Cell-to-cell signaling in cell populations with large cell size variability.

Sizes of multiple cells vary when they communicate with each other. Differences in cell size result in variations in the cell surface area and volume, as well as the number of enzymes and receptors involved in signal transduction. Although heterogeneity in cell size may inhibit uniformity in signaling, cell-to-cell signaling is still possible. The outcome when cell size changes to an extreme degree remains unclear. Hence, we inhibited cell division in Dictyostelium cells, a model organism for signal transduction, to gain insights into the consequences of extreme cell size variations. Measurements of cell signals in this population using fluorescence microscopy indicated that the giant cells can communicate with normal-sized cells by suppressing the signal level. Simulations of signal transduction based on the FitzHugh-Nagumo model also suggested similar results. Our findings suggest that signaling mechanism homogenizes cell-to-cell signaling in response to cell size.

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus.

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

Architecture and Assembly of the Bacterial Flagellar Motor Complex.

One of the central systems responsible for bacterial motility is the flagellum. The bacterial flagellum is a macromolecular protein complex that is more than five times the cell length. Flagella-driven motility is coordinated via a chemosensory signal transduction pathway, and so bacterial cells sense changes in the environment and migrate towards more desirable locations. The flagellum of Salmonella enterica serovar Typhimurium is composed of a bi-directional rotary motor, a universal joint and a helical propeller. The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second. In contrast to an artificial motor, the energy utilized for high-speed flagellar motor rotation is the inward-directed proton flow through a transmembrane proton channel of the stator unit of the flagellar motor. The flagellar motor realizes efficient chemotaxis while performing high-speed movement by an ingenious directional switching mechanism of the motor rotation. To build the universal joint and helical propeller structures outside the cell body, the flagellar motor contains its own protein transporter called a type III protein export apparatus. In this chapter we summarize the structure and assembly of the Salmonella flagellar motor complex.

Direct observation of speed fluctuations of flagellar motor rotation at extremely low load close to zero.

The bacterial flagellar motor accommodates ten stator units around the rotor to produce large torque at high load. But when external load is low, some previous studies showed that a single stator unit can spin the rotor at the maximum speed, suggesting that the maximum speed does not depend on the number of active stator units, whereas others reported that the speed is also dependent on the stator number. To clarify these two controversial observations, much more precise measurements of motor rotation would be required at external load as close to zero as possible. Here, we constructed a Salmonella filament-less mutant that produces a rigid, straight, twice longer hook to efficiently label a 60 nm gold particle and analyzed flagellar motor dynamics at low load close to zero. The maximum motor speed was about 400 Hz. Large speed fluctuations and long pausing events were frequently observed, and they were suppressed by either over-expression of the MotAB stator complex or increase in the external load, suggesting that the number of active stator units in the motor largely fluctuates near zero load. We conclude that the lifetime of the active stator unit becomes much shorter when the motor operates near zero load.

Talin B regulates collective cell migration via PI3K signaling in Dictyostelium discoideum mounds.

Collective cell migration is a key process during the development of multicellular organisms, in which the migrations of individual cells are coordinated through chemical guidance and physical contact between cells. Talin has been implicated in mechanical linkage between actin-based motile machinery and adhesion molecules, but how talin contributes to collective cell migration is unclear. Here we show that talin B is involved in chemical coordination between cells for collective cell migration at the multicellular mound stage in the development of Dictyostelium discoideum. From early aggregation to the mound formation, talB-null cells exhibited collective migration normally with cAMP relay. Subsequently, talB-null cells showed developmental arrest at the mound stage, and at the same time, they had impaired collective migration and cAMP relay, while wild-type cells exhibited rotational cell migration continuously in concert with cAMP relay during the mound stage. Genetic suppression of PI3K activity partially restored talB-null phenotypes in collective cell migration and cAMP relay. Overall, our observations suggest that talin B regulates chemical coordination via PI3K-mediated signaling in a stage-specific manner for the multicellular development of Dictyostelium cells.

Four Cases with Rare Complications of Intramedullary Screw Fixation for Jones Fracture.

Active treatment with intramedullary screw fixation is now common for athletes with Jones fracture. Outcomes are generally good, but complications can occur. We report 4 rare complications of intramedullary screw fixa-tion. Two cases developed osteomyelitis and pseudarthrosis caused by thermal necrosis. In the other two cases, screw-related complications occurred during the insertion of the tapered headless screw. Although thermal necrosis and screw insertion failures are considered rare complications and not widely reported in the litera-ture, they do occur occasionally. Knowing the mechanisms underlying these complications could help prevent them, and knowing their course could lead caregivers to appropriate interventions when they do occur.

Effect of the MotA(M206I) Mutation on Torque Generation and Stator Assembly in the Salmonella H+-Driven Flagellar Motor.

The bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant of Salmonella The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-µm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-µm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCE The bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

Reconstruction of Complete Peroneus Longus and Brevis Tendon Ruptures Using a Semitendinosus and Gracilis Tendon Graft.

The patient was a 40-year-old female who had been treated at our hospital for left peroneal tendonitis due to an ankle sprain 2 years earlier. She re-injured that ankle while dancing. The pain in the lateral left foot soon improved, but she had difficulty standing with the left foot in equinus. Complete peroneus longus and brevis tendon ruptures were diagnosed. The ipsilateral semitendinosus and gracilis tendons were harvested and used to reconstruct the tendons. Three months after surgery, the patient was able to stand in equinus, and at 5 months after surgery she resumed her original level of sports activities.

The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

Retrospective triggering of conscious perception by an interstimulus interaction.

Attention facilitates conscious perception of a visual stimulus at an attended location. Interestingly, a recent study (using the Posner spatial-cueing task) reported that attention facilitated conscious perception even when it was cued after a stimulus was gone (postcued-attention or retroperception effect). Here, we show that this effect can be induced without any contribution of attention. Contrary to previous situations, we fixed a position of a target (Gabor patch) and cue (luminance change of a circle encompassing the target) across trials so that subjects always could allocate their full attention to the target position. The cue (luminance change) improved objective and subjective visibility of the nearby target even when it was given ∼200 ms after the target's offset. This retrospective improvement was diminished when a shape of the cue was changed from a circle to a dot pattern, suggesting that the improvement emerged from a visual interaction (combinations of shapes) between the circular cue and target. Those results indicated that a local visual interaction between the target and cue is sufficient to trigger consciousness of the target, revealing a new type of retroperception effect mediated by sensory (nonattentional) mechanisms.

Load-sensitive coupling of proton translocation and torque generation in the bacterial flagellar motor.

The Salmonella flagellar motor consists of a rotor and about a dozen stator elements. Each stator element, consisting of MotA and MotB, acts as a proton channel to couple proton flow with torque generation. A highly conserved Asp33 residue of MotB is directly involved in the energy coupling mechanism, but it remains unknown how it carries out this function. Here, we show that the MotB(D33E) mutation dramatically alters motor performance in response to changes in external load. Rotation speeds of the MotA/B(D33E) and MotA(V35F)/B(D33E) motors were markedly slower than the wild-type motor and fluctuated considerably at low load but not at high load, whereas the rotation rate of the wild-type motor was stable at any load. At low load, pausing events were frequently observed in both mutant motors. The proton conductivities of these mutant stator channels in their 'unplugged' forms were only half of the conductivity of the wild-type channel. These results suggest that the D33E mutation induces a load-dependent inactivation of the MotA/B complex. We propose that the stator element is a load-sensitive proton channel that efficiently couples proton translocation with torque generation and that Asp33 of MotB is critical for this co-ordinated proton translocation.

Assembly and stoichiometry of FliF and FlhA in Salmonella flagellar basal body.

The bacterial flagellar export apparatus is required for the construction of the bacterial flagella beyond the cytoplasmic membrane. The membrane-embedded part of the export apparatus, which consists of FlhA, FlhB, FliO, FliP, FliQ and FliR, is located in the central pore of the MS ring formed by 26 copies of FliF. The C-terminal cytoplasmic domain of FlhA is located in the centre of the cavity within the C ring made of FliG, FliM and FliN. FlhA interacts with FliF, but its assembly mechanism remains unclear. Here, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the C-termini of FliF and FlhA and investigated their subcellular localization by fluorescence microscopy. The punctate pattern of FliF-YFP localization required FliG but neither FliM, FliN, FlhA, FlhB, FliO, FliP, FliQ nor FliR. In contrast, FlhA-CFP localization required FliF, FliG, FliO, FliP, FliQ and FliR. The number of FlhA-YFP molecules associated with the MS ring was estimated to be about nine. We suggest that FlhA assembles into the export gate along with other membrane components during the MS ring complex formation in a co-ordinated manner.

H(+) and Na(+) are involved in flagellar rotation of the spirochete Leptospira.

Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H(+) is used in Escherichia coli, and Na(+) is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H(+) is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na(+), though it vigorously moved even under Na(+)-free conditions. These results suggest that H(+) is preferentially used and that Na(+) is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment.

A lactose fermentation product produced by Lactococcus lactis subsp. lactis, acetate, inhibits the motility of flagellated pathogenic bacteria.

Many strains of lactic acid bacteria have been used for the production of probiotics. Some metabolites produced by lactic acid bacteria impair the motilities of pathogenic bacteria. Because bacterial motility is strongly associated with virulence, the metabolic activities of lactic acid bacteria are effective for suppressing bacterial infections. Here we show that lactose fermentation by Lactococcus lactis subsp. lactis inhibits the motility of Salmonella enterica serovar Typhimurium. A single-cell tracking and rotation assay for a single flagellum showed that the swimming behaviour of Salmonella was severely but transiently impaired through disruption of flagellar rotation on exposure to media cultivated with Lac. lactis. Using a pH-sensitive fluorescent protein, we observed that the intracellular pH of Salmonella was decreased because of some fermentation products of Lac. lactis. We identified acetate as the lactose fermentation product of Lac. lactis triggering the paralysis of Salmonella flagella. The motilities of Pseudomonas, Vibrio and Leptospira strains were also severely disrupted by lactose utilization by Lac. lactis. These results highlight the potential use of Lac. lactis for preventing infections by multiple bacterial species.

Structural insight into the rotational switching mechanism of the bacterial flagellar motor.

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.

Ion Signaling in Cell Motility and Development in Dictyostelium discoideum.

Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3',5'-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.

Distinct roles of highly conserved charged residues at the MotA-FliG interface in bacterial flagellar motor rotation.

Electrostatic interactions between the stator protein MotA and the rotor protein FliG are important for bacterial flagellar motor rotation. Arg90 and Glu98 of MotA are required not only for torque generation but also for stator assembly around the rotor, but their actual roles remain unknown. Here we analyzed the roles of functionally important charged residues at the MotA-FliG interface in motor performance. About 75% of the motA(R90E) cells and 45% of the motA(E98K) cells showed no fluorescent spots of green fluorescent protein (GFP)-MotB, indicating reduced efficiency of stator assembly around the rotor. The FliG(D289K) and FliG(R281V) mutations, which restore the motility of the motA(R90E) and motA(E98K) mutants, respectively, showed reduced numbers and intensity of GFP-MotB spots as well. The FliG(D289K) mutation significantly recovered the localization of GFP-MotB to the motor in the motA(R90E) mutant, whereas the FliG(R281V) mutation did not recover the GFP-MotB localization in the motA(E98K) mutant. These results suggest that the MotA-Arg90-FliG-Asp289 interaction is critical for the proper positioning of the stators around the rotor, whereas the MotA-Glu98-FliG-Arg281 interaction is more important for torque generation.

Measurements of the Ion Channel Activity of the Transmembrane Stator Complex in the Bacterial Flagellar Motor.

The bacterial flagellum is driven by a rotational motor located at the base of the flagellum. The stator unit complex conducts cations such as protons (H+) and sodium ions (Na+) along the electrochemical potential across the cytoplasmic membrane and interacts with the rotor to generate the rotational force. Escherichia coli and Salmonella have the H+-type stator complex, which serves as a transmembrane H+ channel that couples H+ flow through an ion channel to torque generation whereas Vibrio and some Bacillus species have the Na+-type stator complex. In this chapter, we describe how to measure the ion conductivity of the transmembrane stator complex over-expressed in E. coli cells using fluorescent indicators. Intensity measurements of fluorescent indicators using either a fluorescence spectrophotometer or microscope allow quantitative detection of changes in the intracellular ion concentrations due to the ion channel activity of the transmembrane protein complex.

Visualization of c-di-GMP in multicellular Dictyostelium stages.

The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) is only synthesized and utilized by the cellular slime mold Dictyostelium discoideum among eukaryotes. Dictyostelium cells undergo a transition from a unicellular to a multicellular state, ultimately forming a stalk and spores. While Dictyostelium is known to employ c-di-GMP to induce differentiation into stalk cells, there have been no reports of direct observation of c-di-GMP using fluorescent probes. In this study, we used a fluorescent probe used in bacteria to visualize its localization within Dictyostelium multicellular bodies. Cytosolic c-di-GMP concentrations were significantly higher at the tip of the multicellular body during stalk formation.

Femoral Tunnel Length in Anatomical Double-Bundle Anterior Cruciate Ligament Reconstruction Is Correlated with Body Size and Knee Morphology.

The purpose of this study was to reveal the correlation between anteromedial (AM) and posterolateral (PL) femoral tunnel lengths in anatomical double-bundle anterior cruciate ligament (ACL) reconstruction and body size and knee morphology. Thirty-four subjects undergoing anatomical double-bundle ACL reconstruction were included in this study. Preoperative body size (height, body weight, and body mass index) was measured. Using preoperative magnetic resonance imaging (MRI), quadriceps tendon thickness and the whole anterior-posterior length of the knee were measured. Using postoperative computed tomography (CT), axial and sagittal views of the femoral condyle were evaluated. The correlation between measured intraoperative AM and PL femoral tunnel lengths, and body size and knee morphology using preoperative MRI and postoperative CT parameters was statistically analyzed. Both AM and PL femoral tunnel lengths were significantly correlated with height, body weight, posterior condylar length, and Blumensaat's line length. These results suggest that the femoral ACL tunnel length created using a transportal technique can be estimated preoperatively by measuring the subject's body size and/or the knee morphology using MRI or CT. For clinical relevance, surgeons should be careful to create femoral tunnel of sufficient length when using a transportal technique, especially in knees of subjects with smaller body size and knee morphology. Level of evidence is III.