Faculty Profiles - NARITA, Akihiro

Papers - NARITA, Akihiro

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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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Papers - NARITA, Akihiro