; Koike, R; Nagae, T; Fujiwara, I; Narita, A; Maéda, Y; Ota, M

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS Vol. 77 ( Pt 1 ) page： 13 - 21 2021.1

Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms Reviewed

Morone, N; Usukura, E; Narita, A; Usukura, J

MICROSCOPY Vol. 69 ( 6 ) page： 350 - 359 2020.12

Principal component analysis of data from NMR titration experiment of uniformly <SUP>15</SUP>N labeled amyloid beta (1-42) peptide with osmolytes and phenolic compounds Reviewed

Iwaya, N; Goda, N; Matsuzaki, M; Narita, A; Shigemitsu, Y; Tenno, T; Abe, Y; Hoshi, M; Hiroaki, H

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 690 page： 108446 2020.9

アクチンの機能を構造から理解する Invited Reviewed

成田哲博

生体の科学 Vol. 71 ( 4 ) page： 304 - 309 2020.8

D-Loop Mutation G42A/G46A Decreases Actin Dynamics Reviewed

Matsuzaki, M; Fujiwara, I; Kashima, S; Matsumoto, T; Oda, T; Hayashi, M; Maeda, K; Takiguchi, K; Maéda, Y; Narita, A

BIOMOLECULES Vol. 10 ( 5 ) 2020.5

Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia Reviewed

Yoke, H; Ueno, H; Narita, A; Sakai, T; Horiuchi, K; Shingyoji, C; Hamada, H; Shinohara, K

PLOS GENETICS Vol. 16 ( 3 ) page： e1008664 2020.3

Novel inter-domain Ca 2+-binding site in the gelsolin superfamily protein fragmin Reviewed

Shuichi Takeda, Ikuko Fujiwara, Yasunobu Sugimoto, Toshiro Oda, Akihiro Narita, Yuichiro Maéda

Journal of muscle research and cell motility Vol. 41 page： 153 - 162 2019.12

Dynamic Properties of Human α-Synuclein Related to Propensity to Amyloid Fibril Formation Reviewed

Fujiwara, S; Kono, F; Matsuo, T; Sugimoto, Y; Matsumoto, T; Narita, A; Shibata, K

JOURNAL OF MOLECULAR BIOLOGY Vol. 431 ( 17 ) page： 3229 - 3245 2019.8

Structural Polymorphism of Actin Reviewed

Oda, T; Takeda, S; Narita, A; Maéda, Y

JOURNAL OF MOLECULAR BIOLOGY Vol. 431 ( 17 ) page： 3217 - 3228 2019.8

The structure of a 15-stranded actin-like filament from Clostridium botulinum Reviewed International coauthorship

NATURE COMMUNICATIONS Vol. 10 ( 1 ) page： 2856 2019.6

コフィリンによるアクチン線維切断とその制御 Invited Reviewed

成田哲博田中康太郎

生化学 Vol. 91 ( 1 ) page： 109 - 113 2019

Polymerization and depolymerization of actin with nucleotide states at filament ends Reviewed

Ikuko Fujiwara, Shuichi Takeda, Toshiro Oda, Hajime Honda, Akihiro Narita, Yuichiro Maéda

Biophysical reviews Vol. 10 ( 6 ) page： 1513 - 1519 2018.11

The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation Reviewed

Ikura Teikichi, Tochio Naoya, Kawasaki Ryosuke, Matsuzaki Mizuki, Narita Akihiro, Kikumoto Mahito, Utsunomiya-Tate Naoko, Tate Shin-ichi, Ito Nobutoshi

FEBS LETTERS Vol. 592 ( 18 ) page： 3082-3091 2018.9

クライオ電子顕微鏡法によるアクチン線維の構造解析 Invited Reviewed

成田哲博

実験医学 Vol. 5月号 2018.5

Advances in Structural Biology and the Application to Biological Filament Systems Reviewed International coauthorship

Popp David, Koh Fujiet, Scipion Clement P. M., Ghoshdastider Umesh, Narita Akihiro, Holmes Kenneth C., Robinson Robert C.

BIOESSAYS Vol. 40 ( 4 ) 2018.4

F-Form Actin Crystal Structures: Mechanisms of Actin Assembly and F-Actin ATP-Hydrolysis

Takeda Shuichi, Narita Akihiro, Oda Toshiro, Tanaka Kotaro, Koike Ryotaro, Ota Motonori, Fujiwara Ikuko, Watanabe Nobuhisa, Maeda Yuichiro

BIOPHYSICAL JOURNAL Vol. 114 ( 3 ) page： 381A-381A 2018.2

Keeping the focus on biophysics and actin filaments in Nagoya: A report of the 2016 "now in actin" symposium Invited Reviewed

Fujiwara Ikuko, Narita Akihiro

CYTOSKELETON Vol. 74 ( 12 ) page： 450-464 2017.12

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM). Reviewed

Usukura E, Narita A, Yagi A, Sakai N, Uekusa Y, Imaoka Y, Ito S, Usukura J

Scientific reports Vol. 7 ( 1 ) page： 6462 2017.7

Development of a new type of low-voltage cryo-electron microscope enabling simultaneous imaging of STEM and SEM in biological samples. Invited Reviewed

Usukura J., Narita A., Matsumoto T., Usukura E., Sunaoshi T., Tamba Y., Azuma J., Nagakubo Y., Mizuo T., Osumi M., Nimura K., Tamochi R., Ose Y.

Scientific Instrument News Vol. 2018 Vol10 page： . 2017

A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy. Reviewed

Makihara M, Watanabe T, Usukura E, Kaibuchi K, Narita A, Tanaka N, Usukura J

Microscopy (Oxford, England) Vol. 65 ( 6 ) page： 488-498 2016.12

Structural complexity of filaments formed from the actin and tubulin folds. Reviewed International coauthorship

Jiang S, Ghoshdastider U, Narita A, Popp D, Robinson RC

Communicative & integrative biology Vol. 9 ( 6 ) page： e1242538 2016.11

An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy. Reviewed

Usukura E, Narita A, Yagi A, Ito S, Usukura J

Scientific reports Vol. 6 page： 27472 2016.6

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1-42 in 1,1,1,3,3,3-hexafluoro-2-propanol. Reviewed

Shigemitsu Y, Iwaya N, Goda N, Matsuzaki M, Tenno T, Narita A, Hoshi M, Hiroaki H

Analytical biochemistry Vol. 498 page： 59-67 2016.4

Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation. Reviewed International coauthorship

Jiang S, Narita A, Popp D, Ghoshdastider U, Lee LJ, Srinivasan R, Balasubramanian MK, Oda T, Koh F, Larsson M, Robinson RC

Proceedings of the National Academy of Sciences of the United States of America Vol. 113 ( 9 ) page： E1200-5 2016.3

Dynactin 3D Structure: Implications for Assembly and Dynein Binding Reviewed

Hiroshi Imai, Akihiro Narita, Yuichiro Maéda, Trina A. Schroer

Journal of Molecular Biology Vol. 426 ( 19 ) page： 3262-3271 2014

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion Reviewed

Mueller, J, Pfanzelter, J, Winkler, C, Narita, A, Clainche, CL, Nemethova, M, Carlier, M, Maeda, Y, Welch, MD, Ohkawa, T, Schmeiser, C, Resch, GP, Small, JV

PLOS Biology 2014

Structural analysis for actin filaments in the cell by electron tomography Invited Reviewed

Akihiro Narita

Microscopy Vol. 148 ( 2 ) page： 78-83 2013.8

Authorship：Lead author Language：Japanese

Although electron tomography has been rapidly developed, it is still difficult to analyze fine structures such as protein complexes in the cell. It is mainly because of two reasons: 1. Poor signal to noise ratio in the cell because of cytosol. 2. Missing wedge or missing pyramid caused by limited tilt angles of the sample deform the reconstructed three-dimensional map. We have developed structural analysis methods for filamentous complexes, especially for actin filaments in the cell. John Victor Small's group, our co-researchers, has enhanced the signal to noise ratio by staining the cell negatively. We have developed an image analysis system for analyzing electron tomograms of the negatively stained cells, which enabled us to determine polarity of the actin filaments without any labeling. We review these methods in this article.

Role of the Actin Ala-108–Pro-112 Loop in Actin Polymerization and ATPase Activities Reviewed

Journal of Biological Chemistry Vol. 287 ( 52 ) page： 43270 - 43276 2012.12

Microtubule-like Properties of the Bacterial Actin Homolog ParM-R1 Reviewed International coauthorship

Popp, D. Narita, A. Lee, L. J. Larsson, M. Robinson, R. C.

J Biol Chem Vol. 287 ( 44 ) page： 37078-88 2012.10

Language：English Publishing type：Research paper (scientific journal)

In preparation for mammalian cell division, microtubules repeatedly probe the cytoplasm to capture chromosomes and assemble the mitotic spindle. Critical features of this microtubule system are the formation of radial arrays centered at the centrosomes and dynamic instability, leading to persistent cycles of polymerization and depolymerization. Here, we show that actin homolog, ParM-R1 that drives segregation of the R1 multidrug resistance plasmid from Escherichia coli, can also self-organize in vitro into asters, which resemble astral microtubules. ParM-R1 asters grow from centrosome-like structures consisting of interconnected nodes related by a pseudo 8-fold symmetry. In addition, we show that ParM-R1 is able to perform persistent microtubule-like oscillations of assembly and disassembly. In vitro, a whole population of ParM-R1 filaments is synchronized between phases of growth and shrinkage, leading to prolonged synchronous oscillations even at physiological ParM-R1 concentrations. These results imply that the selection pressure to reliably segregate DNA during cell division has led to common mechanisms within diverse segregation machineries.

Novel actin-like filament structure from Clostridium tetani Reviewed International coauthorship

Popp, D. Narita, A. Lee, L. J. Ghoshdastider, U. Xue, B. Srinivasan, R. Balasubramanian, M. K. Tanaka, T. Robinson, R. C.

J Biol Chem Vol. 287 ( 25 ) page： 21121-9 2012.6

Language：English Publishing type：Research paper (scientific journal)

Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

Merits of the double-stranded form of the actin filament revealed by structures of the filament ends. Invited Reviewed

Akihiro Narita

Communicative and Integrative Biology Vol. 4 ( 69 ) page： 692-695 2011.11

Authorship：Lead author,　Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Actin forms a double-stranded filament, and the majority of actin filaments in the cell undergo the dynamic process of polymerization and depolymerization at both ends. Actin dynamics plays numerous important roles in eukaryotic cells. In order to understand actin dynamics, structural elucidation of the actin filament ends is particularly important because polymerization and depolymerization occurs only at the ends. We have developed original image analysis procedures to determine the structures of the actin filament ends from cryo-electron micrographs, and two structures have been determined. The structures revealed that the actin filament takes advantage of its double-stranded form to regulate its dynamics at both ends by a surprisingly simple mechanism.

Two distinct mechanisms for actin capping protein regulation--steric and allosteric inhibition Reviewed

Takeda, S., Minakata, S., Koike, R., Kawahata, I., Narita, A., Kitazawa, M., Ota, M., Yamakuni, T., Maeda, Y. & Nitanai, Y.

PLoS Biology Vol. 8 page： e1000416 2010.7

Language：English Publishing type：Research paper (scientific journal)

The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the regulation of the barbed end elongation in cells.

Filament structure, organization, and dynamics in MreB sheets. Reviewed

Popp, D., Narita, A., Maeda, K., Fujisawa, T., Ghoshdastider, U., Iwasa, M., Maeda, Y. & Robinson, R. C.

The Journal of Biological Chemistry Vol. 285 page： 15858-65 2010.5

Language：English Publishing type：Research paper (scientific journal)

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mum long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

Suprastructures and dynamic properties of mycobacterium tuberculosis FtsZ. Reviewed

Popp, D., Iwasa, M., Erickson, H. P., Narita, A., Maeda, Y. & Robinson, R. C.

The Journal of Biological Chemistry Vol. 285 page： 11281-9 2010.4

Language：English Publishing type：Research paper (scientific journal)

Tuberculosis causes the most death in humans by any bacterium. Drug targeting of bacterial cytoskeletal proteins requires detailed knowledge of the various filamentous suprastructures and dynamic properties. Here, we have investigated by high resolution electron microscopy the assembly of cell division protein and microtubule homolog FtsZ from Mycobacterium tuberculosis (MtbFtsZ) in vitro in the presence of various monovalent salts, crowding agents and polycations. Supramolecular structures, including two-dimensional rings, three-dimensional toroids, and multistranded helices formed in the presence of molecular crowding, were similar to those observed by fluorescence microscopy in bacteria in vivo. Dynamic properties of MtbFtsZ filaments were visualized by light scattering and real time total internal reflection fluorescence microscopy. Interestingly, MtbFtsZ revealed a form of dynamic instability at steady state. Cation-induced condensation phenomena of bacterial cytomotive polymers have not been investigated in any detail, although it is known that many bacteria can contain high amounts of polycations, which may modulate the prokaryotic cytoskeleton. We find that above a threshold concentration of polycations which varied with the valence of the cation, ionic strength, and pH, MtbFtsZ mainly formed sheets. The general features of these cation-induced condensation phenomena could be explained in the framework of the Manning condensation theory. Chirality and packing defects limited the dimensions of sheets and toroids at steady state as predicted by theoretical models. In first approximation simple physical principles seem to govern the formation of MtbFtsZ suprastructures.

Polymeric structures and dynamic properties of the bacterial actin AlfA Reviewed

Popp, D., Narita, A., Ghoshdastider, U., Maeda, K., Maeda, Y., Oda, T., Fujisawa, T., Onishi, H., Ito, K. & Robinson, R. C.

Journal of Molecular Biology Vol. 397 page： 1031-41 2010.4

Language：English Publishing type：Research paper (scientific journal)

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 A, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 A. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5'-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.

Structure and filament dynamics of the pSK41 actin-like ParM protein: implications for plasmid DNA segregation Reviewed International coauthorship

Vol. 285 ( 13 ) page： 10130 - 10140 2010.3

Molecular mechanism of bundle formation by the bacterial actin ParM. Reviewed

Popp, D., Narita, A., Iwasa, M., Maeda, Y. & Robinson, R. C.

Biochemical and Biophysical Research Communications Vol. 391 page： 1598-603 2010.1

Language：English Publishing type：Research paper (scientific journal)

The actin homolog ParM plays a microtubule-like role in segregating DNA prior to bacterial cell division. Fluorescence and cryo-electron microscopy have shown that ParM forms filament bundles between separating DNA plasmids in vivo. Given the lack of ParM bundling proteins it remains unknown how ParM bundles form at the molecular level. Here we show using time-lapse TIRF microscopy, under in vitro molecular crowding conditions, that ParM-bundle formation consists of two distinct phases. At the onset of polymerization bundle thickness and shape are determined in the form of nuclei of short helically disordered filaments arranged in a liquid-like lattice. These nuclei then undergo an elongation phase whereby they rapidly increase in length. At steady state, ParM bundles fuse into one single large aggregate. This behavior had been predicted by theory but has not been observed for any other cytomotive biopolymer, including F-actin. We employed electron micrographs of ParM rafts, which are 2-D analogs of 3-D bundles, to identify the main molecular interfilament contacts within these suprastructures. The interface between filaments is similar for both parallel and anti-parallel orientations and the distribution of filament polarity is random within a bundle. We suggest that the interfilament interactions are not due to the interactions of specific residues but rather to long-range, counter ion mediated, electrostatic attractive forces. A randomly oriented bundle ensures that the assembly is rigid and that DNA may be captured with equal efficiency at both ends of the bundle via the ParR binding protein.

Single particle analysis for filamentous complexes Invited Reviewed

Narita, A.

Seibutsu Butsuri Vol. 49 ( 6 ) page： 314-317 2009.11

Protofilament formation of ParM mutants. Reviewed

Popp D, Iwasa M, Maeda K, Narita A, Oda T, Maéda Y

J Mol Biol. Vol. 388 ( 2 ) page： 209-17 2009.5

Language：English Publishing type：Research paper (scientific journal)

ParM, an actin homolog, forms left-handed two-start helical filaments that segregate DNA in bacteria prior to cell division. Our recent atomic model obtained from electron microscopy (EM) reconstructions of negatively stained ParM filaments implied that two salt bridges (Glu35-Lys258 and Asp63-Arg262) may be key inter-filament contacts that stabilize the left-handed ParM helix. We made mutations of these amino acids and probed the inter-strand interface of our model experimentally by EM and X-ray fiber diffraction. We found that several mutations, such as ParM single mutants Asp258 and Asp262 and double mutant Asp258/Arg262, were incapable of forming straight filaments in aqueous buffers and appeared ragged and unstructured. However, in the presence of crowding agents, straight filaments or filament bundles formed, which allowed us to elucidate the structure of these mutant filaments. Centrifugation of filaments also resulted in a pellet of straightened filaments that could be oriented in glass capillaries and gave detailed X-ray diffraction patterns. Both EM and X-ray diffraction showed that filaments formed from these ParM mutants were not double-stranded helical filaments but single protofilaments, indicating that these residues are important for formation of the ParM helix. Our data also confirm a major prediction of crowding theory, namely that molecular crowding shifts the equilibrium of even severely impaired, unstructured cytoskeletal polymers toward their structured native and functional state. ParM is the first example of a helical actin homolog that can be induced to form protofilaments.

FtsZ condensates: An in vitro electron microscopy study. Reviewed

Popp D, Iwasa M, Narita A, Erickson HP, Maéda Y.

Biopolymers Vol. 91 ( 5 ) page： 340-50 2009.1

Language：English Publishing type：Research paper (scientific journal)

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto-filaments. Although FtsZ assembles into single proto-filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z-ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy.

Dual roles of Gln137 of actin revealed by recombinant human cardiac muscle alpha-actin mutants. Invited Reviewed

Iwasa M, Maeda K, Narita A, Maéda Y, Oda T.

J.Biol.Chem. Vol. 283 ( 39 ) page： 21045-53 2008.7

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln(137) to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln(137) in these activities, we characterized Gln(137) mutants of human cardiac muscle alpha-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the gamma-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln(137) plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

アクチンフィラメント端の伸長制御機構 Invited

成田哲博

月刊バイオニクス ( ３月号 ) page： 62-69 2007.3

Authorship：Lead author Language：Japanese

細胞運動や細胞分裂、細胞骨格など生命現象の根幹にかかわる機能を担うアクチン。
その機能には、アクチンフィラメントの端でおこる重合、脱落が重要な役割を果たしているが、詳しいメカニズムは不明であった。アクチンフィラメント端とアクチンフィラメント端結合タンパク質CPの３次元構造の決定によって、謎の一端が明かされた。

Concerning the dynamic instability of actin homolog ParM. Reviewed

Popp D, Yamamoto A, Iwasa M, Narita A, Maeda K, Maéda Y.

Biochem Biophys Res Commun. Vol. 353 ( 1 ) page： 109-14 2007.2

Language：English Publishing type：Research paper (scientific journal)

Using in vitro TIRF- and electron-microscopy, we reinvestigated the dynamics of native ParM, a prokaryotic DNA segregation protein and actin homolog. In contrast to a previous study, which used a cysteine ParM mutant, we find that the polymerization process of wild type ATP-ParM filaments consists of a polymerization phase and a subsequent steady state phase, which is dynamically unstable, like that of microtubules. We find that the apparent bidirectional polymerization of ParM, is not due to the intrinsic nature of this filament, but results from ParM forming randomly oriented bundles in the presence of crowding agents. Our results imply, that in the bacterium, ParM filaments spontaneously form bipolar bundles. Due to their intrinsic dynamic instability, ParM bundles can efficiently "search" the cytoplasmic lumen for DNA, bind it equally well at the bipolar ends and segregate it approximately symmetrically, by the insertion of ParM subunits at either end.

Two-dimensional averaged images of the dynactin complex revealed by single particle analysis. Reviewed

Imai H, Narita A, Schroer TA, Maéda Y.

J. Mol. Biol. Vol. 359 ( 4 ) page： 833-9 2006.1

Language：English Publishing type：Research paper (scientific journal)

The dynactin complex interacts with dynein and numerous other proteins to provide for a wide range of subcellular transport functions. A detailed understanding of the structure and subunit organization of dynactin should yield new insights into its function. In the present study, we used single particle analysis to obtain a two-dimensional averaged image of dynactin isolated from chick embryo brains and visualized by negative stain electron microscopy (EM). Each individual image, consisting of the shoulder/sidearm and the rod, closely resembled the previously published quick-freeze deep-etch rotary-shadow electron micrographs. However, the averaged image revealed novel structural features that may have functional significance. The bulky shoulder complex has a triangular shape and is 13 nm wide and 8 nm high. The rod, with an overall length of 40 nm, consists of clearly defined lobes that are apparently grouped into three parts, the pointed-end complex, the middle segment, and the extra lobes at the barbed end. The pointed-end complex reveals the characteristic protrusions and clefts that were previously observed only in the isolated pointed-end complex. In the middle segment, the seven lobes are fitted to the helical symmetry of F-actin. A narrow but prominent gap separates the previously unidentified extra three lobes at the barbed end from the middle segment. The averaged image we obtained contrasts dramatically with the simple Arp1 polymer that was previously reported by single particle analysis of bovine brain dynactin. These apparent structural differences are probably due to the greater stability and integrity of the chick embryo brain dynactin preparation. We propose a new structural model for dynactin, based on our observations.

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