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

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Papers - NARITA, Akihiro