Faculty Profiles - KOJIMA, Seiji

Papers - KOJIMA, Seiji

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Mutations Targeting the C-Terminal Domain of FliG Can Disrupt Motor Assembly in the Na+-Driven Flagella of Vibrio alginolyticus Reviewed

Seiji Kojima, Natsumi Nonoyama, Norihiro Takekawa, Hajime Fukuoka, Michio Homma

JOURNAL OF MOLECULAR BIOLOGY Vol. 414 ( 1 ) page： 62 - 74 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na+-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2011.09.019

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PubMed

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter Reviewed

Yajing Shen, Masahiro Nakajima, Seiji Kojima, Michio Homma, Masaru Kojima, Toshio Fukuda

MEASUREMENT SCIENCE AND TECHNOLOGY Vol. 22 ( 11 ) 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：IOP PUBLISHING LTD

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

DOI： 10.1088/0957-0233/22/11/115802

Web of Science

Sodium-driven motor of the polar flagellum in marine bacteria Vibrio Reviewed

Na Li, Seiji Kojima, Michio Homma

GENES TO CELLS Vol. 16 ( 10 ) page： 985 - 999 2011.10

Language：English Publisher：WILEY-BLACKWELL

The Na+-driven bacterial flagellar motor is a molecular machine powered by an electrochemical potential gradient of sodium ions across the cytoplasmic membrane. The marine bacterium Vibrio alginolyticus has a single polar flagellum that enables it to swim in liquid. The flagellar motor contains a basal body and a stator complexes, which are composed of several proteins. PomA, PomB, MotX, and MotY are thought to be essential components of the stator that are required to generate the torque of the rotation. Several mutations have been investigated to understand the characteristics and function of the ion channel in the stator and the mechanism of its assembly around the rotor to complete the motor. In this review, we summarize recent results of the Na+-driven motor in the polar flagellum of Vibrio.

DOI： 10.1111/j.1365-2443.2011.01545.x

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PubMed

細菌べん毛モーターエネルギー変換タンパク質の構造と機能 Invited Reviewed

寺内尭史、小嶋誠司、本間道夫

生化学 Vol. 83 ( 9 ) page： 822-833 2011.9

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque Reviewed

Takashi Terauchi, Hiroyuki Terashima, Seiji Kojima, Michio Homma

MICROBIOLOGY-SGM Vol. 157 ( Pt 8 ) page： 2422 - 2432 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.

DOI： 10.1099/mic.0.048488-0

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PubMed

Characterization of the Periplasmic Region of PomB, a Na+-Driven Flagellar Stator Protein in Vibrio alginolyticus Reviewed

Na Li, Seiji Kojima, Michio Homma

JOURNAL OF BACTERIOLOGY Vol. 193 ( 15 ) page： 3773 - 3784 2011.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

The stator proteins PomA and PomB form a complex that couples Na+ influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB(C)). To investigate the function of PomB(C), a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB(C) fragment consisting of residues 135 to 315 (PomB(C5)) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB(Delta L)) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na+ influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB(Delta L) stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal alpha-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a "plug" segment is important for the control of Na+ influx through the stator complex as well as for stator assembly.

DOI： 10.1128/JB.00113-11

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PubMed

Design and characterization of nanoknife with buffering beam for in situ single-cell cutting. Reviewed International journal

Shen Y, Nakajima M, Yang Z, Kojima S, Homma M, Fukuda T

Nanotechnology Vol. 22 ( 30 ) page： 305701 - 305701 2011.7

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

A novel nanoknife with a buffering beam is proposed for single-cell cutting. The nanoknife was fabricated from a commercial atomic force microscopy (AFM) cantilever by focused-ion-beam (FIB) etching technique. The material identification of the nanoknife was determined using the energy dispersion spectrometry (EDS) method. It demonstrated that the gallium ion pollution of the nanoknife can be ignored during the etching processes. The buffering beam was used to measure the cutting force based on its deformation. The spring constant of the beam was calibrated based on a referenced cantilever by using a nanomanipulation approach. The tip of the nanoknife was designed with a small edge angle 5° to reduce the compression to the cell during the cutting procedure. For comparison, two other nanoknives with different edge angles, i.e. 25° and 45°, were also prepared. An in situ single-cell cutting experiment was performed using these three nanoknives inside an environmental scanning electron microscope (ESEM). The cutting force and the sample slice angle for each nanoknife were evaluated. It showed the compression to the cell can be reduced when using the nanoknife with a small edge angle 5°. Consequently, the nanoknife was capable for in situ single-cell cutting tasks.

DOI： 10.1088/0957-4484/22/30/305701

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PubMed

Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM Reviewed

Yajing Shen, Masahiro Nakajima, Mohd Ridzuan Ahmad, Seiji Kojima, Michio Homma, Toshio Fukuda

ULTRAMICROSCOPY Vol. 111 ( 8 ) page： 1176 - 1183 2011.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

A novel method for measuring an adhesion force of single yeast cell is proposed based on a nanorobotic manipulation system inside an environmental scanning electron microscope (ESEM). The effect of ambient humidity on a single yeast cell adhesion force was studied. Ambient humidity was controlled by adjusting the chamber pressure and temperature inside the ESEM. It has been demonstrated that a thicker water film was formed at a higher humidity condition. The adhesion force between an atomic force microscopy (AFM) cantilever and a tungsten probe which later on known as a substrate was evaluated at various humidity conditions. A micro-puller was fabricated from an AFM cantilever by use of focused ion beam (FIB) etching. The adhesion force of a single yeast cell (W303) to the substrate was measured using the micro-puller at the three humidity conditions: 100%, 70%, and 40%. The results showed that the adhesion force between the single yeast cell and the substrate is much smaller at higher humidity condition. The yeast cells were still alive after being observed and manipulated inside ESEM based on the result obtained from the re-culturing of the single yeast cell. The results from this work would help us to understand the ESEM system better and its potential benefit to the single cell analysis research. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ultramic.2011.02.008

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PubMed

Study of the time effect on the strength of cell-cell adhesion force by a novel nano-picker Reviewed

Yajing Shen, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Vol. 409 ( 2 ) page： 160 - 165 2011.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Cell's adhesion is important to cell's interaction and activates. In this paper, a novel method for cell-cell adhesion force measurement was proposed by using a nano-picker. The effect of the contact time on the cell-cell adhesion force was studied. The nano-picker was fabricated from an atomic force microscopy (AFM) cantilever by nano fabrication technique. The cell-cell adhesion force was measured based on the deflection of the nano-picker beam. The result suggests that the adhesion force between cells increased with the increasing of contact time at the first few minutes. After that, the force became constant. This measurement methodology was based on the nanorobotic manipulation system inside an environmental scanning electron microscope. It can realize both the observation and manipulation of a single cell at nanoscale. The quantitative and precise cell-cell adhesion force result can be obtained by this method. It would help us to understand the single cell interaction with time and would benefit the research in medical and biological fields potentially. (C) 2011 Published by Elsevier Inc.

DOI： 10.1016/j.bbrc.2011.04.019

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PubMed

M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins Reviewed

Yusuke V. Morimoto, Seiji Kojima, Keiichi Namba, Tohru Minamino

PLOS ONE Vol. 6 ( 5 ) page： e19598 2011.5

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

Background:. Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations.
Methodology/Principal Findings. Here, we we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values.
Conclusions/Significance: Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins.

DOI： 10.1371/journal.pone.0019598

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PubMed

Buckling Nanoneedle for Characterizing Single Cells Mechanics Inside Environmental SEM Reviewed

Mohd Ridzuan Ahmad, Masahiro Nakajima, Seiji Kojima, Michio Homma, Toshio Fukuda

IEEE TRANSACTIONS ON NANOTECHNOLOGY Vol. 10 ( 2 ) page： 226 - 236 2011.3

Language：English Publishing type：Research paper (scientific journal) Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC

We propose a buckling nanoneedle as a force sensor for stiffness characterization of single cells. The buckling nanoneedle was easily fabricated by using focused ion beam etching from a commercialized atomic force microscope cantilever. There are notable advantages of using buckling nanoneedle for single cells stiffness characterizations. First, severe cell damage from an excessive indentation force could be prevented. Second, large variations in single cells stiffness property could be easily detected either from the dented mark on the cell surface after the indentation and/or by comparing the buckling length of the nanoneedle during the indentation. The calibrations of the buckling nanoneedle were done experimentally and numerically. The calibration results from both methods showed a good agreement. The calibration data show the relationship between the indentation force and the buckling length of the nanoneedle. This relationship was used for obtaining force data during a nanoindentation experiment between a buckling nanoneedle and single cells. We performed in situ measurements of mechanical properties of individual W303 wildtype yeast cells by using a buckling nanoneedle inside an integrated SEM (ESEM)-nanomanipulator system. Finer local stiffness property of single cells was compared at different pressure and different temperature ranges. This detection method of the stiffness variations of the single cells could be applied in the future fast disease diagnosis based on single-cell stiffness analysis.

DOI： 10.1109/TNANO.2009.2036997

Web of Science

Sodium-driven motor of the polar flagellum in marine bacteria Vibrio. Reviewed

Li N, Kojima S, Homma M.

Genes to Cells Vol. 16 ( 10 ) page： 985-999 2011

Effect of ambient humidity on the strength of the adhesion force of single yeast cell inside environmental-SEM. Reviewed

Shen Y, Nakajima M, Ahmad MR, Kojima S, Homma M, Fukuda T.

Ultramicroscopy Vol. 111 ( 8 ) page： 1176-1183 2011

Design and characterization of nanoknife with buffering beam for in situ single-cell cutting. Reviewed

Shen Y, Nakajima M, Yang Z, Kojima S, Homma M, Fukuda T.

Nanotechnology Vol. 22 ( 30 ) page： 305701 2011

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque. Reviewed

Terauchi T, Terashima H, Kojima S, Homma M.

Microbiology Vol. 157 page： 2422-2432 2011

Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus. Reviewed

Li N, Kojima S, Homma M.

J Bacteriol Vol. 193 ( 15 ) page： 3773-3784 2011

Study of the time effect on the strength of cell-cell adhesion force by a novel nano-picker. Reviewed

Shen Y, Nakajima M, Kojima S, Homma M, Fukuda T.

Biochem Biophys Res Commun. Vol. 409 ( 2 ) page： 160-165 2011

M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins Reviewed

Morimoto YV, Kojima S, Namba K, Minamino T

PLoS One Vol. 6 ( 5 ) page： e19598 2011

Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the NA +-driven polar flagellum of Vibrio alginolyticus Reviewed

Masafumi Koike, Noriko Nishioka, Seiji Kojima, Michio Homma

Biophysics Vol. 7 page： 59 - 67 2011

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

The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the Cterminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG. © 2011 THE BIOPHYSICAL SOCIETY OF JAPAN.

DOI： 10.2142/biophysics.7.59

Scopus

PubMed

Fabrication and Application of Nanofork for Measuring Single Cells Adhesion Force inside ESEM Reviewed

Mohd Ridzuan Ahmad, Masahiro Nakajima, Masaru Kojima, Seiji Kojima, Michio Homma, Toshio Fukuda

ENABLING SCIENCE AND NANOTECHNOLOGY Vol. 1341 page： 304 - + 2011

Language：English Publishing type：Research paper (international conference proceedings) Publisher：AMER INST PHYSICS

In this paper, single cell adhesion force was measured using a nanofork. The nanofork was used to pick-up a single cell on a line-patterned substrate inside ESEM. The line-patterned substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick-up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line-patterned substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line-patterned substrate were used, i.e. 1 mu m and 2 mu m. Results showed that cells attached on the 1 mu m gap line-patterned substrate required more force to be released as compared to the cells attached on the 2 mu m gap line-patterned substrate.

DOI： 10.1063/1.3587007

Web of Science

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Papers - KOJIMA, Seiji