In this paper, we present a high-speed pick-andplace method for cell-assembly applications. Besides the range of motion and accuracy, the agility of a manipulation system is an important parameter, but it has been so far underrated in the literature. To begin high-speed micromanipulation, obtaining 3D positions of both the target microobject and the end effector rapidly is necessary. Controlling the residual vibration of the end effector, which is greater at high speed, is another arduous task. Successful releasing of objects in micro-scale is also demanding. We propose a new fast detection algorithm for both the target microobject and the end effector, which will enable us to achieve high-speed control of the system. Moreover, to realize stable grasping for very fast movements, the vibration of the system is compensated, and a controllable vibration is applied to the end effectors while performing the releasing task. High-speed control of the microhand system is demonstrated with extensive experiments consisting of pick-and-place actions of 40 to 60 µm microspheres; we aimed at performing the task in 1 second.A comparison with similar studies shows the advantage of the proposed automated high-speed micromanipulation system.

SummaryRotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium-driven motor of Vibrio , have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.

SummaryThe bacterial flagellar motor is an elaborate molecular machine that converts ion-motive force into mechanical force (rotation). One of its remarkable features is its swift switching of the rotational direction or speed upon binding of the response regulator phosphoCheY, which causes the changes in swimming that achieve chemotaxis. Vibrio alginolyticus has dual flagellar systems: the Na

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA-PomB stator complex of Vibrio alginolyticus couples Na(+) influx to torque generation in this supramolecular motor, but little is known about how Na(+) associates with the PomA-PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na(+) to carboxylates in the PomA-PomB complex, including the functionally essential residue Asp24. The Na(+) affinity of Asp24 is estimated to be approximately 85 mM, close to the apparent K(m) value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na(+), but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na(+) interaction sites in the PomA-PomB complex. This work reveals features of the Na(+) pathway in the PomA-PomB Na(+) channel by using vibrational spectroscopy.

Functional chimera of the flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region Yuuki Nishino, Seiji Kojima, Michio Homma (Div. Biol. Sci, Grad. Sch. Of Sci., Nagoya Univ.) We have determined the crystal structure of PomB C5 , the periplasmic region fragment of PomB, which is the stator protein of the flagellar motor of Vibrio. Based on the structural information, we constructed three chimeric proteins between PomB and MotB, named PotB91, PotB129 and PotB138, with various chimeric junctions in addition to PotB. When these chimeric proteins were produced with PomA in the ΔmotAB strain of E. coli or the ΔpomAB and ΔpomABΔmotX strains of Vibrio, their motility was examined. All the chimeras are functional in either E. coli or Vibrio and either with or without MotX that are specific motor proteins for Vibrio though the motilities were very weak in E. coli. We try to find out what caused chimeras to give the different abilities. PomA is a membrane protein essential for torque generation in the bacterial flagellar motor from Vibrio alginolyticus. Previously, we succeeded in expression of the cytoplasm Loop domain (Q54-D148) of PomA using cold shock vector and GB1 tag at N-terminus (Biophysics 2013 Abe-Yoshizumi et al.). However, this mutant has a loose tertiary structure and shows slight dominant negative effect on motility in E. coli. To further characterize the property of the whole cytoplasmic domain, we constructed N-terminal deficient PomA (Q54-E253) with GB1 tag. This construct was expressed in both cytoplasmic and membrane fraction and showed strong dominant negative effect on motility in E. coli. We are currently studying the detailed molecular mechanism of this effect. 3P196 Vibrio alginolyticus 由来べん毛固定子 PomA のみによる複合 体形成 The stator complex in the bacterial flagellar motor forms the specific ion-conducting pathway. Ion flux through the stator through this pathway couples to the interaction between the cytoplasmic region of the stator and the rotor to generate torque. The stator of the Na+-driven motor of Vibrio alginolyticus consists of 4 PomA and 2 PomB molecules. Only the structure of periplasmic region of PomB has been determined. Toward the determination of the whole structure of the stator, we first tried to purify PomA alone. However, against our prediction, purified PomA behaved as the multimer as judged by the size-exclusion chromatography. Currently we are examining the stoichiometry, interaction site and functional meaning of this PomA complex. 3P197 Na+ uptake activity of the plug-deleted Na+-driven stator complex from Vibrio flagellar motor using reconstituted proteoliposome Tetsuya Oba, Seiji Kojima, Michio Homma (Div. of Biol. Sci., Grad. Sch. of Sci., Nagoya Univ.) The PomA/PomB stator complex couples Na+ influx to torque generation. We have been tried to quantify Na+ influx through the PomA/B complex by using not wild type but plug-deleted stator to facilitate detection of Na+ uptake. Last year, we reported a pilot experiment to detect Na+ uptake by reconstitu...

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A noncontact method that can achieve immobilization, transportation, and rotation in the microscale is desired in biological micromanipulation. A multifunctional noncontact micromanipulation method is proposed here based on a vibration‐generated whirling flow. Resonance of a cantilever structure is utilized to extend the straight vibration of a single piezo actuator to the 2D circular vibration of a micropipette. The circular vibration in fluids can generate the whirling flow featured with low pressure in the core area and flow velocity gradient. The low pressure can immobilize the objects nearby and transport them together with the micropipette, and the flow velocity gradient is utilized to form a torque to rotate the immobilized object. Experiments of the microbeads are conducted to evaluate the claimed functions and quantify the key parameters that influence the rotation velocity. The cell spheroid is immobilized and rotated for 3D observation, and by assessing the viability of the cells containing in the spheroid, the proposed method is proved noninvasive to living cells. Finally, another important application in operations of mouse egg cells is shown, which indicates that the proposed method is a potential valuable tool in biological micromanipulation.

Precise regulation of the number and positioning of flagella are critical in order for the mono-polarflagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no flhF cells have flagella, while a very small fraction of flhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of flhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.Key words bacterial flagellum, flagellar localization, flagellar number, polar flagellum.The bacterial flagellum is a locomotive organelle which is composed of a motor and a screw part. The motor is embedded in the cell envelope and driven by ion motive force. The screw part is composed of a helical filament which is connected to the motor via a hook. This hook acts as a universal joint between the two structures. Flagellar assembly begins with formation of the membrane-embedded motor part, after which the hook structure is constructed. Finally the filament is polymerized from the proximal end towards the distal tip. It is believed that formation of an ‡ These authors contributed equally to this work. MS ring by the membrane protein FliF is the first step towards flagellar morphogenesis. After this soluble proteins (FliG, FliM, and FliN) are attached to the cytoplasmic side of the MS ring, forming the C ring. Then a specific apparatus used for flagellar protein export is assembled inside the C ring, which acts as the entrance to the channel for flagellar proteins (1). The number and localization of flagella vary between species (2). For example, V. alginolyticus and V. parahaemolyticus have both peritrichous (or lateral) flagella 76