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Sample records for intact flagellar motor

  1. Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion▿ †

    PubMed Central

    Liu, Jun; Lin, Tao; Botkin, Douglas J.; McCrum, Erin; Winkler, Hanspeter; Norris, Steven J.

    2009-01-01

    The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation. PMID:19429612

  2. Torque generation by the flagellar rotary motor.

    PubMed Central

    Berg, H C

    1995-01-01

    A review is given of the structure and dynamics of the flagellar rotary motor. Force-generating elements in a motor driving a tethered bacterium (a cell fixed to the substratum by a single flagellum) exert forces of order 20 pN while moving at speeds of order 1 micron/s. Force-generating elements in a motor driving a flagellar filament in a bundle exert forces some 10-fold lower but move at speeds more than 10-fold higher. The motor torque-speed relationship has been measured over a wide dynamic range. Motors strongly resist being driven backwards and are easily broken. PMID:7787060

  3. Taking control of the flagellar motor

    NASA Astrophysics Data System (ADS)

    Gauthier, Mathieu; Truchon, Dany; Rainville, Simon

    2008-06-01

    Numerous types of bacteria swim in their environment by rotating long helical filaments. At the base of each filament is a tiny rotary motor called the bacterial flagellar motor. A lot is already known about the structure, assembly and function of this splendid molecular machine of nanoscopic dimensions. Nevertheless many fundamental questions remain open and the study of the flagellar motor is a very exciting area of current research. We are developing an in vitro assay to enable studies of the bacterial flagellar motor in precisely controlled conditions and to gain direct access to the inner components of the motor. We partly squeeze a filamentous E. coli bacterium inside a micropipette, leaving a working flagellar motor outside. We then punch a hole through the cell wall at the end of the bacterium located inside the micropipette using a brief train of ultrashort (~60 fs) laser pulses. This enables us to control the rotation of the motor with an external voltage (for at least 15 minutes). In parallel, new methods to monitor the speed of rotation of the motor in the low load (high speed) regime are being developed using various nanoparticules.

  4. Steps in the Bacterial Flagellar Motor

    PubMed Central

    Mora, Thierry; Yu, Howard; Sowa, Yoshiyuki; Wingreen, Ned S.

    2009-01-01

    The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps. Here we propose a simple physical model, based on the storage of energy in protein springs, that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. We also predict a sublinear speed versus torque relationship for fixed load at low torque, and a peak in rotor diffusion as a function of torque. Our model provides a comprehensive framework for understanding and analyzing stepping behavior in the bacterial flagellar motor and proposes novel, testable predictions. More broadly, the storage of energy in protein springs by the flagellar motor may provide useful general insights into the design of highly efficient molecular machines. PMID:19851449

  5. Structural diversity of bacterial flagellar motors

    PubMed Central

    Chen, Songye; Beeby, Morgan; Murphy, Gavin E; Leadbetter, Jared R; Hendrixson, David R; Briegel, Ariane; Li, Zhuo; Shi, Jian; Tocheva, Elitza I; Müller, Axel; Dobro, Megan J; Jensen, Grant J

    2011-01-01

    The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species. PMID:21673657

  6. How molecular motors shape the flagellar beat

    PubMed Central

    Riedel-Kruse, Ingmar H.; Hilfinger, Andreas; Howard, Jonathon; Jülicher, Frank

    2007-01-01

    Cilia and eukaryotic flagella are slender cellular appendages whose regular beating propels cells and microorganisms through aqueous media. The beat is an oscillating pattern of propagating bends generated by dynein motor proteins. A key open question is how the activity of the motors is coordinated in space and time. To elucidate the nature of this coordination we inferred the mechanical properties of the motors by analyzing the shape of beating sperm: Steadily beating bull sperm were imaged and their shapes were measured with high precision using a Fourier averaging technique. Comparing our experimental data with wave forms calculated for different scenarios of motor coordination we found that only the scenario of interdoublet sliding regulating motor activity gives rise to satisfactory fits. We propose that the microscopic origin of such “sliding control” is the load dependent detachment rate of motors. Agreement between observed and calculated wave forms was obtained only if significant sliding between microtubules occurred at the base. This suggests a novel mechanism by which changes in basal compliance could reverse the direction of beat propagation. We conclude that the flagellar beat patterns are determined by an interplay of the basal properties of the axoneme and the mechanical feedback of dynein motors. PMID:19404446

  7. Shear Stress Transmission Model for the Flagellar Rotary Motor

    PubMed Central

    Mitsui, Toshio; Ohshima, Hiroyuki

    2008-01-01

    Most bacteria that swim are propelled by flagellar filaments, which are driven by a rotary motor powered by proton flux. The mechanism of the flagellar motor is discussed by reforming the model proposed by the present authors in 2005. It is shown that the mean strength of Coulomb field produced by a proton passing the channel is very strong in the Mot assembly so that the Mot assembly can be a shear force generator and induce the flagellar rotation. The model gives clear calculation results in agreement with experimental observations, e g., for the charasteristic torque-velocity relationship of the flagellar rotation. PMID:19325821

  8. Variable symmetry in Salmonella typhimurium flagellar motors.

    PubMed

    Young, Howard S; Dang, Hongyue; Lai, Yimin; DeRosier, David J; Khan, Shahid

    2003-01-01

    Electron cryomicroscopy of rotor complexes of the Salmonella typhimurium flagellar motor, overproduced in a nonmotile Escherichia coli host, has revealed a variation in subunit symmetry of the cytoplasmic ring (C ring) module. C rings with subunit symmetries ranging from 31 to 38 were found. They formed a Gaussian distribution around a mean between 34 and 35, a similar number to that determined for native C rings. C-ring diameter scaled with the number of subunits, indicating that the elliptical-shaped subunits maintained constant intersubunit spacing. Taken together with evidence that the M ring does not correspondingly increase in size, this finding indicates that rotor assembly does not require strict stoichiometric interactions between the M- and C-ring subunits. Implications for motor function are discussed.

  9. Limiting Speed of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Nirody, Jasmine; Berry, Richard; Oster, George

    The bacterial flagellar motor (BFM) drives swimming in a wide variety of bacterial species, making it crucial for several fundamental biological processes including chemotaxis and community formation. Recent experiments have shown that the structure of this nanomachine is more dynamic than previously believed. Specifically, the number of active torque-generating units (stators) was shown to vary across applied loads. This finding invalidates the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we put forward a model for the torque generation mechanism of this motor and propose that the maximum speed of the motor increases as additional torque-generators are recruited. This is contrary to the current widely-held belief that there is a universal upper limit to the speed of the BFM. Our result arises from the assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads.

  10. Exchange of rotor components in functioning bacterial flagellar motor

    SciTech Connect

    Fukuoka, Hajime; Inoue, Yuichi; Terasawa, Shun; Takahashi, Hiroto; Ishijima, Akihiko

    2010-03-26

    The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s{sup -1}, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.

  11. An Element of Determinism in a Stochastic Flagellar Motor Switch

    PubMed Central

    Xie, Li; Altindal, Tuba; Wu, Xiao-Lun

    2015-01-01

    Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δf and backward Δb intervals are investigated herein. We found that the steady-state probability density functions, P(Δf) and P(Δb), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δf) and P(Δb) and found good agreement with the measurements. PMID:26554590

  12. An Element of Determinism in a Stochastic Flagellar Motor Switch.

    PubMed

    Xie, Li; Altindal, Tuba; Wu, Xiao-Lun

    2015-01-01

    Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δf and backward Δb intervals are investigated herein. We found that the steady-state probability density functions, P(Δf) and P(Δb), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δf) and P(Δb) and found good agreement with the measurements. PMID:26554590

  13. Hybrid-fuel bacterial flagellar motors in Escherichia coli.

    PubMed

    Sowa, Yoshiyuki; Homma, Michio; Ishijima, Akihiko; Berry, Richard M

    2014-03-01

    The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H(+) or Na(+) ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H(+)- and Na(+)-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na(+) concentration ([Na(+)]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na(+) and H(+) stator components expressed at high and medium levels, respectively, Na(+) stator units dominate at high [Na(+)] and are replaced by H(+) units when Na(+) is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H(+) and Na(+) stator components sharing a common rotor. PMID:24550452

  14. Resurrection of the flagellar rotary motor near zero load

    PubMed Central

    Yuan, Junhua; Berg, Howard C.

    2008-01-01

    Flagellated bacteria, such as Escherichia coli, are propelled by helical flagellar filaments, each driven at its base by a reversible rotary motor, powered by a transmembrane proton flux. Torque is generated by the interaction of stator proteins, MotA and MotB, with a rotor protein FliG. The physiology of the motor has been studied extensively in the regime of relatively high load and low speed, where it appears to operate close to thermodynamic equilibrium. Here, we describe an assay that allows systematic study of the motor near zero load, where proton translocation and movement of mechanical components are rate limiting. Sixty-nanometer-diameter gold spheres were attached to hooks of cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image plane of a microscope through a small pinhole. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23°C by expression of wild-type MotA, and speeds jumped from zero to a maximum value (≈300 Hz) in one step. Thus, near zero load, the speed of the motor is independent of the number of torque-generating units. Evidently, the units act independently (they do not interfere with one another), and there are no intervals during which a second unit can add to the speed generated by the first (the duty ratio is close to 1). PMID:18202173

  15. The Limiting Speed of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Nirody, Jasmine A.; Berry, Richard M.; Oster, George

    2016-08-01

    Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, is more dynamic than previously believed. Specifically, the number of active torque-generating units (stators) was shown to vary across applied loads. This finding invalidates the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we propose that, contrary to previous assumptions, the maximum speed of the motor is not universal, but rather increases as additional torque-generators are recruited. This result arises from our assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads.

  16. Mechanics of torque generation in the bacterial flagellar motor.

    PubMed

    Mandadapu, Kranthi K; Nirody, Jasmine A; Berry, Richard M; Oster, George

    2015-08-11

    The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator. PMID:26216959

  17. Mechanics of torque generation in the bacterial flagellar motor

    PubMed Central

    Mandadapu, Kranthi K.; Nirody, Jasmine A.; Berry, Richard M.; Oster, George

    2015-01-01

    The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator. PMID:26216959

  18. Dynamics of the bacterial flagellar motor with multiple stators

    PubMed Central

    Meacci, Giovanni; Tu, Yuhai

    2009-01-01

    The bacterial flagellar motor drives the rotation of flagellar filaments and enables many species of bacteria to swim. Torque is generated by interaction of stator units, anchored to the peptidoglycan cell wall, with the rotor. Recent experiments [Yuan J, Berg HC (2008) Proc Natl Acad Sci USA 105:1182–1185] show that at near-zero load the speed of the motor is independent of the number of stators. Here, we introduce a mathematical model of the motor dynamics that explains this behavior based on a general assumption that the stepping rate of a stator depends on the torque exerted by the stator on the rotor. We find that the motor dynamics can be characterized by two timescales: the moving-time interval for the mechanical rotation of the rotor and the waiting-time interval determined by the chemical transitions of the stators. We show that these two timescales depend differently on the load, and that their cross-over provides the microscopic explanation for the existence of two regimes in the torque-speed curves observed experimentally. We also analyze the speed fluctuation for a single motor by using our model. We show that the motion is smoothed by having more stator units. However, the mechanism for such fluctuation reduction is different depending on the load. We predict that the speed fluctuation is determined by the number of steps per revolution only at low load and is controlled by external noise for high load. Our model can be generalized to study other molecular motor systems with multiple power-generating units. PMID:19234112

  19. Model Studies of the Dynamics of Bacterial Flagellar Motors

    SciTech Connect

    Bai, F; Lo, C; Berry, R; Xing, J

    2009-03-19

    The Bacterial Flagellar Motor is a rotary molecular machine that rotates the helical filaments which propel swimming bacteria. Extensive experimental and theoretical studies exist on the structure, assembly, energy input, power generation and switching mechanism of the motor. In our previous paper, we explained the general physics underneath the observed torque-speed curves with a simple two-state Fokker-Planck model. Here we further analyze this model. In this paper we show (1) the model predicts that the two components of the ion motive force can affect the motor dynamics differently, in agreement with the latest experiment by Lo et al.; (2) with explicit consideration of the stator spring, the model also explains the lack of dependence of the zero-load speed on stator number in the proton motor, recently observed by Yuan and Berg; (3) the model reproduces the stepping behavior of the motor even with the existence of the stator springs and predicts the dwelling time distribution. Predicted stepping behavior of motors with two stators is discussed, and we suggest future experimental verification.

  20. The Gearbox of the Bacterial Flagellar Motor Switch.

    PubMed

    Pandini, Alessandro; Morcos, Faruck; Khan, Shahid

    2016-07-01

    Switching of flagellar motor rotation sense dictates bacterial chemotaxis. Multi-subunit FliM-FliG rotor rings couple signal protein binding in FliM with reversal of a distant FliG C-terminal (FliGC) helix involved in stator contacts. Subunit dynamics were examined in conformer ensembles generated by molecular simulations from the X-ray structures. Principal component analysis extracted collective motions. Interfacial loop immobilization by complex formation coupled elastic fluctuations of the FliM middle (FliMM) and FliG middle (FliGM) domains. Coevolved mutations captured interfacial dynamics as well as contacts. FliGM rotation was amplified via two central hinges to the FliGC helix. Intrinsic flexibility, reported by the FliGMC ensembles, reconciled conformers with opposite FliGC helix orientations. FliG domain stacking deformed the inter-domain linker and reduced flexibility; but conformational changes were not triggered by engineered linker deletions that cause a rotation-locked phenotype. These facts suggest that binary rotation states arise from conformational selection by stacking interactions. PMID:27345932

  1. The Gearbox of the Bacterial Flagellar Motor Switch.

    PubMed

    Pandini, Alessandro; Morcos, Faruck; Khan, Shahid

    2016-07-01

    Switching of flagellar motor rotation sense dictates bacterial chemotaxis. Multi-subunit FliM-FliG rotor rings couple signal protein binding in FliM with reversal of a distant FliG C-terminal (FliGC) helix involved in stator contacts. Subunit dynamics were examined in conformer ensembles generated by molecular simulations from the X-ray structures. Principal component analysis extracted collective motions. Interfacial loop immobilization by complex formation coupled elastic fluctuations of the FliM middle (FliMM) and FliG middle (FliGM) domains. Coevolved mutations captured interfacial dynamics as well as contacts. FliGM rotation was amplified via two central hinges to the FliGC helix. Intrinsic flexibility, reported by the FliGMC ensembles, reconciled conformers with opposite FliGC helix orientations. FliG domain stacking deformed the inter-domain linker and reduced flexibility; but conformational changes were not triggered by engineered linker deletions that cause a rotation-locked phenotype. These facts suggest that binary rotation states arise from conformational selection by stacking interactions.

  2. Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

    PubMed

    Baker, Matthew A B; Hynson, Robert M G; Ganuelas, Lorraine A; Mohammadi, Nasim Shah; Liew, Chu Wai; Rey, Anthony A; Duff, Anthony P; Whitten, Andrew E; Jeffries, Cy M; Delalez, Nicolas J; Morimoto, Yusuke V; Stock, Daniela; Armitage, Judith P; Turberfield, Andrew J; Namba, Keiichi; Berry, Richard M; Lee, Lawrence K

    2016-03-01

    Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings. PMID:26854663

  3. Populational Heterogeneity vs. Temporal Fluctuation in Escherichia coli Flagellar Motor Switching

    PubMed Central

    Bai, Fan; Che, Yong-Suk; Kami-ike, Nobunori; Ma, Qi; Minamino, Tohru; Sowa, Yoshiyuki; Namba, Keiichi

    2013-01-01

    The dynamic switching of the bacterial flagellar motor regulates cell motility in bacterial chemotaxis. It has been reported under physiological conditions that the switching bias of the flagellar motor undergoes large temporal fluctuations, which reflects noise propagating in the chemotactic signaling network. On the other hand, nongenetic heterogeneity is also observed in flagellar motor switching, as a large group of switching motors show different switching bias and frequency under the same physiological condition. In this work, we present simultaneous measurement of groups of Escherichia coli flagellar motor switching and compare them to long time recording of single switching motors. Consistent with previous studies, we observed temporal fluctuations in switching bias in long time recording experiments. However, the variability in switching bias at the populational level showed much higher volatility than its temporal fluctuation. These results suggested stable individuality in E. coli motor switching. We speculate that uneven expression of key regulatory proteins with amplification by the ultrasensitive response of the motor can account for the observed populational heterogeneity and temporal fluctuations. PMID:24209857

  4. Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

    PubMed

    Paulick, Anja; Delalez, Nicolas J; Brenzinger, Susanne; Steel, Bradley C; Berry, Richard M; Armitage, Judith P; Thormann, Kai M

    2015-06-01

    The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels.

  5. Bacterial Flagellar Motor Switch in Response to CheY-P Regulation and Motor Structural Alterations.

    PubMed

    Ma, Qi; Sowa, Yoshiyuki; Baker, Matthew A B; Bai, Fan

    2016-03-29

    The bacterial flagellar motor (BFM) is a molecular machine that rotates the helical filaments and propels the bacteria swimming toward favorable conditions. In our previous works, we built a stochastic conformational spread model to explain the dynamic and cooperative behavior of BFM switching. Here, we extended this model to test whether it can explain the latest experimental observations regarding CheY-P regulation and motor structural adaptivity. We show that our model predicts a strong correlation between rotational direction and the number of CheY-Ps bound to the switch complex, in agreement with the latest finding from Fukuoka et al. It also predicts that the switching sensitivity of the BFM can be fine-tuned by incorporating additional units into the switch complex, as recently demonstrated by Yuan et al., who showed that stoichiometry of FliM undergoes dynamic change to maintain ultrasensitivity in the motor switching response. In addition, by locking some rotor switching units on the switch complex into the stable clockwise-only conformation, our model has accurately simulated recent experiments expressing clockwise-locked FliG(ΔPAA) into the switch complex and reproduced the increased switching rate of the motor.

  6. Bio-Hybrid Micro/Nanodevices Powered by Flagellar Motor: Challenges and Strategies

    PubMed Central

    Kim, Jin-Woo; Tung, Steve

    2015-01-01

    Molecular motors, which are precision engineered by nature, offer exciting possibilities for bio-hybrid engineered systems. They could enable real applications ranging from micro/nano fluidics, to biosensing, to medical diagnoses. This review describes the fundamental biological insights and fascinating potentials of these remarkable sensing and actuation machines, in particular, bacterial flagellar motors, as well as their engineering perspectives with regard to applications in bio-engineered hybrid systems. PMID:26284237

  7. A novel type bacterial flagellar motor that can use divalent cations as a coupling ion

    PubMed Central

    Imazawa, Riku; Takahashi, Yuka; Aoki, Wataru; Sano, Motohiko; Ito, Masahiro

    2016-01-01

    The bacterial flagellar motor is a sophisticated nanomachine embedded in the cell envelope and powered by an electrochemical gradient of H+, Na+, or K+across the cytoplasmic membrane. Here we describe a new member of the bacterial flagellar stator channel family (MotAB1 of Paenibacillus sp. TCA20 (TCA-MotAB1)) that is coupled to divalent cations (Ca2+and Mg2+). In the absence of divalent cations of alkaline earth metals, no swimming was observed in Paenibacillus sp. TCA20, which grows optimally in Ca2+-rich environments. This pattern was confirmed by swimming assays of a stator-free Bacillus subtilis mutant expressing TCA-MotAB1. Both a stator-free and major Mg2+uptake system-deleted B. subtilis mutant expressing TCA-MotAB1 complemented both growth and motility deficiency under low Mg2+conditions and exhibited [Mg2+]in identical to that of the wild-type. This is the first report of a flagellar motor that can use Ca2+and Mg2+as coupling ions. These findings will promote the understanding of the operating principles of flagellar motors and molecular mechanisms of ion selectivity. PMID:26794857

  8. How Biophysics May Help Us Understand the Flagellar Motor of Bacteria Which Cause Infections.

    PubMed

    Baker, Matthew A B

    2016-01-01

    Motor proteins are molecules which convert chemical energy to mechanical work and are responsible for motility across all levels: for transport within a cell, for the motion of an individual cell in its surroundings, and for movement in multicellular aggregates, such as muscles. The bacterial flagellar motor is one of the canonical examples of a molecular complex made from several motor proteins, which self-assembles on demand and provides the locomotive force for bacteria. This locomotion provides a key aspect of bacteria's prevalence. Here, we outline the biophysics behind the assembly, the energetics, the switching and the rotation of this remarkable nanoscale electric motor that is Nature's first wheel.

  9. How Biophysics May Help Us Understand the Flagellar Motor of Bacteria Which Cause Infections.

    PubMed

    Baker, Matthew A B

    2016-01-01

    Motor proteins are molecules which convert chemical energy to mechanical work and are responsible for motility across all levels: for transport within a cell, for the motion of an individual cell in its surroundings, and for movement in multicellular aggregates, such as muscles. The bacterial flagellar motor is one of the canonical examples of a molecular complex made from several motor proteins, which self-assembles on demand and provides the locomotive force for bacteria. This locomotion provides a key aspect of bacteria's prevalence. Here, we outline the biophysics behind the assembly, the energetics, the switching and the rotation of this remarkable nanoscale electric motor that is Nature's first wheel. PMID:27193546

  10. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.

    PubMed

    Beeby, Morgan; Ribardo, Deborah A; Brennan, Caitlin A; Ruby, Edward G; Jensen, Grant J; Hendrixson, David R

    2016-03-29

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. PMID:26976588

  11. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

    PubMed Central

    Ribardo, Deborah A.; Brennan, Caitlin A.; Ruby, Edward G.; Jensen, Grant J.; Hendrixson, David R.

    2016-01-01

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. PMID:26976588

  12. Structure and Function of the Bi-Directional Bacterial Flagellar Motor

    PubMed Central

    Morimoto, Yusuke V.; Minamino, Tohru

    2014-01-01

    The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor–stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor. PMID:24970213

  13. Modeling Torque Versus Speed, Shot Noise, and Rotational Diffusion of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Mora, Thierry; Yu, Howard; Wingreen, Ned S.

    2009-12-01

    We present a minimal physical model for the flagellar motor that enables bacteria to swim. Our model explains the experimentally measured torque-speed relationship of the proton-driven E. coli motor at various pH and temperature conditions. In particular, the dramatic drop of torque at high rotation speeds (the “knee”) is shown to arise from saturation of the proton flux. Moreover, we show that shot noise in the proton current dominates the diffusion of motor rotation at low loads. This suggests a new way to probe the discreteness of the energy source, analogous to measurements of charge quantization in superconducting tunnel junctions.

  14. Rotational speed control of Na +-driven flagellar motor by dual pipettes.

    PubMed

    Nogawa, Kousuke; Kojima, Masaru; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2009-12-01

    Single cell analysis has attracted much attention to reveal the detailed and localized biological information. Local environmental control technique is desired to analyze the detailed and localized properties of single cells. In this paper, we propose the local environmental control system with nano/micro dual pipettes to control the local reagent concentration dynamically and arbitrarily. Local environmental control by dual pipettes is applied to the rotational speed control of bacterial flagellar motor, which is a rotary molecular machine. We demonstrate quick response and iterative rotational speed control of Na (+)-driven flagellar motor in both accelerating and relaxing directions by switching the local spout between Na (+)-containing and Na (+) -free solutions with dual pipettes. It is shown that the rotational speed might be controllable by changing the spouting velocity of Na (+)-containing solution with multiplying the applied dc voltage. PMID:19887330

  15. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching.

    PubMed

    Lee, Lawrence K; Ginsburg, Michael A; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

    2010-08-19

    The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation. PMID:20676082

  16. Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching

    SciTech Connect

    Lee, Lawrence K.; Ginsburg, Michael A.; Crovace, Claudia; Donohoe, Mhairi; Stock, Daniela

    2010-09-13

    The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.

  17. Torque generated by the flagellar motor of Escherichia coli.

    PubMed Central

    Berg, H C; Turner, L

    1993-01-01

    Cells of the bacterium Escherichia coli were tethered and spun in a high-frequency rotating electric field at a series of discrete field strengths. This was done first at low field strengths, then at field strengths generating speeds high enough to disrupt motor function, and finally at low field strengths. Comparison of the initial and final speed versus applied-torque plots yielded relative motor torque. For backward rotation, motor torque rose steeply at speeds close to zero, peaking, on average, at about 2.2 times the stall torque. For forward rotation, motor torque remained approximately constant up to speeds of about 60% of the zero-torque speed. Then the torque dropped linearly with speed, crossed zero, and reached a minimum, on average, at about -1.7 times the stall torque. The zero-torque speed increased with temperature (about 90 Hz at 11 degrees C, 140 Hz at 16 degrees C, and 290 Hz at 23 degrees C), while other parameters remained approximately constant. Sometimes the motor slipped at either extreme (delivered constant torque over a range of speeds), but eventually it broke. Similar results were obtained whether motors broke catastrophically (suddenly and completely) or progressively or were de-energized by brief treatment with an uncoupler. These results are consistent with a tightly coupled ratchet mechanism, provided that elastic deformation of force-generating elements is limited by a stop and that mechanical components yield at high applied torques. PMID:8298044

  18. A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

    PubMed Central

    Pilizota, Teuta; Brown, Mostyn T.; Leake, Mark C.; Branch, Richard W.; Berry, Richard M.; Armitage, Judith P.

    2009-01-01

    Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides—using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27–28 discrete angles, locked in place by a relatively high torque, approximately 2–3 times its stall torque. PMID:19571004

  19. The effect of flagellar motor-rotor complexes on twitching motility in P. aeruginosa

    NASA Astrophysics Data System (ADS)

    Zhao, Kun; Utada, Andrew; Gibiansky, Maxsim; Xian, Wujing; Wong, Gerard

    2013-03-01

    P. aeruginosa is an opportunistic bacterium responsible for a broad range of biofilm infections. In order for biofilms to form, P. aeruginosa uses different types of surface motility. In the current understanding, flagella are used for swarming motility and type IV pili are used for twitching motility. The flagellum also plays important roles in initial surface attachment and in shaping the architectures of mature biofilms. Here we examine how flagella and pili interact during surface motility, by using cell tracking techniques. We show that the pili driven twitching motility of P. aeruginosa can be affected by the motor-rotor complexes of the flagellar system.

  20. Electrostatic interactions between rotor and stator in the bacterial flagellar motor

    PubMed Central

    Zhou, Jiadong; Lloyd, Scott A.; Blair, David F.

    1998-01-01

    Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor–stator interface. PMID:9600984

  1. Electrostatic interactions between rotor and stator in the bacterial flagellar motor.

    PubMed

    Zhou, J; Lloyd, S A; Blair, D F

    1998-05-26

    Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface. PMID:9600984

  2. Electrostatic Interactions between Rotor and Stator in the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Zhou, Jiadong; Lloyd, Scott A.; Blair, David F.

    1998-05-01

    Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.

  3. Coupling between Switching Regulation and Torque Generation in Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Bai, Fan; Minamino, Tohru; Wu, Zhanghan; Namba, Keiichi; Xing, Jianhua

    2012-04-01

    The bacterial flagellar motor plays a crucial role in both bacterial locomotion and chemotaxis. Recent experiments reveal that the switching dynamics of the motor depend on the rotation speed of the motor, and thus the motor torque, nonmonotonically. Here we present a unified mathematical model which treats motor torque generation based on experimental torque-speed curves and the torque-dependent switching based on the conformational spread model. The model successfully reproduces the observed switching rate as a function of the rotation speed, and provides a generic physical explanation independent of most details. A stator affects the switching dynamics through two mechanisms: accelerating the conformational flipping rate of individual rotor-switching units, which contributes most when the stator works at a high torque and thus a low speed; and influencing a larger number of rotor-switching units within unit time, whose contribution is the greatest when the motor rotates at a high speed. Consequently, the switching rate shows a maximum at intermediate speed, where the above two mechanisms find an optimal output. The load-switching relation may serve as a mechanism for sensing the physical environment, similar to the chemotaxis mechanism for sensing the chemical environment. It may also coordinate the switch dynamics of motors within the same cell.

  4. Mechanism and kinetics of a sodium-driven bacterial flagellar motor

    PubMed Central

    Lo, Chien-Jung; Sowa, Yoshiyuki; Pilizota, Teuta; Berry, Richard M.

    2013-01-01

    The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque–speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque–speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a “powerstroke” in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration. PMID:23788659

  5. The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backward

    PubMed Central

    Lele, Pushkar P.; Roland, Thibault; Shrivastava, Abhishek; Chen, Yihao; Berg, Howard C.

    2016-01-01

    Caulobacter crescentus, a monotrichous bacterium, swims by rotating a single right-handed helical filament. CW motor rotation thrusts the cell forward 1, a mode of motility known as the pusher mode; CCW motor rotation pulls the cell backward, a mode of motility referred to as the puller mode 2. The situation is opposite in E. coli, a peritrichous bacterium, where CCW rotation of multiple left-handed filaments drives the cell forward. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells 3,4. However, monotrichous bacteria including C. crescentus swim forward and backward at similar speeds, prompting the assumption that motor torques in the two modes are the same 5,6. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque-generation is similar in two species, despite the differences in filament handedness and motor bias (probability of CW rotation). PMID:27499800

  6. Switching of Bacterial Flagellar Motors Is Triggered by Mutant FliG

    PubMed Central

    Lele, Pushkar P.; Berg, Howard C.

    2015-01-01

    Binding of the chemotaxis response regulator CheY-P promotes switching between rotational states in flagellar motors of the bacterium Escherichia coli. Here, we induced switching in the absence of CheY-P by introducing copies of a mutant FliG locked in the clockwise (CW) conformation (FliGCW). The composition of the mixed FliG ring was estimated via fluorescence imaging, and the probability of CW rotation (CWbias) was determined from the rotation of tethered cells. The results were interpreted in the framework of a 1D Ising model. The data could be fit by assuming that mutant subunits are more stable in the CW conformation than in the counterclockwise conformation. We found that CWbias varies depending on the spatial arrangement of the assembled subunits in the FliG ring. This offers a possible explanation for a previous observation of hysteresis in the switch function in analogous mixed FliM motors—in motors containing identical fractions of mutant FliMCW in otherwise wild-type motors, the CWbias differed depending on whether mutant subunits were expressed in strains with native motors or native subunits were expressed in strains with mutant motors. PMID:25762339

  7. The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards

    NASA Astrophysics Data System (ADS)

    Lele, Pushkar P.; Roland, Thibault; Shrivastava, Abhishek; Chen, Yihao; Berg, Howard C.

    2016-02-01

    The bacterium Caulobacter crescentus swims by rotating a single right-handed helical filament. These cells have two swimming modes: a pusher mode, in which clockwise (CW) rotation of the filament thrusts the cell body forwards, and a puller mode, in which counterclockwise (CCW) rotation pulls it backwards. The situation is reversed in Escherichia coli, a bacterium that rotates several left-handed filaments CCW to drive the cell body forwards. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells. However, C. crescentus and other bacteria with single filaments swim forwards and backwards at similar speeds, prompting the assumption that motor torques in the two modes are the same. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque generation is similar in the two species, despite the differences in filament handedness and motor bias.

  8. Fluctuations in rotation rate of the flagellar motor of Escherichia coli.

    PubMed Central

    Kara-Ivanov, M; Eisenbach, M; Caplan, S R

    1995-01-01

    The purpose of this work was to study the changes in rotation rate of the bacterial motor and to try to discriminate between various sources of these changes with the aim of understanding the mechanism of force generation better. To this end Escherichia coli cells were tethered and videotaped with brief stroboscopic light flashes. The records were scanned by means of a computerized motion analysis system, yielding cell size, radius of rotation, and accumulated angle of rotation as functions of time for each cell selected. In conformity with previous studies, fluctuations in the rotation rate of the flagellar motor were invariably found. Employing an exclusively counterclockwise rotating mutant ("gutted" RP1091 strain) and using power spectral density, autocorrelation and residual mean square angle analysis, we found that a simple superposition of rotational diffusion on a steady rotary motion is insufficient to describe the observed rotation. We observed two additional rotational components, one fluctuating (0.04-0.6 s) and one oscillating (0.8-7 s). However, the effective rotational diffusion coefficient obtained after taking these two components into account generally exceeded that calculated from external friction by two orders of magnitude. This is consistent with a model incorporating association and dissociation of force-generating units. PMID:7669902

  9. New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas

    PubMed Central

    2013-01-01

    Background The building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein. Methods We used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis. We examined the effect of these mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC). Results The strains, fla18 and fla24, have mutations in kinesin-2 and cytoplasmic dynein, respectively. The fla18 mutation alters the same glutamic acid (E24G) mutated in the fla10-14 allele (E24K). The fla18 strain loses flagella at 32?C more rapidly than the E24K allele but less rapidly than the fla10-1 allele. The fla18 mutant loses its flagella by detachment rather than by shortening. The fla24 mutation falls in cytoplasmic dynein and changes a completely conserved amino acid (L3243P) in an alpha helix in the AAA5 domain. The fla24 mutant loses its flagella by shortening within 6 hours at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at 21?C compared to wild-type cells. We identified two pseudorevertants (L3243S and L3243R), which remain flagellated at 32?C. Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein localization is dramatically altered. Instead of localizing at the basal body and along the flagella, IFT81 is concentrated at the proximal end of the flagella. The pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end localization of IFT81 at 32?C. Conclusions The change in the AAA5 domain of the cytoplasmic dynein in fla24 may block the recycling of IFT

  10. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor

    PubMed Central

    Pandini, Alessandro; Kleinjung, Jens; Rasool, Shafqat; Khan, Shahid

    2015-01-01

    Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) “torque” helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could

  11. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

    PubMed

    Pandini, Alessandro; Kleinjung, Jens; Rasool, Shafqat; Khan, Shahid

    2015-01-01

    Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be

  12. Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load.

    PubMed

    Lo, Chien-Jung; Leake, Mark C; Pilizota, Teuta; Berry, Richard M

    2007-07-01

    Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.

  13. Nonequivalence of Membrane Voltage and Ion-Gradient as Driving Forces for the Bacterial Flagellar Motor at Low Load

    PubMed Central

    Lo, Chien-Jung; Leake, Mark C.; Pilizota, Teuta; Berry, Richard M.

    2007-01-01

    Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (Vm) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure Vm in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated Vm using the ratio of intracellular/extracellular dye concentration. We found Vm = −140 ± 14 mV in Escherichia coli at external pH 7.0 (pHex), decreasing to −85 ± 10 mV at pHex 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of Vm and intracellular sodium concentration. We were able to vary the SMF between −187 ± 15 mV and −53 ± 15 mV by varying pHex in the range 7.0–5.0 and extracellular sodium concentration in the range 1–85 mM. Rotation rates for 0.35-μm- and 1-μm-diameter beads attached to Na+-driven chimeric flagellar motors varied linearly with Vm. For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration. PMID:17416615

  14. Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

    PubMed Central

    Nishihara, Yasutaka; Kitao, Akio

    2015-01-01

    The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H3O+ permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor. PMID:26056313

  15. Effect of the MotB(D33N) mutation on stator assembly and rotation of the proton-driven bacterial flagellar motor.

    PubMed

    Nakamura, Shuichi; Minamino, Tohru; Kami-Ike, Nobunori; Kudo, Seishi; Namba, Keiichi

    2014-01-01

    The bacterial flagellar motor generates torque by converting the energy of proton translocation through the transmembrane proton channel of the stator complex formed by MotA and MotB. The MotA/B complex is thought to be anchored to the peptidoglycan (PG) layer through the PG-binding domain of MotB to act as the stator. The stator units dynamically associate with and dissociate from the motor during flagellar motor rotation, and an electrostatic interaction between MotA and a rotor protein FliG is required for efficient stator assembly. However, the association and dissociation mechanism of the stator units still remains unclear. In this study, we analyzed the speed fluctuation of the flagellar motor of Salmonella enterica wild-type cells carrying a plasmid encoding a nonfunctional stator complex, MotA/B(D33N), which lost the proton conductivity. The wild-type motor rotated stably but the motor speed fluctuated considerably when the expression level of MotA/B(D33N) was relatively high compared to MotA/B. Rapid accelerations and decelerations were frequently observed. A quantitative analysis of the speed fluctuation and a model simulation suggested that the MotA/B(D33N) stator retains the ability to associate with the motor at a low affinity but dissociates more rapidly than the MotA/B stator. We propose that the stator dissociation process depends on proton translocation through the proton channel. PMID:27493496

  16. Function of the Histone-Like Protein H-NS in Motility of Escherichia coli: Multiple Regulatory Roles Rather than Direct Action at the Flagellar Motor

    PubMed Central

    Kim, Eun A

    2015-01-01

    ABSTRACT A number of investigations of Escherichia coli have suggested that the DNA-binding protein H-NS, in addition to its well-known functions in chromosome organization and gene regulation, interacts directly with the flagellar motor to modulate its function. Here, in a study initially aimed at characterizing the H-NS/motor interaction further, we identify problems and limitations in the previous work that substantially weaken the case for a direct H-NS/motor interaction. Null hns mutants are immotile, largely owing to the downregulation of the flagellar master regulators FlhD and FlhC. We, and others, previously reported that an hns mutant remains poorly motile even when FlhDC are expressed constitutively. In the present work, we use better-engineered strains to show that the motility defect in a Δhns, FlhDC-constitutive strain is milder than that reported previously and does not point to a direct action of H-NS at the motor. H-NS regulates numerous genes and might influence motility via a number of regulatory molecules besides FlhDC. To examine the sources of the motility defect that persists in an FlhDC-constitutive Δhns mutant, we measured transcript levels and overexpression effects of a number of genes in candidate regulatory pathways. The results indicate that H-NS influences motility via multiple regulatory linkages that include, minimally, the messenger molecule cyclic di-GMP, the biofilm regulatory protein CsgD, and the sigma factors σS and σF. The results are in accordance with the more standard view of H-NS as a regulator of gene expression rather than a direct modulator of flagellar motor performance. IMPORTANCE Data from a number of previous studies have been taken to indicate that the nucleoid-organizing protein H-NS influences motility not only by its well-known DNA-based mechanisms but also by binding directly to the flagellar motor to alter function. In this study, H-NS is shown to influence motility through diverse regulatory pathways

  17. Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I.

    PubMed Central

    McCain, D A; Amici, L A; Spudich, J L

    1987-01-01

    Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II). PMID:3654583

  18. Defects in the Flagellar Motor Increase Synthesis of Poly-γ-Glutamate in Bacillus subtilis

    PubMed Central

    Chan, Jia Mun; Guttenplan, Sarah B.

    2014-01-01

    Bacillus subtilis swims in liquid media and swarms over solid surfaces, and it encodes two sets of flagellar stator homologs. Here, we show that B. subtilis requires only the MotA/MotB stator during swarming motility and that the residues required for stator force generation are highly conserved from the Proteobacteria to the Firmicutes. We further find that mutants that abolish stator function also result in an overproduction of the extracellular polymer poly-γ-glutamate (PGA) to confer a mucoid colony phenotype. PGA overproduction appeared to be the result of an increase in the expression of the pgs operon that encodes genes for PGA synthesis. Transposon mutagenesis was conducted to identify insertions that abolished colony mucoidy and disruptions in known transcriptional regulators of PGA synthesis (Com and Deg two-component systems) as well as mutants defective in transcription-coupled DNA repair (Mfd)-reduced expression of the pgs operon. A final class of insertions disrupted proteins involved in the assembly of the flagellar filament (FliD, FliT, and FlgL), and these mutants did not reduce expression of the pgs operon, suggesting a second mechanism of PGA control. PMID:24296669

  19. Helicobacter pylori CheZ(HP) and ChePep form a novel chemotaxis-regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor.

    PubMed

    Lertsethtakarn, Paphavee; Howitt, Michael R; Castellon, Juan; Amieva, Manuel R; Ottemann, Karen M

    2015-09-01

    Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ(HP), localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ(HP) localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ(HP) for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ(HP) determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ(HP) and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities.

  20. Helicobacter pylori CheZHP and ChePep form a novel chemotaxis-regulatory complex distinct from the core chemotaxis signaling proteins and the flagellar motor

    PubMed Central

    Lertsethtakarn, Paphavee; Howitt, Michael R.; Castellon, Juan; Amieva, Manuel R.; Ottemann, Karen M.

    2015-01-01

    Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZHP, localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZHP localization depends on the chemotaxis regulatory protein ChePep and reciprocally, ChePep requires CheZHP for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZHP determined the polar localization motif lies within the central domain of the protein, and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZHP and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins’ activities. PMID:26061894

  1. Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts

    PubMed Central

    Sultan, Syed Z.; Sekar, Padmapriya; Zhao, Xiaowei; Manne, Akarsh; Liu, Jun; Wooten, R. Mark

    2015-01-01

    Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts. PMID:25690096

  2. Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor.

    PubMed

    Morimoto, Yusuke V; Nakamura, Shuichi; Kami-ike, Nobunori; Namba, Keiichi; Minamino, Tohru

    2010-12-01

    MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotA(C) ), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotA(C) with FliG is required for the efficient assembly of the stators around the rotor. PMID:21091499

  3. Contribution of many charged residues at the stator-rotor interface of the Na+-driven flagellar motor to torque generation in Vibrio alginolyticus.

    PubMed

    Takekawa, Norihiro; Kojima, Seiji; Homma, Michio

    2014-04-01

    In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na(+)-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na(+)-driven motor than in the MotA-FliG interactions in the H(+)-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na(+)-driven motor seem to be more complex than those previously proposed in the H(+)-driven motor. PMID:24464458

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

    PubMed

    Morimoto, Yusuke V; Nakamura, Shuichi; Hiraoka, Koichi D; Namba, Keiichi; Minamino, Tohru

    2013-02-01

    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. PMID:23161029

  5. Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics.

    PubMed

    Morse, Michael; Bell, Jordan; Li, Guanglai; Tang, Jay X

    2015-11-01

    A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli.

  6. Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics

    NASA Astrophysics Data System (ADS)

    Morse, Michael; Bell, Jordan; Li, Guanglai; Tang, Jay X.

    2015-11-01

    A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli.

  7. Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics.

    PubMed

    Morse, Michael; Bell, Jordan; Li, Guanglai; Tang, Jay X

    2015-11-01

    A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli. PMID:26588419

  8. Serine 26 in the PomB Subunit of the Flagellar Motor Is Essential for Hypermotility of Vibrio cholerae

    PubMed Central

    Halang, Petra; Vorburger, Thomas; Steuber, Julia

    2015-01-01

    Vibrio cholerae is motile by means of its single polar flagellum which is driven by the sodium-motive force. In the motor driving rotation of the flagellar filament, a stator complex consisting of subunits PomA and PomB converts the electrochemical sodium ion gradient into torque. Charged or polar residues within the membrane part of PomB could act as ligands for Na+, or stabilize a hydrogen bond network by interacting with water within the putative channel between PomA and PomB. By analyzing a large data set of individual tracks of swimming cells, we show that S26 located within the transmembrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB at pH 7.0, but fast swimming was restored by decreasing the H+ concentration of the external medium. Our study identifies S26 as a second important residue besides D23 in the PomB channel. It is proposed that S26, together with D23 located in close proximity, is important to perturb the hydration shell of Na+ before its passage through a constriction within the stator channel. PMID:25874792

  9. The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium

    PubMed Central

    Takekawa, Norihiro; Terahara, Naoya; Kato, Takayuki; Gohara, Mizuki; Mayanagi, Kouta; Hijikata, Atsushi; Onoue, Yasuhiro; Kojima, Seiji; Shirai, Tsuyoshi; Namba, Keiichi; Homma, Michio

    2016-01-01

    Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator. PMID:27531865

  10. Torque and switching in the bacterial flagellar motor. An electrostatic model.

    PubMed Central

    Berry, R M

    1993-01-01

    A model is presented for the rotary motor that drives bacterial flagella, using the electrochemical gradient of protons across the cytoplasmic membrane. The model unifies several concepts present in previous models. Torque is generated by proton-conducting particles around the perimeter of the rotor at the base of the flagellum. Protons in channels formed by these particles interact electrostatically with tilted lines of charges on the rotor, providing "loose coupling" between proton flux and rotation of the flagellum. Computer simulations of the model correctly predict the experimentally observed dynamic properties of the motor. Unlike previous models, the motor presented here may rotate either way for a given direction of the protonmotive force. The direction of rotation only depends on the level of occupancy of the proton channels. This suggests a novel and simple mechanism for the switching between clockwise and counterclockwise rotation that is the basis of bacterial chemotaxis. Images FIGURE 2 PMID:7684268

  11. Torque and switching in the bacterial flagellar motor. An electrostatic model.

    PubMed

    Berry, R M

    1993-04-01

    A model is presented for the rotary motor that drives bacterial flagella, using the electrochemical gradient of protons across the cytoplasmic membrane. The model unifies several concepts present in previous models. Torque is generated by proton-conducting particles around the perimeter of the rotor at the base of the flagellum. Protons in channels formed by these particles interact electrostatically with tilted lines of charges on the rotor, providing "loose coupling" between proton flux and rotation of the flagellum. Computer simulations of the model correctly predict the experimentally observed dynamic properties of the motor. Unlike previous models, the motor presented here may rotate either way for a given direction of the protonmotive force. The direction of rotation only depends on the level of occupancy of the proton channels. This suggests a novel and simple mechanism for the switching between clockwise and counterclockwise rotation that is the basis of bacterial chemotaxis. PMID:7684268

  12. Observed frequency-independent torque in flagellar bacterial motors optimizes space exploration.

    PubMed

    Di Salvo, Mario E; Condat, C A

    2012-12-01

    A surprising feature of many bacterial motors is the apparently conserved form of their torque-frequency relation. Experiments indicate that the torque provided by the bacterial rotary motor is approximately constant over a large range of angular speeds. This is observed in both monotrichous and peritrichous bacteria, independently of whether they are propelled by a proton flux or by a Na(+) ion flux. If the relation between angular speed ω and swimming speed is linear, a ω-independent torque implies that the power spent in active motion is proportional to the instantaneous bacterial speed. Using realistic values of the relevant parameters, we show that a constant torque maximizes the volume of the region explored by a bacterium in a resource-depleted medium. Given that nutrients in the ocean are often concentrated in separate, ephemeral patches, we propose that the observed constancy of the torque may be a trait evolved to maximize bacterial survival in the ocean.

  13. Dynamism and regulation of the stator, the energy conversion complex of the bacterial flagellar motor.

    PubMed

    Kojima, Seiji

    2015-12-01

    Many motile bacteria swim by rotating their motility organ, the flagellum. Rotation of the flagellum is driven by a motor at its base, and torque is generated by the rotor-stator interaction coupled with the specific ion flow through the channel in the stator. Because the stator works as an energy-conversion complex in the motor, understanding the functional mechanism of the stator is critically important. But its characterization has been hampered due to the difficulty in isolating the functional stator complex from the membrane. Recently, successful new approaches for studying the stator have been reported that reveal its novel properties. Two of those, visualization of the in vivo behavior of stator units using fluorescently tagged proteins and structure-guided functional analyses of the soluble region in the stator, are summarized in this short review. PMID:26457925

  14. Internal and external components of the bacterial flagellar motor rotate as a unit.

    PubMed

    Hosu, Basarab G; Nathan, Vedavalli S J; Berg, Howard C

    2016-04-26

    Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook.

  15. Internal and external components of the bacterial flagellar motor rotate as a unit

    PubMed Central

    Hosu, Basarab G.; Nathan, Vedavalli S. J.; Berg, Howard C.

    2016-01-01

    Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook. PMID:27071081

  16. An electrostatic mechanism closely reproducing observed behavior in the bacterial flagellar motor.

    PubMed

    Walz, D; Caplan, S R

    2000-02-01

    A mechanism coupling the transmembrane flow of protons to the rotation of the bacterial flagellum is studied. The coupling is accomplished by means of an array of tilted rows of positive and negative charges around the circumference of the rotor, which interacts with a linear array of proton binding sites in channels. We present a rigorous treatment of the electrostatic interactions using minimal assumptions. Interactions with the transition states are included, as well as proton-proton interactions in and between channels. In assigning values to the parameters of the model, experimentally determined structural characteristics of the motor have been used. According to the model, switching and pausing occur as a consequence of modest conformational changes in the rotor. In contrast to similar approaches developed earlier, this model closely reproduces a large number of experimental findings from different laboratories, including the nonlinear behavior of the torque-frequency relation in Escherichia coli, the stoichiometry of the system in Streptococcus, and the pH-dependence of swimming speed in Bacillus subtilis. PMID:10653777

  17. An electrostatic mechanism closely reproducing observed behavior in the bacterial flagellar motor.

    PubMed Central

    Walz, D; Caplan, S R

    2000-01-01

    A mechanism coupling the transmembrane flow of protons to the rotation of the bacterial flagellum is studied. The coupling is accomplished by means of an array of tilted rows of positive and negative charges around the circumference of the rotor, which interacts with a linear array of proton binding sites in channels. We present a rigorous treatment of the electrostatic interactions using minimal assumptions. Interactions with the transition states are included, as well as proton-proton interactions in and between channels. In assigning values to the parameters of the model, experimentally determined structural characteristics of the motor have been used. According to the model, switching and pausing occur as a consequence of modest conformational changes in the rotor. In contrast to similar approaches developed earlier, this model closely reproduces a large number of experimental findings from different laboratories, including the nonlinear behavior of the torque-frequency relation in Escherichia coli, the stoichiometry of the system in Streptococcus, and the pH-dependence of swimming speed in Bacillus subtilis. PMID:10653777

  18. Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA.

    PubMed

    Takekawa, Norihiro; Terauchi, Takashi; Morimoto, Yusuke V; Minamino, Tohru; Lo, Chien-Jung; Kojima, Seiji; Homma, Michio

    2013-05-01

    PomA and PomB form the stator complex, which functions as a Na(+) channel, in the Na(+)-driven flagellar motor of Vibrio alginolyticus. The plug region of PomB is thought to regulate the Na(+) flow and to suppress massive ion influx through the stator channel. In this study, in order to measure the Na(+) conductivity of the unplugged stator, we over-produced a plug-deleted stator of the Na(+)-driven flagellar motor in Escherichia coli. The over-production of the plug-deleted stator in E. coli cells caused more severe growth inhibition than in Vibrio cells and that growth inhibition depended on the Na(+) concentration in the growth medium. Measurement of intracellular Na(+) concentration by flame photometry and fluorescent analysis with a Na(+) indicator, Sodium Green, revealed that over-production of the plug-deleted stator increased the Na(+) concentration in cell. Some mutations in the channel region of PomB or in the cytoplasmic region of PomA suppressed both the growth inhibition and the increase in intracellular Na(+) concentration. These results suggest that the level of growth inhibition correlates with the intracellular Na(+) concentration, probably due to the Na(+) conductivity through the stator due to the mutations. PMID:23420849

  19. Mutations alter the sodium versus proton use of a Bacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors.

    PubMed

    Terahara, Naoya; Krulwich, Terry A; Ito, Masahiro

    2008-09-23

    Bacterial flagella contain membrane-embedded stators, Mot complexes, that harness the energy of either transmembrane proton or sodium ion gradients to power motility. Use of sodium ion gradients is associated with elevated pH and sodium concentrations. The Mot complexes studied to date contain channels that use either protons or sodium ions, with some bacteria having only one type and others having two distinct Mot types with different ion-coupling. Here, alkaliphilic Bacillus clausii KSM-K16 was shown to be motile in a pH range from 7 to 11 although its genome encodes only one Mot (BCl-MotAB). Assays of swimming as a function of pH, sodium concentration, and ion-selective motility inhibitors showed that BCl-MotAB couples motility to sodium at the high end of its pH range but uses protons at lower pH. This pattern was confirmed in swimming assays of a statorless Bacillus subtilis mutant expressing either BCl-MotAB or one of the two B. subtilis stators, sodium-coupled Bs-MotPS or proton-coupled Bs-MotAB. Pairs of mutations in BCl-MotB were identified that converted the naturally bifunctional BCl-MotAB to stators that preferentially use either protons or sodium ions across the full pH range. We then identified trios of mutations that added a capacity for dual-ion coupling on the distinct B. subtilis Bs-MotAB and Bs-MotPS motors. Determinants that alter the specificity of bifunctional and single-coupled flagellar stators add to insights from studies of other ion-translocating transporters that use both protons and sodium ions. PMID:18796609

  20. Corticospinal activity evoked and modulated by non-invasive stimulation of the intact human motor cortex.

    PubMed

    Di Lazzaro, Vincenzo; Rothwell, John C

    2014-10-01

    A number of methods have been developed recently that stimulate the human brain non-invasively through the intact scalp. The most common are transcranial magnetic stimulation (TMS), transcranial electric stimulation (TES) and transcranial direct current stimulation (TDCS). They are widely used to probe function and connectivity of brain areas as well as therapeutically in a variety of conditions such as depression or stroke. They are much less focal than conventional invasive methods which use small electrodes placed on or in the brain and are often thought to activate all classes of neurones in the stimulated area. However, this is not true. A large body of evidence from experiments on the motor cortex shows that non-invasive methods of brain stimulation can be surprisingly selective and that adjusting the intensity and direction of stimulation can activate different classes of inhibitory and excitatory inputs to the corticospinal output cells. Here we review data that have elucidated the action of TMS and TES, concentrating mainly on the most direct evidence available from spinal epidural recordings of the descending corticospinal volleys. The results show that it is potentially possible to test and condition specific neural circuits in motor cortex that could be affected differentially by disease, or be used in different forms of natural behaviour. However, there is substantial interindividual variability in the specificity of these protocols. Perhaps in the future it will be possible, with the advances currently being made to model the electrical fields induced in individual brains, to develop forms of stimulation that can reliably target more specific populations of neurones, and open up the internal circuitry of the motor cortex for study in behaving humans. PMID:25172954

  1. Reconstitution of flagellar sliding.

    PubMed

    Alper, Joshua; Geyer, Veikko; Mukundan, Vikram; Howard, Jonathon

    2013-01-01

    The motile structure within eukaryotic cilia and flagella is the axoneme. This structure typically consists of nine doublet microtubules arranged around a pair of singlet microtubules. The axoneme contains more than 650 different proteins that have structural, force-generating, and regulatory functions. Early studies on sea urchin sperm identified the force-generating components, the dynein motors. It was shown that dynein can slide adjacent doublet microtubules in the presence of ATP. How this sliding gives rise to the beating of the axoneme is still unknown. Reconstitution assays provide a clean system, free from cellular effects, to elucidate the underlying beating mechanisms. These assays can be used to identify the components that are both necessary and sufficient for the generation of flagellar beating. PMID:23498749

  2. Reconstitution of flagellar sliding.

    PubMed

    Alper, Joshua; Geyer, Veikko; Mukundan, Vikram; Howard, Jonathon

    2013-01-01

    The motile structure within eukaryotic cilia and flagella is the axoneme. This structure typically consists of nine doublet microtubules arranged around a pair of singlet microtubules. The axoneme contains more than 650 different proteins that have structural, force-generating, and regulatory functions. Early studies on sea urchin sperm identified the force-generating components, the dynein motors. It was shown that dynein can slide adjacent doublet microtubules in the presence of ATP. How this sliding gives rise to the beating of the axoneme is still unknown. Reconstitution assays provide a clean system, free from cellular effects, to elucidate the underlying beating mechanisms. These assays can be used to identify the components that are both necessary and sufficient for the generation of flagellar beating.

  3. The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation

    PubMed Central

    DiBella, Linda M.; Sakato, Miho; Patel-King, Ramila S.; Pazour, Gregory J.; King, Stephen M.

    2004-01-01

    Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins. PMID:15304520

  4. Combining d-cycloserine with motor training does not result in improved general motor learning in neurologically intact people or in people with stroke.

    PubMed

    Cherry, Kendra M; Lenze, Eric J; Lang, Catherine E

    2014-06-15

    Neurological rehabilitation involving motor training has resulted in clinically meaningful improvements in function but is unable to eliminate many of the impairments associated with neurological injury. Thus there is a growing need for interventions that facilitate motor learning during rehabilitation therapy, to optimize recovery. d-Cycloserine (DCS), a partial N-methyl-d-aspartate (NMDA) receptor agonist that enhances neurotransmission throughout the central nervous system (Ressler KJ, Rothbaum BO, Tannenbaum L, Anderson P, Graap K, Zimand E, Hodges L, Davis M. Arch Gen Psychiatry 61: 1136-1144, 2004), has been shown to facilitate declarative and emotional learning. We therefore tested whether combining DCS with motor training facilitates motor learning after stroke in a series of two experiments. Forty-one healthy adults participated in experiment I, and twenty adults with stroke participated in experiment II of this two-session, double-blind study. Session one consisted of baseline assessment, subject randomization, and oral administration of DCS or placebo (250 mg). Subjects then participated in training on a balancing task, a simulated feeding task, and a cognitive task. Subjects returned 1-3 days later for posttest assessment. We found that all subjects had improved performance from pretest to posttest on the balancing task, the simulated feeding task, and the cognitive task. Subjects who were given DCS before motor training, however, did not show enhanced learning on the balancing task, the simulated feeding task, or the associative recognition task compared with subjects given placebo. Moreover, training on the balancing task did not generalize to a similar, untrained balance task. Our findings suggest that DCS does not enhance motor learning or motor skill generalization in neurologically intact adults or in adults with stroke.

  5. Combining d-cycloserine with motor training does not result in improved general motor learning in neurologically intact people or in people with stroke

    PubMed Central

    Cherry, Kendra M.; Lenze, Eric J.

    2014-01-01

    Neurological rehabilitation involving motor training has resulted in clinically meaningful improvements in function but is unable to eliminate many of the impairments associated with neurological injury. Thus there is a growing need for interventions that facilitate motor learning during rehabilitation therapy, to optimize recovery. d-Cycloserine (DCS), a partial N-methyl-d-aspartate (NMDA) receptor agonist that enhances neurotransmission throughout the central nervous system (Ressler KJ, Rothbaum BO, Tannenbaum L, Anderson P, Graap K, Zimand E, Hodges L, Davis M. Arch Gen Psychiatry 61: 1136–1144, 2004), has been shown to facilitate declarative and emotional learning. We therefore tested whether combining DCS with motor training facilitates motor learning after stroke in a series of two experiments. Forty-one healthy adults participated in experiment I, and twenty adults with stroke participated in experiment II of this two-session, double-blind study. Session one consisted of baseline assessment, subject randomization, and oral administration of DCS or placebo (250 mg). Subjects then participated in training on a balancing task, a simulated feeding task, and a cognitive task. Subjects returned 1–3 days later for posttest assessment. We found that all subjects had improved performance from pretest to posttest on the balancing task, the simulated feeding task, and the cognitive task. Subjects who were given DCS before motor training, however, did not show enhanced learning on the balancing task, the simulated feeding task, or the associative recognition task compared with subjects given placebo. Moreover, training on the balancing task did not generalize to a similar, untrained balance task. Our findings suggest that DCS does not enhance motor learning or motor skill generalization in neurologically intact adults or in adults with stroke. PMID:24671538

  6. Synergistic plasticity of intrinsic conductance and electrical coupling restores synchrony in an intact motor network.

    PubMed

    Lane, Brian J; Samarth, Pranit; Ransdell, Joseph L; Nair, Satish S; Schulz, David J

    2016-01-01

    Motor neurons of the crustacean cardiac ganglion generate virtually identical, synchronized output despite the fact that each neuron uses distinct conductance magnitudes. As a result of this variability, manipulations that target ionic conductances have distinct effects on neurons within the same ganglion, disrupting synchronized motor neuron output that is necessary for proper cardiac function. We hypothesized that robustness in network output is accomplished via plasticity that counters such destabilizing influences. By blocking high-threshold K(+) conductances in motor neurons within the ongoing cardiac network, we discovered that compensation both resynchronized the network and helped restore excitability. Using model findings to guide experimentation, we determined that compensatory increases of both GA and electrical coupling restored function in the network. This is one of the first direct demonstrations of the physiological regulation of coupling conductance in a compensatory context, and of synergistic plasticity across cell- and network-level mechanisms in the restoration of output. PMID:27552052

  7. Flagellar waveform analysis of swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, Huseyin; Johnson, Karl; Gollub, Jerry

    2011-11-01

    The twin flagella of the green alga Chlamydomas reinhardtii are driven by dynein molecular motors to oscillate at about 50-60 Hz in a breaststroke motion. For decades, Chlamydomas has been used as a model organism for studies of flagellar motility, and of genetic disorders of ciliary motion. However, little is known experimentally about the flagellar waveforms, and the resulting time-dependent force distribution along the 250 nm diameter flagella. Here, we study flagellar dynamics experimentally by confining cells in quasi-2D liquid films. From simultaneous measurements of the cell body velocity and the time-dependent velocities along the center lines of the two flagella, we determine the drag coefficients, and estimate the power expended by the body and the flagella, comparing our findings with measurements based on the induced fluid flow field. We contrast the results for the quite different beating patterns of synchronous and asynchronous flagella, respectively. Supported by NSF Grant DMR-0803153.

  8. Synergistic plasticity of intrinsic conductance and electrical coupling restores synchrony in an intact motor network

    PubMed Central

    Lane, Brian J; Samarth, Pranit; Ransdell, Joseph L; Nair, Satish S; Schulz, David J

    2016-01-01

    Motor neurons of the crustacean cardiac ganglion generate virtually identical, synchronized output despite the fact that each neuron uses distinct conductance magnitudes. As a result of this variability, manipulations that target ionic conductances have distinct effects on neurons within the same ganglion, disrupting synchronized motor neuron output that is necessary for proper cardiac function. We hypothesized that robustness in network output is accomplished via plasticity that counters such destabilizing influences. By blocking high-threshold K+ conductances in motor neurons within the ongoing cardiac network, we discovered that compensation both resynchronized the network and helped restore excitability. Using model findings to guide experimentation, we determined that compensatory increases of both GA and electrical coupling restored function in the network. This is one of the first direct demonstrations of the physiological regulation of coupling conductance in a compensatory context, and of synergistic plasticity across cell- and network-level mechanisms in the restoration of output. DOI: http://dx.doi.org/10.7554/eLife.16879.001 PMID:27552052

  9. Intact Acquisition and Short-Term Retention of Non-Motor Procedural Learning in Parkinson's Disease.

    PubMed

    Panouillères, Muriel T N; Tofaris, George K; Brown, Peter; Jenkinson, Ned

    2016-01-01

    Procedural learning is a form of memory where people implicitly acquire a skill through repeated practice. People with Parkinson's disease (PD) have been found to acquire motor adaptation, a form of motor procedural learning, similarly to healthy older adults but they have deficits in long-term retention. A similar pattern of normal learning on initial exposure with a deficit in retention seen on subsequent days has also been seen in mirror-reading, a form of non-motor procedural learning. It is a well-studied fact that disrupting sleep will impair the consolidation of procedural memories. Given the prevalence of sleep disturbances in PD, the lack of retention on following days seen in these studies could simply be a side effect of this well-known symptom of PD. Because of this, we wondered whether people with PD would present with deficits in the short-term retention of a non-motor procedural learning task, when the test of retention was done the same day as the initial exposure. The aim of the present study was then to investigate acquisition and retention in the immediate short term of cognitive procedural learning using the mirror-reading task in people with PD. This task involved two conditions: one where triads of mirror-inverted words were always new that allowed assessing the learning of mirror-reading skill and another one where some of the triads were presented repeatedly during the experiment that allowed assessing the word-specific learning. People with PD both ON and OFF their normal medication were compared to healthy older adults and young adults. Participants were re-tested 50 minutes break after initial exposure to probe for short-term retention. The results of this study show that all groups of participants acquired and retained the two skills (mirror-reading and word-specific) similarly. These results suggest that neither healthy ageing nor the degeneration within the basal ganglia that occurs in PD does affect the mechanisms that underpin the

  10. Simultaneous measurement of bacterial flagellar rotation rate and swimming speed.

    PubMed Central

    Magariyama, Y; Sugiyama, S; Muramoto, K; Kawagishi, I; Imae, Y; Kudo, S

    1995-01-01

    Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C. A roughly linear relation between swimming speed and flagellar rotation rate was observed. The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation. At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate. That is, the cell with a faster-rotating flagellum did not always swim faster. To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered. The model could be analytically solved, and it qualitatively explained the experimental results. The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20%. The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force. Images FIGURE 1 FIGURE 4 PMID:8580359

  11. Motor learning in children with spina bifida: intact learning and performance on a ballistic task.

    PubMed

    Dennis, Maureen; Jewell, Derryn; Edelstein, Kim; Brandt, Michael E; Hetherington, Ross; Blaser, Susan E; Fletcher, Jack M

    2006-09-01

    Learning and performance on a ballistic task were investigated in children with spina bifida meningomyelocele (SBM), with either upper level spinal lesions (n = 21) or lower level spinal lesions (n = 81), and in typically developing controls (n = 35). Participants completed three phases (20 trials each) of an elbow goniometer task that required a ballistic arm movement to move a cursor to one of two target positions on a screen, including (1) an initial learning phase, (2) an adaptation phase with a gain change such that recalibration of the ballistic arm movement was required, and (3) a learning reactivation phase under the original gain condition. Initial error rate, asymptotic error rate, and learning rate did not differ significantly between the SBM and control groups. Relative to controls, the SBM group had reduced volumes in the cerebellar hemispheres and pericallosal gray matter (the region including the basal ganglia), although only the pericallosal gray matter was significantly correlated with motor adaptation. Congenital cerebellar dysmorphology is associated with preserved motor skill learning on voluntary, nonreflexive tasks in children with SBM, in whom the relative roles of the cerebellum and basal ganglia may differ from those in the adult brain.

  12. Undiscovered regions on the molecular landscape of flagellar assembly.

    PubMed

    Altegoer, Florian; Bange, Gert

    2015-12-01

    The bacterial flagellum is a motility structure and one of the most complicated motors in the biosphere. A flagellum consists of several dozens of building blocks in different stoichiometries and extends from the cytoplasm to the extracellular space. Flagellar biogenesis follows a strict spatio-temporal regime that is guided by a plethora of flagellar assembly factors and chaperones. The goal of this review is to summarize our current structural and mechanistic knowledge of this intricate process and to identify the undiscovered regions on the molecular landscape of flagellar assembly. PMID:26490009

  13. Motor recovery after stroke depends on intact sustained attention: a 2-year follow-up study.

    PubMed

    Robertson, I H; Ridgeway, V; Greenfield, E; Parr, A

    1997-04-01

    The functional recovery of 47 right-brain-damaged stroke patients was studied over a 2-year period. The researchers hypothesized that sustained attention capacity should predict the degree of motor and functional recovery over this period because of a proposed privileged role of sustained attention in learning-based recovery of function. As predicted, significant correlations were found between sustained attention capacity at 2 months and functional status (including the Barthel Index) at 2 years. This relationship was shown to exist independently of 2-month functional status. Furthermore, compared with a left-brain-damaged group of cerebrovascular accident (CVA) patients, the right-brain CVA group did not recover functional ability as well over the 2-year period. This increasing difference in functional status over a 2-year period was mirrored by an emerging difference in sustained attention capacity, in favor of the left-brain CVA group.

  14. Active Phase and Amplitude Fluctuations of Flagellar Beating

    NASA Astrophysics Data System (ADS)

    Ma, Rui; Klindt, Gary S.; Riedel-Kruse, Ingmar H.; Jülicher, Frank; Friedrich, Benjamin M.

    2014-07-01

    The eukaryotic flagellum beats periodically, driven by the oscillatory dynamics of molecular motors, to propel cells and pump fluids. Small but perceivable fluctuations in the beat of individual flagella have physiological implications for synchronization in collections of flagella as well as for hydrodynamic interactions between flagellated swimmers. Here, we characterize phase and amplitude fluctuations of flagellar bending waves using shape mode analysis and limit-cycle reconstruction. We report a quality factor of flagellar oscillations Q =38.0±16.7 (mean±s.e.). Our analysis shows that flagellar fluctuations are dominantly of active origin. Using a minimal model of collective motor oscillations, we demonstrate how the stochastic dynamics of individual motors can give rise to active small-number fluctuations in motor-cytoskeleton systems.

  15. Active phase and amplitude fluctuations of flagellar beating.

    PubMed

    Ma, Rui; Klindt, Gary S; Riedel-Kruse, Ingmar H; Jülicher, Frank; Friedrich, Benjamin M

    2014-07-25

    The eukaryotic flagellum beats periodically, driven by the oscillatory dynamics of molecular motors, to propel cells and pump fluids. Small but perceivable fluctuations in the beat of individual flagella have physiological implications for synchronization in collections of flagella as well as for hydrodynamic interactions between flagellated swimmers. Here, we characterize phase and amplitude fluctuations of flagellar bending waves using shape mode analysis and limit-cycle reconstruction. We report a quality factor of flagellar oscillations Q = 38.0 ± 16.7 (mean ± s.e.). Our analysis shows that flagellar fluctuations are dominantly of active origin. Using a minimal model of collective motor oscillations, we demonstrate how the stochastic dynamics of individual motors can give rise to active small-number fluctuations in motor-cytoskeleton systems.

  16. Regulation of Eukaryotic Flagellar Motility

    NASA Astrophysics Data System (ADS)

    Mitchell, David R.

    2005-03-01

    The central apparatus is essential for normal eukaryotic flagellar bend propagation as evidenced by the paralysis associated with mutations that prevent central pair (CP) assembly. Interactions between doublet-associated radial spokes and CP projections are thought to modulate spoke-regulated protein kinases and phosphatases on outer doublets, and these enzymes in turn modulate dynein activity. To better understand CP control mechanisms, we determined the three-dimensional structure of the Chlamydomonas reinhardtii CP complex and analyzed CP orientation during formation and propagation of flagellar bending waves. We show that a single CP microtubule, C1, is near the outermost doublet in curved regions of the flagellum, and this orientation is maintained by twists between successive principal and reverse bends. The Chlamydomonas CP is inherently twisted; twists are not induced by bend formation, and do not depend on forces or signals transmitted through spoke-central pair interactions. We hypothesize that CP orientation passively responds to bend formation, and that bend propagation drives rotation of the CP and maintains a constant CP orientation in bends, which in turn permits signal transduction between specific CP projections and specific doublet-associated dyneins through radial spokes. The central pair kinesin, Klp1, although essential for normal motility, is therefore not the motor that drives CP rotation. The CP also acts as a scaffold for enzymes that maintain normal intraflagellar ATP concentration.

  17. Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides, a bacterium with a unidirectional, stop-start flagellum.

    PubMed

    Shah, D S; Sockett, R E

    1995-09-01

    Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation stops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli, a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli. MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB. Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a sigma 54-type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of the R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.

  18. Studies on the mechanism of bacterial flagellar rotation and the flagellar number regulation.

    PubMed

    Kojima, Seiji

    2016-01-01

    Many motile bacteria have the motility organ, the flagellum. It rotates by the rotary motor driven by the ion-motive force and is embedded in the cell surface at the base of each flagellar filament. Many researchers have been studying its rotary mechanism for years, but most of the energy conversion processes have been remained in mystery. We focused on the flagellar stator, which works at the core process of energy conversion, and found that the periplasmic region of the stator changes its conformation to be activated only when the stator units are incorporated into the motor and anchored at the cell wall. Meanwhile, the physiologically important supramolecular complex is localized in the cell at the right place and the right time with a proper amount. How the cell achieves such a proper localization is the fundamental question for life science, and we undertake this problem by analyzing the mechanism for biogenesis of a single polar flagellum of Vibrio alginolyticus. Here I describe the molecular mechanism of how the flagellum is generated at the specific place with a proper number, and also how the flagellar stator is incorporated into the motor to complete the functional motor assembly, based on our studies. PMID:27581279

  19. The flagellar cytoskeleton of the spirochetes.

    PubMed

    Wolgemuth, Charles W; Charon, Nyles W; Goldstein, Stuart F; Goldstein, Raymond E

    2006-01-01

    The recent discoveries of prokaryotic homologs of all three major eukaryotic cytoskeletal proteins (actin, tubulin, intermediate filaments) have spurred a resurgence of activity in the field of bacterial morphology. In spirochetes, however, it has long been known that the flagellar filaments act as a cytoskeletal protein structure, contributing to their shape and conferring motility on this unique phylum of bacteria. Therefore, revisiting the spirochete cytoskeleton may lead to new paradigms for exploring general features of prokaryotic morphology. This review discusses the role that the periplasmic flagella in spirochetes play in maintaining shape and producing motility. We focus on four species of spirochetes: Borrelia burgdorferi, Treponema denticola, Treponema phagedenis and Leptonema (formerly Leptospira) illini. In spirochetes, the flagella reside in the periplasmic space. Rotation of the flagella in the above species by a flagellar motor induces changes in the cell morphology that drives motility. Mutants that do not produce flagella have a markedly different shape than wild-type cells. PMID:16983197

  20. Flagellar membranes are rich in raft-forming phospholipids

    PubMed Central

    Serricchio, Mauro; Schmid, Adrien W.; Steinmann, Michael E.; Sigel, Erwin; Rauch, Monika; Julkowska, Daria; Bonnefoy, Serge; Fort, Cécile; Bastin, Philippe; Bütikofer, Peter

    2015-01-01

    ABSTRACT The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei. PMID:26276100

  1. Crystallization and preliminary X-ray analysis of MotY, a stator component of the Vibrio alginolyticus polar flagellar motor.

    PubMed

    Shinohara, Akari; Sakuma, Mayuko; Yakushi, Toshiharu; Kojima, Seiji; Namba, Keiichi; Homma, Michio; Imada, Katsumi

    2007-02-01

    The polar flagellum of Vibrio alginolyticus is rotated by the sodium motor. The stator unit of the sodium motor consists of four different proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium motor, form the T-ring structure attached to the LP ring in the periplasmic space. MotY has a putative peptidoglycan-binding motif in its C-terminal region and MotX is suggested to interact with PomB. Thus, MotX and MotY are thought to be required for incorporation and stabilization of the PomA/B complex. In this study, mature MotY composed of 272 amino-acid residues and its SeMet derivative were expressed with a C-terminal hexahistidine-tag sequence, purified and crystallized. Native crystals were grown in the hexagonal space group P6(1)22/P6(5)22, with unit-cell parameters a = b = 104.1, c = 132.6 A. SeMet-derivative crystals belonged to the same space group with the same unit-cell parameters as the native crystals. Anomalous difference Patterson maps of the SeMet derivative showed significant peaks in their Harker sections, indicating that the derivatives are suitable for structure determination.

  2. FLAGELLAR REGENERATION IN PROTOZOAN FLAGELLATES

    PubMed Central

    Rosenbaum, Joel L.; Child, F. M.

    1967-01-01

    The flagella of populations of three protozoan species (Ochromonas, Euglena, and Astasia) were amputated and allowed to regenerate. The kinetics of regeneration in all species were characterized by a lag phase during which there was no apparent flagellar elongation; this phase was followed by elongation at a rate which constantly decelerated as the original length was regained. Inhibition by cycloheximide applied at the time of flagellar amputation showed that flagellar regeneration was dependent upon de novo protein synthesis. This was supported by evidence showing that a greater amount of leucine was incorporated into the proteins of regenerating than nonregenerating flagella. The degree of inhibition of flagellar elongation observed with cycloheximide depended on how soon after flagellar amputation it was applied: when applied to cells immediately following amputation, elongation was almost completely inhibited, but its application at various times thereafter permitted considerable elongation to occur prior to complete inhibition of flagellar elongation. Hence, a sufficient number of precursors were synthesized and accumulated prior to addition of cycloheximide so that their assembly (elongation) could occur for a time under conditions in which protein synthesis had been inhibited. Evidence that the site of this assembly may be at the tip of the elongating flagellum was obtained from radioautographic studies in which the flagella of Ochromonas were permitted to regenerate part way in the absence of labeled leucine and to complete their regeneration in the presence of the isotope. Possible mechanisms which may be operating to control flagellar regeneration are discussed in light of these and other observations. PMID:6033540

  3. Second-Chance Signal Transduction Explains Cooperative Flagellar Switching

    PubMed Central

    Zot, Henry G.; Hasbun, Javier E.; Van Minh, Nguyen

    2012-01-01

    The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating stationary unit, or stator (motor unit). To explain the steeply cooperative ligand-induced switching, present models propose allosteric interactions between subunits of the rotor, but do not address the possibility of a reaction that stimulates a bidirectional motor unit to reverse direction of torque. During flagellar motion, the binding of a ligand-bound switch complex at the dwell site could excite a motor unit. The probability that another switch complex of the rotor, moving according to steady-state rotation, will reach the same dwell site before that motor unit returns to ground state will be determined by the independent decay rate of the excited-state motor unit. Here, we derive an analytical expression for the energy coupling between a switch complex and a motor unit of the stator complex of a flagellum, and demonstrate that this model accounts for the cooperative switching response without the need for allosteric interactions. The analytical result can be reproduced by simulation when (1) the motion of the rotor delivers a subsequent ligand-bound switch to the excited motor unit, thereby providing the excited motor unit with a second chance to remain excited, and (2) the outputs from multiple independent motor units are constrained to a single all-or-none event. In this proposed model, a motor unit and switch complex represent the components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and is regulated by stochastic ligand binding. Mathematical derivation of the model shows the analytical function to be a general form of the Hill equation (Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv–vii). PMID:22844429

  4. Effects of the dynein inhibitor ciliobrevin on the flagellar motility of sea urchin spermatozoa.

    PubMed

    Wada, Yuuko; Baba, Shoji A; Kamimura, Shinji

    2015-04-01

    Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 μM of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin.

  5. Intact Acquisition and Short-Term Retention of Non-Motor Procedural Learning in Parkinson’s Disease

    PubMed Central

    Panouillères, Muriel T. N.; Tofaris, George K.; Brown, Peter; Jenkinson, Ned

    2016-01-01

    Procedural learning is a form of memory where people implicitly acquire a skill through repeated practice. People with Parkinson’s disease (PD) have been found to acquire motor adaptation, a form of motor procedural learning, similarly to healthy older adults but they have deficits in long-term retention. A similar pattern of normal learning on initial exposure with a deficit in retention seen on subsequent days has also been seen in mirror-reading, a form of non-motor procedural learning. It is a well-studied fact that disrupting sleep will impair the consolidation of procedural memories. Given the prevalence of sleep disturbances in PD, the lack of retention on following days seen in these studies could simply be a side effect of this well-known symptom of PD. Because of this, we wondered whether people with PD would present with deficits in the short-term retention of a non-motor procedural learning task, when the test of retention was done the same day as the initial exposure. The aim of the present study was then to investigate acquisition and retention in the immediate short term of cognitive procedural learning using the mirror-reading task in people with PD. This task involved two conditions: one where triads of mirror-inverted words were always new that allowed assessing the learning of mirror-reading skill and another one where some of the triads were presented repeatedly during the experiment that allowed assessing the word-specific learning. People with PD both ON and OFF their normal medication were compared to healthy older adults and young adults. Participants were re-tested 50 minutes break after initial exposure to probe for short-term retention. The results of this study show that all groups of participants acquired and retained the two skills (mirror-reading and word-specific) similarly. These results suggest that neither healthy ageing nor the degeneration within the basal ganglia that occurs in PD does affect the mechanisms that underpin the

  6. Intact neurogenesis is required for benefits of exercise on spatial memory but not motor performance or contextual fear conditioning in C57BL/6J mice.

    PubMed

    Clark, P J; Brzezinska, W J; Thomas, M W; Ryzhenko, N A; Toshkov, S A; Rhodes, J S

    2008-09-01

    The mammalian hippocampus continues to generate new neurons throughout life. Experiences such as exercise, anti-depressants, and stress regulate levels of neurogenesis. Exercise increases adult hippocampal neurogenesis and enhances behavioral performance on rotarod, contextual fear and water maze in rodents. To directly test whether intact neurogenesis is required for gains in behavioral performance from exercise in C57BL/6J mice, neurogenesis was reduced using focal gamma irradiation (3 sessions of 5 Gy). Two months after treatment, mice (total n=42 males and 42 females) (Irradiated or Sham), were placed with or without running wheels (Runner or Sedentary) for 54 days. The first 10 days mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. The last 14 days mice were tested on water maze (two trials per day for 5 days, then 1 h later probe test), rotarod (four trials per day for 3 days), and contextual fear conditioning (2 days), then measured for neurogenesis using immunohistochemical detection of BrdU and neuronal nuclear protein (NeuN) mature neuronal marker. Consistent with previous studies, in Sham animals, running increased neurogenesis fourfold and gains in performance were observed for the water maze (spatial learning and memory), rotarod (motor performance), and contextual fear (conditioning). These positive results provided the reference to determine whether gains in performance were blocked by irradiation. Irradiation reduced neurogenesis by 50% in both groups, Runner and Sedentary. Irradiation did not affect running or baseline performance on any task. Minimal changes in microglia associated with inflammation (using immunohistochemical detection of cd68) were detected at the time of behavioral testing. Irradiation did not reduce gains in performance on rotarod or contextual fear, however it eliminated gain in performance on the water maze. Results support the hypothesis that intact exercise-induced hippocampal neurogenesis

  7. Analysis of flagellar protein ubiquitination.

    PubMed

    Long, Huan; Huang, Kaiyao

    2013-01-01

    Flagella/cilia are conserved organelles existing in unicellular protists and multicellular animals, where they perform essential motile and sensory functions. Their assembly and disassembly are coordinated with the cell cycle, and recent evidence shows that posttranslational modifications such as phosphorylation, methylation, and ubiquitination are involved in these two processes, perhaps through interacting with intraflagellar transport (IFT), a specialized intracellular transport that is required for the assembly and maintenance of flagella/cilia. In this chapter, we summarize the components of the ubiquitination system published in proteomic databases of flagella/cilia. Furthermore, we describe procedures to analyze the ubiquitin-conjugating system in Chlamydomonas flagella and to analyze flagellar protein ubiquitination during flagellar shortening and the mating process. These results and tools will be valuable for the characterization of substrates of ubiquitination and their roles in flagellar disassembly and in regulating signal transduction pathways in flagella/cilia.

  8. Flagellar apparatus structure of choanoflagellates.

    PubMed

    Karpov, Sergey A

    2016-01-01

    Phylum choanoflagellata is the nearest unicellular neighbor of metazoa at the phylogenetic tree. They are single celled or form the colonies, can be presented by naked cells or live in theca or lorica, but in all cases they have a flagellum surrounded by microvilli of the collar. They have rather uniform and peculiar flagellar apparatus structure with flagellar basal body (FB) producing a flagellum, and non-flagellar basal body (NFB) lying orthogonal to the FB. Long flagellar transition zone contains a unique structure among eukaryotes, the central filament, which connects central microtubules to the transversal plate. Both basal bodies are composed of triplets and interconnected with fibrillar bridge. They also contain the internal arc-shaped connectives between the triplets. The FB has prominent transitional fibers similar to those of chytrid zoospores and choanocytes of sponges, and a radial microtubular root system. The ring-shaped microtubule organizing center (MTOC) produces radial root microtubules, but in some species a MTOC is represented by separate foci. The NFB has a narrow fibrillar root directed towards the Golgi apparatus in association with membrane-bounded sac. Prior to cell division, the basal bodies replicate and migrate to poles of elongated nucleus. The basal bodies serve as MTOCs for the spindle microtubules during nuclear division by semiopen orthomitosis. PMID:27148446

  9. Flagellar kinesins in protists.

    PubMed

    Marande, William; Kohl, Linda

    2011-02-01

    Cilia and flagella are organelles of the cell body present in many eukaryotic cells. Although their basic structure is well conserved from unicellular organisms to mammals, they show amazing diversity in number, structure, molecular composition, disposition and function. These complex organelles are generally assembled by the action of intraflagellar transport, which is powered by kinesin and dynein motor proteins. Several types of kinesins can function in flagella. They all have a well-conserved motor domain with characteristic signatures, but display exhaustive diversification of some domains. This diversity can be explained by the multitude of functions fulfilled by these proteins (transport of cargoes along microtubules, polymerization and depolymerization of microtubules). Functional and phylogenetic analyses reveal that at least seven kinesin families are involved in flagellum assembly and function. In protists, where cilia and flagella fulfill many essential roles, this diversity of function is also observed. PMID:21366422

  10. Flagellar kinesins in protists.

    PubMed

    Marande, William; Kohl, Linda

    2011-02-01

    Cilia and flagella are organelles of the cell body present in many eukaryotic cells. Although their basic structure is well conserved from unicellular organisms to mammals, they show amazing diversity in number, structure, molecular composition, disposition and function. These complex organelles are generally assembled by the action of intraflagellar transport, which is powered by kinesin and dynein motor proteins. Several types of kinesins can function in flagella. They all have a well-conserved motor domain with characteristic signatures, but display exhaustive diversification of some domains. This diversity can be explained by the multitude of functions fulfilled by these proteins (transport of cargoes along microtubules, polymerization and depolymerization of microtubules). Functional and phylogenetic analyses reveal that at least seven kinesin families are involved in flagellum assembly and function. In protists, where cilia and flagella fulfill many essential roles, this diversity of function is also observed.

  11. Flagellar membrane proteins in kinetoplastid parasites.

    PubMed

    Landfear, Scott M; Tran, Khoa D; Sanchez, Marco A

    2015-09-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

  12. Flagellar membrane proteins in kinetoplastid parasites.

    PubMed

    Landfear, Scott M; Tran, Khoa D; Sanchez, Marco A

    2015-09-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted. PMID:26599841

  13. Flagellar Membrane Proteins in Kinetoplastid Parasites

    PubMed Central

    Landfear, Scott M.; Tran, Khoa D.; Sanchez, Marco A.

    2015-01-01

    All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. While flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted. PMID:26599841

  14. Successive incorporation of force-generating units in the bacterial rotary motor

    NASA Astrophysics Data System (ADS)

    Block, Steven M.; Berg, Howard C.

    1984-05-01

    Mot mutants of Escherichia coli are paralysed: their flagella appear to be intact but do not rotate1 . The motA and motB gene products are found in the cytoplasmic membrane2; they do not co-purify with flagellar basal bodies isolated in neutral detergents1. Silverman et al. found that mot mutants could be `resurrected' through protein synthesis directed by λ transducing phages carrying the wild-type genes2. Here, we have studied this activation at the level of a single flagellar motor. Cells of a motB strain carrying plasmids in which transcription of the wild-type motB gene was controlled by the lac promoter were tethered to a glass surface by a single flagellum. These cells began to spin within several minutes after the addition of a lac inducer, and their rotational speed changed in a series of equally spaced steps. As many as 7 steps were seen in individual cells and, from the final speeds attained, as many as 16 steps could be inferred. These experiments show that each flagellar motor contains several independent force-generating units comprised, at least in part, of motB protein.

  15. Flagellar root contraction and nuclear movement during flagellar regeneration in Chlamydomonas reinhardtii

    PubMed Central

    1987-01-01

    When Chlamydomonas cells are deflagellated by pH shock or mechanical shear the nucleus rapidly moves toward the flagellar basal apparatus at the anterior end of the cell. During flagellar regeneration the nucleus returns to a more central position within the cell. The nucleus is connected to the flagellar apparatus by a system of fibers, the flagellar roots (rhizoplasts), which undergo a dramatic contraction that coincides with anterior nuclear movement. A corresponding extension of the root system, back to its preshock configuration is observed as the nucleus retracts to a central position. Anterior displacement of the nucleus and flagellar root contraction require free calcium in the medium. Nuclear movement and flagellar root contraction and extension are not sensitive to inhibitors of protein synthesis (cycloheximide), or drugs that influence either microtubules (colchicine) or actin-based microfilaments (cytochalasin D). Detergent- extracted cell models contract and extend their flagellar roots and move their nuclei in response to alterations of free calcium levels in the medium. Cycles of nuclear movement in detergent-extracted models require ATP to potentiate the contractile mechanism for subsequent calcium-induced contraction. Flagellar root contraction and nuclear movement in Chlamydomonas may be causally related to signaling of induction of flagellar precursor genes or to the transport of flagellar precursors or their messages to sites of synthesis or assembly near the basal apparatus of the cell. PMID:3667698

  16. Flagellar flows around bacterial swarms

    NASA Astrophysics Data System (ADS)

    Dauparas, Justas; Lauga, Eric

    2016-08-01

    Flagellated bacteria on nutrient-rich substrates can differentiate into a swarming state and move in dense swarms across surfaces. A recent experiment measured the flow in the fluid around an Escherichia coli swarm [Wu, Hosu, and Berg, Proc. Natl. Acad. Sci. USA 108, 4147 (2011)], 10.1073/pnas.1016693108. A systematic chiral flow was observed in the clockwise direction (when viewed from above) ahead of the swarm with flow speeds of about 10 μ m /s , about 3 times greater than the radial velocity at the edge of the swarm. The working hypothesis is that this flow is due to the action of cells stalled at the edge of a colony that extend their flagellar filaments outward, moving fluid over the virgin agar. In this work we quantitatively test this hypothesis. We first build an analytical model of the flow induced by a single flagellum in a thin film and then use the model, and its extension to multiple flagella, to compare with experimental measurements. The results we obtain are in agreement with the flagellar hypothesis. The model provides further quantitative insight into the flagella orientations and their spatial distributions as well as the tangential speed profile. In particular, the model suggests that flagella are on average pointing radially out of the swarm and are not wrapped tangentially.

  17. Rhythmicity, Recurrence, and Recovery of Flagellar Beating

    NASA Astrophysics Data System (ADS)

    Wan, Kirsty Y.; Goldstein, Raymond E.

    2014-12-01

    The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the alga C. reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating.

  18. Rhythmicity, recurrence, and recovery of flagellar beating.

    PubMed

    Wan, Kirsty Y; Goldstein, Raymond E

    2014-12-01

    The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the alga C. reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating.

  19. Structure of the microtubule-binding domain of flagellar dynein.

    PubMed

    Kato, Yusuke S; Yagi, Toshiki; Harris, Sarah A; Ohki, Shin-ya; Yura, Kei; Shimizu, Youské; Honda, Shinya; Kamiya, Ritsu; Burgess, Stan A; Tanokura, Masaru

    2014-11-01

    Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

  20. Bacterial Flagellar Microhydrodynamics: Laminar Flow over Complex Flagellar Filaments, Analog Archimedean Screws and Cylinders, and Its Perturbations

    PubMed Central

    Trachtenberg, Shlomo; Fishelov, Dalia; Ben-Artzi, Matania

    2003-01-01

    The flagellar filament, the bacterial organelle of motility, is the smallest rotary propeller known. It consists of 1), a basal body (part of which is the proton driven rotary motor), 2), a hook (universal joint—allowing for off-axial transmission of rotary motion), and 3), a filament (propeller—a long, rigid, supercoiled helical assembly allowing for the conversion of rotary motion into linear thrust). Helically perturbed (so-called “complex”) filaments have a coarse surface composed of deep grooves and ridges following the three-start helical lines. These surface structures, reminiscent of a turbine or Archimedean screw, originate from symmetry reduction along the six-start helical lines due to dimerization of the flagellin monomers from which the filament self assembles. Using high-resolution electron microscopy and helical image reconstruction methods, we calculated three-dimensional density maps of the complex filament of Rhizobium lupini H13-3 and determined its surface pattern and boundaries. The helical symmetry of the filament allows viewing it as a stack of identical slices spaced axially and rotated by constant increments. Here we use the closed outlines of these slices to explore, in two dimensions, the hydrodynamic effect of the turbine-like boundaries of the flagellar filament. In particular, we try to determine if, and under what conditions, transitions from laminar to turbulent flow (or perturbations of the laminar flow) may occur on or near the surface of the bacterial propeller. To address these questions, we apply the boundary element method in a manner allowing the handling of convoluted boundaries. We tested the method on several simple, well-characterized cylindrical structures before applying it to real, highly convoluted biological surfaces and to simplified mechanical analogs. Our results indicate that under extreme structural and functional conditions, and at low Reynolds numbers, a deviation from laminar flow might occur on the

  1. Functional state of the axonemal dyneins during flagellar bend propagation.

    PubMed Central

    Woolley, D M; Vernon, G G

    2002-01-01

    When mouse spermatozoa swim in media of high viscosity, additional waves of bending are superimposed on the primary traveling wave. The additional (secondary) waves are relatively small in scale and high in frequency. They originate in the proximal part of the interbend regions. The initiation of secondary bending happens only in distal parts of the flagellum. The secondary waves propagate along the interbends and then tend to die out as they encounter the next-most-distal bend of the primary wave, if that bend exceeds a certain angle. The principal bends of the primary wave, being of greater angle than the reverse bends, strongly resist invasion by the secondary waves; when a principal bend of the primary wave propagates off the flagellar tip, the secondary wave behind it suddenly increases in amplitude. We claim that the functional state of the dynein motors in relation to the primary wave can be deduced from their availability for recruitment into secondary wave activity. Therefore, only the dyneins in bends are committed functionally to the maintenance and propagation of the flagellar wave; dyneins in interbend regions are not functionally committed in this way. We equate functional commitment with tension-generating activity, although we argue that the regions of dynein thus engaged nevertheless permit sliding displacements between the doublets. PMID:12324433

  2. Analysis of unstable modes distinguishes mathematical models of flagellar motion

    PubMed Central

    Bayly, P. V.; Wilson, K. S.

    2015-01-01

    The mechanisms underlying the coordinated beating of cilia and flagella remain incompletely understood despite the fundamental importance of these organelles. The axoneme (the cytoskeletal structure of cilia and flagella) consists of microtubule doublets connected by passive and active elements. The motor protein dynein is known to drive active bending, but dynein activity must be regulated to generate oscillatory, propulsive waveforms. Mathematical models of flagellar motion generate quantitative predictions that can be analysed to test hypotheses concerning dynein regulation. One approach has been to seek periodic solutions to the linearized equations of motion. However, models may simultaneously exhibit both periodic and unstable modes. Here, we investigate the emergence and coexistence of unstable and periodic modes in three mathematical models of flagellar motion, each based on a different dynein regulation hypothesis: (i) sliding control; (ii) curvature control and (iii) control by interdoublet separation (the ‘geometric clutch’ (GC)). The unstable modes predicted by each model are used to critically evaluate the underlying hypothesis. In particular, models of flagella with ‘sliding-controlled’ dynein activity admit unstable modes with non-propulsive, retrograde (tip-to-base) propagation, sometimes at the same parameter values that lead to periodic, propulsive modes. In the presence of these retrograde unstable modes, stable or periodic modes have little influence. In contrast, unstable modes of the GC model exhibit switching at the base and propulsive base-to-tip propagation. PMID:25833248

  3. A solid-state control system for dynein-based ciliary/flagellar motility

    PubMed Central

    2013-01-01

    Ciliary and flagellar beating requires the coordinated action of multiple dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm dynein essential for waveform determination. PMID:23569213

  4. DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

    PubMed Central

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M.; Sanderson, Michael J.; Nicastro, Daniela; Witman, George B.

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform. PMID:26063732

  5. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

    PubMed Central

    Reck, Jaimee; Schauer, Alexandria M.; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A.; Porter, Mary E.

    2016-01-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  6. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas.

    PubMed

    Reck, Jaimee; Schauer, Alexandria M; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A; Porter, Mary E

    2016-08-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  7. Rhythmicity, recurrence, and recovery of flagellar beating

    NASA Astrophysics Data System (ADS)

    Wan, Kirsty; Goldstein, Raymond

    2015-03-01

    The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the unicellular alga Chlamydomonas reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating. Financial support is acknowledged from the EPSRC, ERC Advanced Investigator Grant No. 247333, and a Senior Investigator Award from the Wellcome Trust.

  8. Mesoscopic modeling of bacterial flagellar microhydrodynamics.

    PubMed

    Gebremichael, Yeshitila; Ayton, Gary S; Voth, Gregory A

    2006-11-15

    A particle-based hybrid method of elastic network model and smooth-particle hydrodynamics has been employed to describe the propulsion of bacterial flagella in a viscous hydrodynamic environment. The method explicitly models the two aspects of bacterial propulsion that involve flagellar flexibility and long-range hydrodynamic interaction of low-Reynolds-number flow. The model further incorporates the molecular organization of the flagellar filament at a coarse-grained level in terms of the 11 protofilaments. Each of these protofilaments is represented by a collection of material points that represent the flagellin proteins. A computational model of a single flexible helical segment representing the filament of a bacterial flagellum is presented. The propulsive dynamics and the flow fields generated by the motion of the model filament are examined. The nature of flagellar deformation and the influence of hydrodynamics in determining the shape of deformations are examined based on the helical filament.

  9. Regulation of Flagellar Gene Expression in Bacteria.

    PubMed

    Osterman, I A; Dikhtyar, Yu Yu; Bogdanov, A A; Dontsova, O A; Sergiev, P V

    2015-11-01

    The flagellum of a bacterium is a supramolecular structure of extreme complexity comprising simultaneously both a unique system of protein transport and a molecular machine that enables the bacterial cell movement. The cascade of expression of genes encoding flagellar components is closely coordinated with the steps of molecular machine assembly, constituting an amazing regulatory system. Data on structure, assembly, and regulation of flagellar gene expression are summarized in this review. The regulatory mechanisms and correlation of the process of regulation of gene expression and flagellum assembly known from the literature are described. PMID:26615435

  10. Flagellar Synchronization Independent of Hydrodynamic Interactions

    NASA Astrophysics Data System (ADS)

    Friedrich, Benjamin M.; Jülicher, Frank

    2012-09-01

    Inspired by the coordinated beating of the flagellar pair of the green algae Chlamydomonas, we study theoretically a simple, mirror-symmetric swimmer, which propels itself at low Reynolds number by a revolving motion of a pair of spheres. We show that perfect synchronization between these two driven spheres can occur due to the motion of the swimmer and local hydrodynamic friction forces. Hydrodynamic interactions, though crucial for net propulsion, contribute little to synchronization for this free-moving swimmer.

  11. Regulation of flagellar motility during biofilm formation

    PubMed Central

    Guttenplan, Sarah B.; Kearns, Daniel B.

    2013-01-01

    Many bacteria swim in liquid or swarm over solid surfaces by synthesizing rotary flagella. The same bacteria that are motile also commonly form non-motile multicellular aggregates held together by an extracellular matrix called biofilms. Biofilms are an important part of the lifestyle of pathogenic bacteria and it is assumed that there is a motility-to-biofilm transition wherein the inhibition of motility promotes biofilm formation. The transition is largely inferred from regulatory mutants that reveal the opposite regulation of the two phenotypes. Here we review the regulation of motility during biofilm formation in Bacillus, Pseudomonas, Vibrio, and Escherichia, and we conclude that the motility-to-biofilm transition, if necessary, likely involves two steps. In the short term, flagella are functionally regulated to either inhibit rotation or modulate the basal flagellar reversal frequency. Over the long term, flagellar gene transcription is inhibited and in the absence of de novo synthesis, flagella are likely diluted to extinction through growth. Both short term and long term control is likely important to the motility-to-biofilm transition to stabilize aggregates and optimize resource investment. We emphasize the newly discovered classes of flagellar functional regulators and speculate that others await discovery in the context of biofilm formation. PMID:23480406

  12. Studies on flagellar shortening in Chlamydomonas reinhardtii

    SciTech Connect

    Cherniack, J.

    1985-01-01

    Flagellar shortening of Chlamydomonas reinhardtii was promoted by sodium chloride, pyrophosphate (sodium, potassium and ammonium salts), EDTA and EGTA, succinate, citrate and oxalate (sodium salts), caffeine and aminophylline. Removal of calcium from the medium potentiated the effects of these agents in inducing shortening. Investigations of the release of phosphorylated compounds to the medium during pyrophosphate-induced flagellar shortening of cells pre-labelled with /sup 32/P, revealed an as yet unidentified /sup 32/P-labelled compound with distinct chromatographic properties. Chromatography and electrophoresis indicates that it is a small, highly polar molecule with a high charge to mass ratio, containing thermo- and acid-labile phosphate linkages. Investigations showed of the release of /sup 35/S-labelled protein to the medium from cells pre-labelled with /sup 35/S-sulfate showed that flagellated cells released two prominent polypeptides which comigrated with ..cap alpha..- and ..beta..-flagellar tubulin on SDS polyacrylamide gel electrophoresis, while deflagellated cells did not.

  13. Aeromonas hydrophila Lateral Flagellar Gene Transcriptional Hierarchy

    PubMed Central

    Wilhelms, Markus; Gonzalez, Victor; Merino, Susana

    2013-01-01

    Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ54/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. PMID:23335410

  14. Inhibitio of flagellar coordination in Spirillum volutans.

    PubMed

    Krieg, N R; Tomelty, J P; Wells, J S

    1967-11-01

    The motility of Spirillum volutans is caused by the rotation of each polar flagellar fascicle in a direction opposite to that of the more slowly rotating cell. Both flagella form cones of revolution oriented in the same direction. When the cell reverses its motion, both fascicles simultaneously reverse their rotation and also the orientation of their cones of revolution, with the tail fascicle becoming the head and vice versa. Chloral hydrate and phenol were found to cause uncoordination, with both fascicles becoming the head type; MgSO(4), Mg(NO(3))(2), NiSO(4), NiCl(2), CuSO(4), and CuCl(2) also caused uncoordination, with both fascicles becoming the tail type. In all cases, the flagellar fascicles remained highly active but the cells were motionless because of the opposing propulsion; the rotation of the fascicles was in a constant direction without reversal. Uncoordinated states could be maintained for 30 to 60 min. Neutralization of the dual-tail flagellation caused by NiSO(4) could be accomplished with chloral hydrate. At the null point, the flagellar orientation was intermediate between head and tail; the fascicles continually reversed direction of rotation, and, now coordinated, caused the cells to move back and forth. Higher concentrations of chloral hydrate completely overcame the effect of NiSO(4) and caused dual-head flagellation. Optimal concentrations of test compounds were determined with the use of pure cultures and a reproducible growth medium. PMID:6057800

  15. Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

    NASA Astrophysics Data System (ADS)

    Namdeo, S.; Onck, P. R.

    2016-10-01

    Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

  16. A role for the membrane in regulating Chlamydomonas flagellar length.

    PubMed

    Dentler, William

    2013-01-01

    Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.

  17. Programmed synthesis of flagellar tubulin during cell differentiation in Naegleria.

    PubMed

    Fulton, C; Kowit, J D

    1975-06-30

    Amebae of Naegleria gruberi differentiate into flagellates when transferred from growth medium to non-nutrient buffer. This differentiation, which requires 48 min at 28 degrees C, is dependent on transcription and translation. Tubulin of the flagellar outer doublets comprises about 0.15% of the protein of flagellate, and only about 1-2% of the total tubulin. An antiserum to flagellar (outer-doublet) tubulin contains antibodies that react selectively with flagellar tubulin. Measurements using this antiserum have shown that 97-98% of the flagellar tubulin antigen appears during differentiation. The appearance of tubulin antigen is sensitive to actinomycin D and cycloheximide. Isotope dilution experiments using [35S]methione demonstrated that at least 70% of the flagellar tubulin is synthesized from amino acids during differentiation. Experiments using both the specific antiserum and isotopes have shown that flagellar tubulin synthesis begins about one-third of the way through differentiation, before any morphological change has occurred. These experiments demonstrate that most, if not all, of the flagellar tubulin is synthesized de novo during differentiation, and that cells selectively use a specific subpopulation of tubulin in assembling the outer doub)lets. The results bring into focus major unsolved questions about the synthesis and assembly of flagellar tubulin. PMID:1056749

  18. Dynamics of a tightly coupled mechanism for flagellar rotation. Bacterial motility, chemiosmotic coupling, protonmotive force.

    PubMed

    Meister, M; Caplan, S R; Berg, H C

    1989-05-01

    The bacterial flagellar motor is a molecular engine that couples the flow of protons across the cytoplasmic membrane to rotation of the flagellar filament. We analyze the steady-state behavior of an explicit mechanical model in which a fixed number of protons carries the filament through one revolution. Predictions of this model are compared with experimentally determined relationships between protonmotive force, proton flux, torque, and speed. All such tightly coupled mechanisms produce the same torque when the motor is stalled but vary greatly in their behavior at high speed. The speed at zero load predicted by our model is limited by the rates of association and dissociation of protons at binding sites on the rotor and by the mobility of force generators containing transmembrane channels that interact with these sites. Our analysis suggests that more could be learned about the motor if it were driven by an externally applied torque backwards (at negative speed) or forwards at speeds greater than the zero-load speed. PMID:2720081

  19. Locomotive consequences of non-axisymmetric flagellar configurations

    NASA Astrophysics Data System (ADS)

    Fu, Henry; Marcos, Marcos; Hyon, Yunkyong; Powers, Thomas; Stocker, Roman

    2011-11-01

    Although peritrichous bacteria can form flagellar bundles at many attachment points and directions relative to the cell body, locomotion of these bacteria is often modeled as arising from a polar bundle oriented along the cell body axis. We discuss the consequences of non-axisymmetric flagellar configurations for bacterial locomotion and implications for bacterial behavior using a boundary element method (BEM) based on the method of regularized Stokeslets. We validate our BEM by comparing to analytic results for spheres and ellipsoids, as well as results in the literature for axisymmetric flagella with spherical and ellipsoidal heads obtained from other boundary element methods and slender body theory. Non-axisymmetric flagellar configurations generically lead to wobbling cell bodies and wiggling helical cell trajectories, both of which have been observed experimentally. We compare experimental and numerically calculated wiggling trajectories to deduce information about flagellar geometries of swimming B. subtilis. We discuss the implications of off-axis flagellar geometries for bacterial rheotaxis and chemotaxis.

  20. A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides▿ †

    PubMed Central

    Poggio, Sebastian; Abreu-Goodger, Cei; Fabela, Salvador; Osorio, Aurora; Dreyfus, Georges; Vinuesa, Pablo; Camarena, Laura

    2007-01-01

    Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other α-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster. PMID:17293429

  1. Polar features in the flagellar propulsion of E. coli bacteria

    NASA Astrophysics Data System (ADS)

    Bianchi, S.; Saglimbeni, F.; Lepore, A.; Di Leonardo, R.

    2015-06-01

    E. coli bacteria swim following a run and tumble pattern. In the run state all flagella join in a single helical bundle that propels the cell body along approximately straight paths. When one or more flagellar motors reverse direction the bundle unwinds and the cell randomizes its orientation. This basic picture represents an idealization of a much more complex dynamical problem. Although it has been shown that bundle formation can occur at either pole of the cell, it is still unclear whether these two run states correspond to asymmetric propulsion features. Using holographic microscopy we record the 3D motions of individual bacteria swimming in optical traps. We find that most cells possess two run states characterized by different propulsion forces, total torque, and bundle conformations. We analyze the statistical properties of bundle reversal and compare the hydrodynamic features of forward and backward running states. Our method is naturally multi-particle and opens up the way towards controlled hydrodynamic studies of interacting swimming cells.

  2. Flagellar synchronization through direct hydrodynamic interactions

    PubMed Central

    Brumley, Douglas R; Wan, Kirsty Y; Polin, Marco; Goldstein, Raymond E

    2014-01-01

    Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties. DOI: http://dx.doi.org/10.7554/eLife.02750.001 PMID:25073925

  3. Flagellar synchronization through direct hydrodynamic interactions.

    PubMed

    Brumley, Douglas R; Wan, Kirsty Y; Polin, Marco; Goldstein, Raymond E

    2014-01-01

    Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties.DOI: http://dx.doi.org/10.7554/eLife.02750.001. PMID:25073925

  4. Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

    PubMed Central

    Osorio-Valeriano, Manuel; de la Mora, Javier; Camarena, Laura

    2015-01-01

    ABSTRACT The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCE Despite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled. PMID:26574514

  5. Characterization of Escherichia coli Flagellar Mutants That are Insensitive to Catabolite Repression

    PubMed Central

    Silverman, Michael; Simon, Melvin

    1974-01-01

    In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3′,5′-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene. Images PMID:4373438

  6. Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas

    PubMed Central

    1982-01-01

    We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta- tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools. PMID:7118994

  7. The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili.

    PubMed

    Cohen-Krausz, Sara; Trachtenberg, Shlomo

    2002-08-16

    Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin. The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament. Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is

  8. Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

    PubMed Central

    Yang, Yong; Cochran, Deborah A.; Gargano, Mary D.; King, Iryna; Samhat, Nayef K.; Burger, Benjain P.; Sabourin, Katherine R.; Hou, Yuqing; Awata, Junya; Parry, David A.D.; Marshall, Wallace F.; Witman, George B.; Lu, Xiangyi

    2011-01-01

    Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia. PMID:21289096

  9. Flagellar oscillation: a commentary on proposed mechanisms.

    PubMed

    Woolley, David M

    2010-08-01

    Eukaryotic flagella and cilia have a remarkably uniform internal 'engine' known as the '9+2' axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive 'switching' event is assumed to occur; there are several proposals regarding the nature of the 'switch' and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The 'provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force-generating cycles and dynein-to-dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self-triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem. PMID

  10. Flagellar oscillation: a commentary on proposed mechanisms.

    PubMed

    Woolley, David M

    2010-08-01

    Eukaryotic flagella and cilia have a remarkably uniform internal 'engine' known as the '9+2' axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive 'switching' event is assumed to occur; there are several proposals regarding the nature of the 'switch' and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The 'provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force-generating cycles and dynein-to-dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self-triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.

  11. Computer simulation of flagellar movement X: doublet pair splitting and bend propagation modeled using stochastic dynein kinetics.

    PubMed

    Brokaw, Charles J

    2014-04-01

    Experimental observations on cyclic splitting and bending by a flagellar doublet pair are modeled using forces obtained from a model for dynein mechanochemistry, based on ideas introduced by Andrew Huxley and Terrill Hill and extended previously for modeling flagellar movements. The new feature is elastic attachment of dynein to the A doublet, which allows movement perpendicular to the A doublet and provides adhesive force that can strain attached dyneins. This additional strain influences the kinetics of dynein attachment and detachment. Computations using this dynein model demonstrate that very simple and realistic ideas about dynein mechanochemistry are sufficient for explaining the separation and reattachment seen experimentally with flagellar doublet pairs. Additional simulations were performed after adding a "super-adhesion" elasticity. This elastic component is intended to mimic interdoublet connections, normally present in an intact axoneme, that would prevent visible splitting but allow sufficient separation to cause dynein detachment and cessation of shear force generation. This is the situation envisioned by Lindemann's "geometric clutch" hypothesis for control of dynein function in flagella and cilia. The simulations show abrupt disengagement of the "clutch" at one end of a bend, and abrupt reengagement of the "clutch" at the other end of a bend, ensuring that active sliding is only operating where it will cause bend propagation from base to tip.

  12. Stoichiometry and turnover of the bacterial flagellar switch protein FliN.

    PubMed

    Delalez, Nicolas J; Berry, Richard M; Armitage, Judith P

    2014-07-01

    Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments. Importance: The flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single

  13. Comprehensive Mapping of the Escherichia coli Flagellar Regulatory Network

    PubMed Central

    Fitzgerald, Devon M.; Bonocora, Richard P.; Wade, Joseph T.

    2014-01-01

    Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process. PMID:25275371

  14. Sequential Assembly of Flagellar Radial Spokes

    PubMed Central

    Diener, Dennis R.; Yang, Pinfen; Geimer, Stefan; Cole, Douglas G.; Sale, Winfield S.; Rosenbaum, Joel L.

    2013-01-01

    The unicellular alga Chlamydomonas can assemble two 10 μm flagella in one hour from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella. On the axoneme, radial spokes are “T” shaped structures with a head of 5 proteins and a stalk of 18 proteins that sediment together at 20S. In the cell body, radial spokes are partially assembled; about half of the radial spoke proteins (RSPs) form a 12S complex. In mutants lacking a single radial spoke protein, smaller spoke subassemblies were identified. When extracts from two such mutants were mixed in vitro the 12S complex was assembled from several smaller complexes demonstrating that portions of the stepwise assembly of radial spoke assembly can be carried out in vitro to elucidate the order of spoke assembly in the cell body. PMID:21692193

  15. Architecture of a flagellar apparatus in the fast-swimming magnetotactic bacterium MO-1

    PubMed Central

    Ruan, Juanfang; Kato, Takayuki; Santini, Claire-Lise; Miyata, Tomoko; Kawamoto, Akihiro; Zhang, Wei-Jia; Bernadac, Alain; Wu, Long-Fei; Namba, Keiichi

    2012-01-01

    The bacterial flagellum is a motility organelle that consists of a rotary motor and a helical propeller. The flagella usually work individually or by forming a loose bundle to produce thrust. However, the flagellar apparatus of marine bacterium MO-1 is a tight bundle of seven flagellar filaments enveloped in a sheath, and it has been a mystery as to how the flagella rotate smoothly in coordination. Here we have used electron cryotomography to visualize the 3D architecture of the sheathed flagella. The seven filaments are enveloped with 24 fibrils in the sheath, and their basal bodies are arranged in an intertwined hexagonal array similar to the thick and thin filaments of vertebrate skeletal muscles. This complex and exquisite architecture strongly suggests that the fibrils counter-rotate between flagella in direct contact to minimize the friction of high-speed rotation of individual flagella in the tight bundle within the sheath to enable MO-1 cells to swim at about 300 µm/s. PMID:23184985

  16. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles

    PubMed Central

    2016-01-01

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  17. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles.

    PubMed

    Maier, Alexander M; Weig, Cornelius; Oswald, Peter; Frey, Erwin; Fischer, Peer; Liedl, Tim

    2016-02-10

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials.

  18. Mechanoregulation of molecular motors in flagella

    NASA Astrophysics Data System (ADS)

    Gadelha, Hermes

    2014-11-01

    Molecular motors are nano-biological machines responsible for exerting forces that drive movement in living organisms, from cargo transport to cell division and motility. Interestingly, despite the inherent complexity of many interacting motors, order and structure may arise naturally, as exemplified by the harmonic, self-organized undulatory motion of the flagellum. The real mechanisms behind this collective spontaneous oscillation are still unknown, and it is challenging task to measure experimentally the molecular motor dynamics within the flagellar structure in real time. In this talk we will explore different competing hypotheses that are capable of generating flagellar bending waves that ``resemble'' in-vitro observations, emphasizing the need for further mathematical analysis and model validation. It also highlight that this is a fertile and challenging area of inter-disciplinary research for applied mathematicians and demonstrates the importance of future observational and theoretical studies in understanding the underlying mechanics of these motile cell appendages.

  19. Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas.

    PubMed

    Kubo, Tomohiro; Hirono, Masafumi; Aikawa, Takumi; Kamiya, Ritsu; Witman, George B

    2015-08-01

    Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, α-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly.

  20. Divalent Cation Control of Flagellar Motility in African Trypanosomes

    NASA Astrophysics Data System (ADS)

    Westergard, Anna M.; Hutchings, Nathan R.

    2005-03-01

    Changes in calcium concentration have been shown to dynamically affect flagellar motility in several eukaryotic systems. The African trypanosome is a monoflagellated protozoan parasite and the etiological agent of sleeping sickness. Although cell motility has been implicated in disease progression, very little is currently known about biochemical control of the trypanosome flagellum. In this study, we assess the effects of extracellular changes in calcium and nickel concentration on trypanosome flagellar movement. Using a flow through chamber, we determine the relative changes in motility in individual trypanosomes in response to various concentrations of calcium and nickel, respectively. Extracellular concentrations of calcium and nickel (as low as 100 micromolar) significantly inhibit trypanosome cell motility. The effects are reversible, as indicated by the recovery of motion after removal of the calcium or nickel from the chamber. We are currently investigating the specific changes in flagellar oscillation and coordination that result from calcium and nickel, respectively. These results verify the presence of a calcium-responsive signaling mechanism(s) that regulates flagellar beat in trypanosomes.

  1. Functional Activation of the Flagellar Type III Secretion Export Apparatus

    PubMed Central

    Phillips, Andrew M.; Calvo, Rebecca A.; Kearns, Daniel B.

    2015-01-01

    Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. PMID:26244495

  2. Flagellar motility is necessary for Aeromonas hydrophila adhesion.

    PubMed

    Qin, Yingxue; Lin, Guifang; Chen, Wenbo; Xu, Xiaojin; Yan, Qingpi

    2016-09-01

    Adhesion to host surface or cells is the initial step in bacterial pathogenesis, and the adhesion mechanisms of the fish pathogenic bacteria Aeromonas hydrophila were investigated in this study. First, a mutagenesis library of A. hydrophila that contained 332 random insertion mutants was constructed via mini-Tn10 Km mutagenesis. Four mutants displayed the most attenuated adhesion. Sequence analysis revealed that the mini-Tn10 insertion sites in the four mutant strains were flgC(GenBank accession numbers KX261880), cytb4(GenBank accession numbers JN133621), rbsR(GenBank accession numbers KX261881) and flgE(GenBank accession numbers JQ974982). To further study the roles of flgC and flgE in the adhesion of A. hydrophila, some biological characteristics of the wild-type strain B11, the mutants M121 and M240, and the complemented strains C121 and C240 were investigated. The results showed that the mutation in flgC or flgE led to the flagellar motility of A. hydrophila significant reduction or abolishment. flgC was not necessary for flagellar biosynthesis but was necessary for the full motility of A. hydrophila, flgE was involved in both flagellar biosynthesis and motility. The flagellar motility is necessary for A. hydrophila to adhere to the host mucus, which suggests flagellar motility plays crucial roles in the early infection process of this bacterium. PMID:27432325

  3. (Photosynthesis in intact plants)

    SciTech Connect

    Not Available

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  4. Flagellar Length Control System: Testing a Simple Model Based on Intraflagellar Transport and Turnover

    PubMed Central

    Marshall, Wallace F.; Qin, Hongmin; Brenni, Mónica Rodrigo; Rosenbaum, Joel L.

    2005-01-01

    Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle. PMID:15496456

  5. Flagellar regeneration in the scaly green flagellate Tetraselmis striata (Prasinophyceae): regeneration kinetics and effect of inhibitors

    NASA Astrophysics Data System (ADS)

    Reize, I. B.; Melkonian, M.

    1987-06-01

    Flagellar regeneration after experimental amputation was studied in synchronized axenic cultures of the scaly green flagellate Tetraselmis striata (Prasinophyceae). After removal of flagella by mechanical shearing, 95% of the cells regrow all four flagella (incl. the scaly covering) to nearly full length with a linear velocity of 50 nm/min under standard conditions. Flagellar regeneration is independent of photosynthesis (no effect of DCMU; the same regeneration rate in the light or in the dark), but depends on de novo protein synthesis: cycloheximide at a low concentration (0.35 μM) blocks flagellar regeneration reversibly. No pool of flagellar precursors appears to be present throughout the flagellated phase of the cell cycle. A transient pool of flagellar precursors, sufficient to generate 2.5 μm of flagellar length, however, develops during flagellar regeneration. Tunicamycin (2 μg/ml) inhibits flagellar regeneration only after a second flagellar amputation, when flagella reach only one third the length of the control. Flagellar regeneration in T. striata differs considerably from that of Chlamydomonas reinhardtii and represents an excellent model system for the study of synchronous Golgi apparatus (GA) activation, and transport and exocytosis of GA-derived macromolecules (scales).

  6. The glycosylphosphatidylinositol-PLC in Trypanosoma brucei forms a linear array on the exterior of the flagellar membrane before and after activation.

    PubMed

    Hanrahan, Orla; Webb, Helena; O'Byrne, Robert; Brabazon, Elaine; Treumann, Achim; Sunter, Jack D; Carrington, Mark; Voorheis, H Paul

    2009-06-01

    Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC.

  7. The Glycosylphosphatidylinositol-PLC in Trypanosoma brucei Forms a Linear Array on the Exterior of the Flagellar Membrane Before and After Activation

    PubMed Central

    Hanrahan, Orla; Webb, Helena; O'Byrne, Robert; Brabazon, Elaine; Treumann, Achim; Sunter, Jack D.; Carrington, Mark; Voorheis, H. Paul

    2009-01-01

    Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC. PMID:19503825

  8. The physics of intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter; Griffiths, D. J.; Albee, A. L.

    1994-01-01

    The ability to capture projectiles intact at hypervelocities in underdense media open a new area of study in physics. Underdense material behaves markedly different than solid, liquid, or gas upon hypervelocity impact. This new phenomenon enables applications in science that would either not be possible or would be very costly by other means. This phenomenon has been fully demonstrated in the laboratory and validated in space. Even more interesting is the fact that this hypervelocity intact capture was accomplished passively. A better understanding of the physics of intact capture will lead to improvements in intact capture. A collection of physical observations of this phenomenon is presented here.

  9. Energy complexes are apparently associated with the switch-motor complex of bacterial flagella†

    PubMed Central

    Zarbiv, Gabriel; Li, Hui; Wolf, Amnon; Cecchini, Gary; Caplan, S. Roy; Sourjik, Victor; Eisenbach, Michael

    2015-01-01

    Recently the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer (FRET) in vivo and pull-down assays in vitro we provided evidence for the interaction of FoF1 ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of FoF1 ATP synthase with FliG is important for the function of the switch of bacterial flagella. PMID:22210351

  10. Chemical screening methods for flagellar phenotypes in Chlamydomonas.

    PubMed

    Avasthi, Prachee; Marshall, Wallace F

    2013-01-01

    Cilia and flagella are important organelles used for sensing the external cellular environment or for motility. Abnormalities in ciliary structure or function can have devastating pathological consequences ranging from sinusitis and obesity to polycystic kidney disease, retinal degeneration, and mental retardation. Chlamydomonas flagella are excellent models to study the regulation and normal function of cilia. We utilized the 1280 compound Sigma LOPAC annotated library to screen for phenotypes in Chlamydomonas flagellar length, motility, deflagellation, and cellular toxicity. Phenotypes were assessed by quantitation from direct microscopic visualization and custom-designed motility/viability assays. Compounds were clustered based on data across all assays to facilitate the identification of novel pathways regulating flagella in Chlamydomonas. These methods can both aid our understanding of the basic biology of flagellar regulation and provide useful points of therapeutic intervention for cilia-related disorders.

  11. Regulation of flagellar biogenesis by a calcium dependent protein kinase in Chlamydomonas reinhardtii.

    PubMed

    Liang, Yinwen; Pan, Junmin

    2013-01-01

    Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs--CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas.

  12. Direct evidence of flagellar synchronization through hydrodynamic interactions

    NASA Astrophysics Data System (ADS)

    Brumley, Douglas; Polin, Marco; Wan, Kirsty; Goldstein, Raymond

    2013-11-01

    Eukaryotic cilia and flagella exhibit striking coordination, from the synchronous beating of two flagella in Chlamydomonas to the metachronal waves and large-scale flows displayed by carpets of cilia. However, the precise mechanisms responsible for flagellar synchronization remain unclear. We perform a series of experiments involving two individual flagella in a quiescent fluid. Cells are isolated from the colonial alga Volvox carteri, held in place at a fixed distance d, and oriented so that their flagellar beating planes coincide. In this fashion, we are able to explicitly assess the role of hydrodynamics in achieving synchronization. For closely separated cells, the flagella are capable of exhibiting a phase-locked state for thousands of beats at a time, despite significant differences in their intrinsic frequencies. For intermediate values of d, synchronous periods are interrupted by brief phase slips, while for d >> 1 the flagellar phase difference drifts almost linearly with time. The coupling strength extracted through analysis of the synchronization statistics exhibits excellent agreement with hydrodynamic predictions. This study unambiguously reveals that flagella coupled only through hydrodynamics are capable of exhibiting robust synchrony.

  13. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility.

    PubMed

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2016-01-01

    Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues. PMID:27468805

  14. Escherichia coli swimming is robust against variations in flagellar number

    PubMed Central

    Mears, Patrick J; Koirala, Santosh; Rao, Chris V; Golding, Ido; Chemla, Yann R

    2014-01-01

    Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell’s flagella cooperatively determine whether the cell ‘runs’ or ‘tumbles’ remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell’s run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an ‘effective number’ of flagella—smaller than the actual number—enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella. DOI: http://dx.doi.org/10.7554/eLife.01916.001 PMID:24520165

  15. Approaches for functional analysis of flagellar proteins in African trypanosomes.

    PubMed

    Oberholzer, Michael; Lopez, Miguel A; Ralston, Katherine S; Hill, Kent L

    2009-01-01

    The eukaryotic flagellum is a highly conserved organelle serving motility, sensory, and transport functions. Although genetic, genomic, and proteomic studies have led to the identification of hundreds of flagellar and putative flagellar proteins, precisely how these proteins function individually and collectively to drive flagellum motility and other functions remains to be determined. In this chapter we provide an overview of tools and approaches available for studying flagellum protein function in the protozoan parasite Trypanosoma brucei. We begin by outlining techniques for in vitro cultivation of both T. brucei life cycle stages, as well as transfection protocols for the delivery of DNA constructs. We then describe specific assays used to assess flagellum function including flagellum preparation and quantitative motility assays. We conclude the chapter with a description of molecular genetic approaches for manipulating gene function. In summary, the availability of potent molecular tools, as well as the health and economic relevance of T. brucei as a pathogen, combine to make the parasite an attractive and integral experimental system for the functional analysis of flagellar proteins. PMID:20409810

  16. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility

    PubMed Central

    Fan, Yongqiang; Evans, Christopher R.; Ling, Jiqiang

    2016-01-01

    Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues. PMID:27468805

  17. Probing flagellar promoter occupancy in wild-type and mutant Caulobacter crescentus by chromatin immunoprecipitation.

    PubMed

    Davis, Nicole J; Viollier, Patrick H

    2011-06-01

    In the asymmetric predivisional cell of Caulobacter crescentus, TipF and TipN mark the cellular pole for future flagellar development. TipF is essential for motility and contains a cyclic-di-GMP phosphodiesterase-like (EAL) domain that is necessary for proper function. TipN is localized to the flagellar pole before TipF and is essential for the proper placement of the flagellum in C. crescentus. Using β-galactosidase promoter-probe assays and quantitative chromatin immunoprecipitation, we investigated the influence of the C. crescentus flagellar assembly regulator TipF on flagellar gene transcription. We compared the transcriptional activity of class II-fliF-lacZ, class III-flgE-lacZ, and class IV-fljL-lacZ fusions in a ΔtipF mutant with that of other flagellar mutants and the wild-type strain. We subsequently verified the in vivo occupancy of the fliF, flgE, and fljL flagellar promoters by the flagellar regulators CtrA, FlbD, and FliX in addition to RNA polymerase. We deduce that TipF contributes to proper expression of flagellar genes in C. crescentus by acting both within and outside of the canonical flagellar gene expression hierarchy.

  18. Space research with intact organisms

    NASA Technical Reports Server (NTRS)

    Phillips, Robert W.; Haddy, Francis J.

    1992-01-01

    Effects of space exposure on intact organisms are briefly reviewed, and examples of future experiments that might provide new information on the role of gravity in the evolution of life are suggested. It is noted that long term experiments with intact plant and animals for studying gravitational thresholds will provide important new insights.

  19. Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

    PubMed Central

    McKeithan, TW; Rosenbaum, JL

    1981-01-01

    The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins. PMID:7309786

  20. The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

    PubMed Central

    Albertini, A M; Caramori, T; Crabb, W D; Scoffone, F; Galizzi, A

    1991-01-01

    We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase. PMID:1828465

  1. The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly.

    PubMed

    Pazour, G J; Dickert, B L; Witman, G B

    1999-02-01

    Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

  2. Both sequence and context are important for flagellar targeting of a glucose transporter

    PubMed Central

    Tran, Khoa D.; Rodriguez-Contreras, Dayana; Shinde, Ujwal; Landfear, Scott M.

    2012-01-01

    Summary Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81–113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins. PMID:22467850

  3. Both sequence and context are important for flagellar targeting of a glucose transporter.

    PubMed

    Tran, Khoa D; Rodriguez-Contreras, Dayana; Shinde, Ujwal; Landfear, Scott M

    2012-07-15

    Many of the cilia- and flagella-specific integral membrane proteins identified to date function to sense the extracellular milieu, and there is considerable interest in defining pathways for targeting such proteins to these sensory organelles. The flagellar glucose transporter of Leishmania mexicana, LmxGT1, is targeted selectively to the flagellar membrane, whereas two other isoforms, LmxGT2 and LmxGT3, are targeted to the pellicular plasma membrane of the cell body. To define the flagellar targeting signal, deletions and point mutations were generated in the N-terminal hydrophilic domain of LmxGT1, which mediates flagellar localization. Three amino acids, N95-P96-M97, serve critical roles in flagellar targeting, resulting in strong mistargeting phenotypes when mutagenized. However, to facilitate flagellar targeting of other non-flagellar membrane proteins, it was necessary to attach a larger region surrounding the NPM motif containing amino acids 81-113. Molecular modeling suggests that this region might present the critical NPM residues at the surface of the N-terminal domain. It is likely that the NPM motif is recognized by currently unknown protein-binding partners that mediate flagellar targeting of membrane-associated proteins. PMID:22467850

  4. Visualization of the flagellar surface of protists by atomic force microscopy.

    PubMed

    Rocha, Gustavo Miranda; Miranda, Kildare; Weissmüller, Gilberto; Bisch, Paulo Mascarello; de Souza, Wanderley

    2010-12-01

    In many cells, motility is mediated by flagellar beating. Protist parasites are capable of highly coordinated motility which contributes to their pathogenicity in mammalian hosts. Understanding the structural aspects of the flagellum may be important to the identification of novel targets for therapeutic intervention. Our group used atomic force microscopy (AFM) to examine the ultrastructure of Trypanosoma cruzi, obtaining valuable information on the organisation of the flagellar sub-structure. AFM images revealed novel flagellar components such as the presence of periodically-spaced protrusions organised along a flagellar furrow and oriented through the major flagellar axis between the axoneme and the paraflagellar rod. The nature and functional role of this structure are still unknown, although the hypothesis that the furrow might physically separate the two distinct domains of the flagellar membrane that comprise the surface of the axoneme and the paraflagellar rod (PFR) has been raised. To test whether the furrow was present or not only in PFR-bearing flagella, different protists containing or lacking the PFR, were analysed by AFM. Analysis of T. cruzi, Trypanosoma brucei and Herpetomonas megaseliae, which present distinct PFRs, showed similar and equivalent furrows along the main axis of their flagella, whereas Crithidia deanei, Giardia lamblia and Tritrichomonas foetus (in which the PFR is reduced or absent) lacked a furrow. Our results strongly suggest that the flagellar furrow is a characteristic feature of PFR-containing flagella and opens new perspectives for its functional role in the definition of sub-domains on the flagellar membrane.

  5. The Three-Dimensional Structure of the Flagellar Rotor from a Clockwise-Locked Mutant of Salmonella enterica Serovar Typhimurium

    PubMed Central

    Thomas, Dennis R.; Francis, Noreen R.; Xu, Chen; DeRosier, David J.

    2006-01-01

    Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM. PMID:17015643

  6. Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

    PubMed

    Kage, Azusa; Mogami, Yoshihiro

    2015-08-01

    Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii.

  7. Efficient spatiotemporal analysis of the flagellar waveform of Chlamydomonas reinhardtii.

    PubMed

    Bayly, P V; Lewis, B L; Kemp, P S; Pless, R B; Dutcher, S K

    2010-01-01

    The 9 + 2 axoneme is a microtubule-based machine that powers the oscillatory beating of cilia and flagella. Its highly regulated movement is essential for the normal function of many organs; ciliopathies cause congenital defects, chronic respiratory tract infections and infertility. We present an efficient method to obtain a quantitative description of flagellar motion, with high spatial and temporal resolution, from high speed video recording of bright field images. This highly automated technique provides the shape, shear angle, curvature, and bend propagation speeds along the length of the flagellum, with approximately 200 temporal samples per beat. We compared the waveforms of uniflagellated wild-type and ida3 mutant cells, which lack the I1 inner dynein complex. Video images were captured at 350 fps. Rigid-body motion was eliminated by fast Fourier transform (FFT)-based registration, and the Cartesian (x-y) coordinates of points on the flagellum were identified. These x-y "point clouds" were embedded in two data dimensions using Isomap, a nonlinear dimension reduction method, and sorted by phase in the flagellar cycle. A smooth surface was fitted to the sorted point clouds, which provides high-resolution estimates of shear angle and curvature. Wild-type and ida3 cells exhibit large differences in shear amplitude, but similar maximum and minimum curvature values. In ida3 cells, the reverse bend begins earlier and travels more slowly relative to the principal bend, than in wild-type cells. The regulation of flagellar movement must involve I1 dynein in a manner consistent with these results.

  8. Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

    PubMed

    Kage, Azusa; Mogami, Yoshihiro

    2015-08-01

    Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii. PMID:26245228

  9. Flagellar waveform dynamics of freely swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, H.; Tam, D.; Hosoi, A. E.; Johnson, K. A.; Gollub, J. P.

    2013-07-01

    We present quantitative measurements of time-dependent flagellar waveforms for freely swimming biflagellated algal cells, for both synchronous and asynchronous beating. We use the waveforms in conjunction with resistive force theory as well as a singularity method to predict a cell's time-dependent velocity for comparison with experiments. While net propulsion is thought to arise from asymmetry between the power and recovery strokes, we show that hydrodynamic interactions between the flagella and cell body on the return stroke make an important contribution to enhance net forward motion.

  10. Intact capture of hypervelocity particles

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    1986-01-01

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  11. Intact capture of hypervelocity particles

    NASA Astrophysics Data System (ADS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  12. Quorum sensing positively regulates flagellar motility in pathogenic Vibrio harveyi.

    PubMed

    Yang, Qian; Defoirdt, Tom

    2015-04-01

    Vibrios belonging to the Harveyi clade are among the major pathogens of aquatic organisms. Quorum sensing (QS) is essential for virulence of V. harveyi towards different hosts. However, most virulence factors reported to be controlled by QS to date are negatively regulated by QS, therefore suggesting that their impact on virulence is limited. In this study, we report that QS positively regulates flagellar motility. We found that autoinducer synthase mutants showed significantly lower swimming motility than the wild type, and the swimming motility could be restored by adding synthetic signal molecules. Further, motility of a luxO mutant with inactive QS (LuxO D47E) was significantly lower than that of the wild type and of a luxO mutant with constitutively maximal QS activity (LuxO D47A). Furthermore, we found that the expression of flagellar genes (both early, middle and late genes) was significantly lower in the luxO mutant with inactive QS when compared with wild type and the luxO mutant with maximal QS activity. Motility assays and gene expression also revealed the involvement of the quorum-sensing master regulator LuxR in the QS regulation of motility. Finally, the motility inhibitor phenamil significantly decreased the virulence of V. harveyi towards gnotobiotic brine shrimp larvae. PMID:24528485

  13. Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei

    PubMed Central

    Höög, Johanna L; Lacomble, Sylvain; O’Toole, Eileen T; Hoenger, Andreas; McIntosh, J Richard; Gull, Keith

    2014-01-01

    Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects. DOI: http://dx.doi.org/10.7554/eLife.01479.001 PMID:24448408

  14. Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei.

    PubMed

    Höög, Johanna L; Lacomble, Sylvain; O'Toole, Eileen T; Hoenger, Andreas; McIntosh, J Richard; Gull, Keith

    2014-01-01

    Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects. DOI: http://dx.doi.org/10.7554/eLife.01479.001. PMID:24448408

  15. Gains of Bacterial Flagellar Motility in a Fungal World

    PubMed Central

    Pion, Martin; Bshary, Redouan; Bindschedler, Saskia; Filippidou, Sevasti; Wick, Lukas Y.; Job, Daniel

    2013-01-01

    The maintenance of energetically costly flagella by bacteria in non-water-saturated media, such as soil, still presents an evolutionary conundrum. Potential explanations have focused on rare flooding events allowing dispersal. Such scenarios, however, overlook bacterial dispersal along mycelia as a possible transport mechanism in soils. The hypothesis tested in this study is that dispersal along fungal hyphae may lead to an increase in the fitness of flagellated bacteria and thus offer an alternative explanation for the maintenance of flagella even in unsaturated soils. Dispersal along fungal hyphae was shown for a diverse array of motile bacteria. To measure the fitness effect of dispersal, additional experiments were conducted in a model system mimicking limited dispersal, using Pseudomonas putida KT2440 and its nonflagellated (ΔfliM) isogenic mutant in the absence or presence of Morchella crassipes mycelia. In the absence of the fungus, flagellar motility was beneficial solely under conditions of water saturation allowing dispersal, while under conditions limiting dispersal, the nonflagellated mutant exhibited a higher level of fitness than the wild-type strain. In contrast, in the presence of a mycelial network under conditions limiting dispersal, the flagellated strain was able to disperse using the mycelial network and had a higher level of fitness than the mutant. On the basis of these results, we propose that the benefit of mycelium-associated dispersal helps explain the persistence of flagellar motility in non-water-saturated environments. PMID:23995942

  16. Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei.

    PubMed

    Höög, Johanna L; Lacomble, Sylvain; O'Toole, Eileen T; Hoenger, Andreas; McIntosh, J Richard; Gull, Keith

    2014-01-01

    Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects. DOI: http://dx.doi.org/10.7554/eLife.01479.001.

  17. Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise

    PubMed Central

    Wakabayashi, Ken-ichi; King, Stephen M.

    2006-01-01

    Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+-binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility. PMID:16754958

  18. Bacterial flagellar capping proteins adopt diverse oligomeric states

    PubMed Central

    Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H; Beckett, Dorothy; Wintrode, Patrick L; Sundberg, Eric J

    2016-01-01

    Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD from Pseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find that Pseudomonas FliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies. DOI: http://dx.doi.org/10.7554/eLife.18857.001 PMID:27664419

  19. Automated methods for estimation of sperm flagellar bending parameters.

    PubMed

    Brokaw, C J

    1984-01-01

    Parameters to describe flagellar bending patterns can be obtained by a microcomputer procedure that uses a set of parameters to synthesize model bending patterns, compares the model bending patterns with digitized and filtered data from flagellar photographs, and uses the Simplex method to vary the parameters until a solution with minimum root mean square differences between the model and the data is found. Parameters for Chlamydomonas bending patterns have been obtained from comparison of shear angle curves for the model and the data. To avoid the determination of the orientation of the basal end of the flagellum, which is required for calculation of shear angles, parameters for sperm flagella have been obtained by comparison of curves of curvature as a function of length for the model and for the data. A constant curvature model, modified from that originally used for Chlamydomonas flagella, has been used for obtaining parameters from sperm flagella, but the methods can be applied using other models for synthesizing the model bending patterns.

  20. Intact capture of cosmic dust

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1991-01-01

    The focus of this development effort is to capture dust particles at hypervelocities intact and unmelted in order to preserve volatile organics. At the same time, the capture process must minimize any organic elemental or compound contamination to prevent any compromise of exobiological analyses. Inorganic silicate aerogel has been developed as a successful capture medium to satisfy both requirements of intact capture and minimal organic contamination. Up to 6 km/s, silicate projectiles from a few microns up to 100 microns have been captured intact without any melting and with minimal loss of mass. Carbon in silicate aerogel can be reduced to less than 1 part in 1000 and hydrogen 3 parts in 1000 when baked in air. Under controlled inert gas environments, additional hydrocarbon reduction can be achieved.

  1. Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion

    PubMed Central

    Lovewell, Rustin R.; Collins, Ryan M.; Acker, Julie L.; O'Toole, George A.; Wargo, Matthew J.; Berwin, Brent

    2011-01-01

    Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria. PMID:21949654

  2. Swarming motility and the control of master regulators of flagellar biosynthesis

    PubMed Central

    Patrick, Joyce E.; Kearns, Daniel B.

    2011-01-01

    Swarming motility is the movement of bacteria over a solid surface powered by rotating flagella. The expression of flagellar biosynthesis genes is governed by species-specific master regulator transcription factors. Mutations that reduce or enhance master regulator activity have a commensurate effect on swarming motility. Here we review what is known about the proteins that modulate swarming motility and appear to act upstream of the master flagellar regulators in diverse swarming bacteria. We hypothesize that environmental control of the master regulators is important to the swarming phenotype perhaps at the level of controlling flagellar number. PMID:22092493

  3. A Programmable Optical Angle Clamp for Rotary Molecular Motors

    PubMed Central

    Pilizota, Teuta; Bilyard, Thomas; Bai, Fan; Futai, Masamitsu; Hosokawa, Hiroyuki; Berry, Richard M.

    2007-01-01

    Optical tweezers are widely used for experimental investigation of linear molecular motors. The rates and force dependence of steps in the mechanochemical cycle of linear motors have been probed giving detailed insight into motor mechanisms. With similar goals in mind for rotary molecular motors we present here an optical trapping system designed as an angle clamp to study the bacterial flagellar motor and F1-ATPase. The trap position was controlled by a digital signal processing board and a host computer via acousto-optic deflectors, the motor position via a three-dimensional piezoelectric stage and the motor angle using a pair of polystyrene beads as a handle for the optical trap. Bead-pair angles were detected using back focal plane interferometry with a resolution of up to 1°, and controlled using a feedback algorithm with a precision of up to 2° and a bandwidth of up to 1.6 kHz. Details of the optical trap, algorithm, and alignment procedures are given. Preliminary data showing angular control of F1-ATPase and angular and speed control of the bacterial flagellar motor are presented. PMID:17434937

  4. Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar 'switch complex' in Vibrio cholerae.

    PubMed

    Khamrui, Susmita; Biswas, Maitree; Sen, Udayaditya; Dasgupta, Jhimli

    2010-08-01

    Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1-CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ;switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 A3 Da(-1) (47% solvent content) assuming the presence of one molecule per asymmetric unit.

  5. Flagellar swimmers oscillate between pusher- and puller-type swimming

    NASA Astrophysics Data System (ADS)

    Klindt, Gary S.; Friedrich, Benjamin M.

    2015-12-01

    Self-propulsion of cellular microswimmers generates flow signatures, commonly classified as pusher and puller type, which characterize hydrodynamic interactions with other cells or boundaries. Using experimentally measured beat patterns, we compute that the flagellated green alga Chlamydomonas oscillates between pusher and puller, rendering it an approximately neutral swimmer, when averaging over its full beat cycle. Beyond a typical distance of 100 μ m from the cell, inertia attenuates oscillatory microflows. We show that hydrodynamic interactions between cells oscillate in time and are of similar magnitude as stochastic swimming fluctuations. From our analysis, we also find that the rate of hydrodynamic dissipation varies in time, which implies that flagellar beat patterns are not optimized with respect to this measure.

  6. Antiphase Synchronization in a Flagellar-Dominance Mutant of Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Leptos, Kyriacos C.; Wan, Kirsty Y.; Polin, Marco; Tuval, Idan; Pesci, Adriana I.; Goldstein, Raymond E.

    2013-10-01

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  7. Composition and sensory function of the trypanosome flagellar membrane

    PubMed Central

    Maric, Danijela; Epting, Conrad L.; Engman, David M.

    2010-01-01

    Summary A cilium is an extension of the cell that contains an axonemal complex of microtubules and associated proteins bounded by a membrane which is contiguous with the cell body membrane. Cilia may be nonmotile or motile, the latter having additional specific roles in cell or fluid movement. The term flagellum refers to the motile cilium of free-living single cells (e.g., bacteria, archaea, spermatozoa and protozoa). In eukaryotes, both nonmotile and motile cilia possess sensory functions. The ciliary interior (cilioplasm) is separated from the cytoplasm by a selective barrier that prevents passive diffusion of molecules between the two domains. The sensory functions of cilia reside largely in the membrane and signals generated in the cilium are transduced into a variety of cellular responses. In this review we discuss the structure and biogenesis of the cilium, with special attention to the trypanosome flagellar membrane, its lipid and protein composition and its proposed roles in sensing and signaling. PMID:20580599

  8. Flagellar generated flow mediates attachment of Giardia lamblia

    NASA Astrophysics Data System (ADS)

    Urbach, Jeffrey; Luo, Haibei; Picou, Theodore; McAllister, Ryan; Elmendorf, Heidi

    2011-03-01

    Giardia lamblia is a protozoan parasite responsible for widespread diarrheal disease in humans and animals worldwide. Attachment to the host intestinal mucosa and resistance to peristalsis is necessary for establishing infection, but the physical basis for this attachment is poorly understood. We report results from TIRF and confocal fluorescence microscopy that demonstrate that the regular beating of the posterior flagella generate a flow through the ventral disk, a suction-cup shaped structure that is against the substrate during attachment. Finite element simulations are used to compare the negative pressure generated by the flow to the measured attachment force and the expected performance of the flagellar pump. NIH grant 1R21AI062934-0.

  9. The effects of translation and rotation on flagellar synchronization

    NASA Astrophysics Data System (ADS)

    Tu, Jonathan H.; Arcak, Murat; Maharbiz, Michel

    2014-11-01

    Synchrony is often observed in studies of swimming microorganisms. Examples include collective behavior in large populations of microswimmers, metachronal waves passing through arrays of cilia, and flagellar bundling. In this work, we focus on the hydrodynamic interactions that occur between flagella in close proximity. Specifically, we use the method of regularized Stokeslets to numerically investigate the precise mechanisms through which phase synchrony occurs in a pair of side-by-side rigid helices. Because our ``end-pinned'' model enforces restoring forces at a single end of each helix, we are able to isolate and compare the respective effects of translational and rotational motions. We find that while certain degrees of freedom promote synchrony, others promote anti-synchrony or have little effect. Funded by ONR Grant N000141310551.

  10. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    PubMed Central

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-01-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes. PMID:25778677

  11. Single-file diffusion of flagellin in flagellar filaments.

    PubMed

    Stern, Alan S; Berg, Howard C

    2013-07-01

    A bacterial flagellar filament is a cylindrical crystal of a protein known as flagellin. Flagellin subunits travel from the cytoplasm through a 2 nm axial pore and polymerize at the filament's distal end. They are supplied by a pump in the cell membrane powered by a proton-motive force. In a recent experiment, it was observed that growth proceeded at a rate of approximately one subunit every 2 s. Here, we asked whether transport of subunits through the pore at this rate could be effected by single-file diffusion, which we simulated by a random walk on a one-dimensional lattice. Assuming that the subunits are α-helical, the answer is yes, by a comfortable margin.

  12. Antiphase synchronization in a flagellar-dominance mutant of Chlamydomonas.

    PubMed

    Leptos, Kyriacos C; Wan, Kirsty Y; Polin, Marco; Tuval, Idan; Pesci, Adriana I; Goldstein, Raymond E

    2013-10-11

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  13. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    NASA Astrophysics Data System (ADS)

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-03-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes.

  14. Flagellar motility confers epiphytic fitness advantages upon Pseudomonas syringae

    SciTech Connect

    Haefele, D.M.; Lindow, S.E.

    1987-10-01

    The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain.

  15. Flagellar Motility Confers Epiphytic Fitness Advantages upon Pseudomonas syringae

    PubMed Central

    Haefele, Douglas M.; Lindow, Steven E.

    1987-01-01

    The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain. PMID:16347469

  16. Intact Procedural Motor Sequence Learning in Developmental Coordination Disorder

    ERIC Educational Resources Information Center

    Lejeune, Caroline; Catale, Corinne; Willems, Sylvie; Meulemans, Thierry

    2013-01-01

    The purpose of the present study was to explore the possibility of a procedural learning deficit among children with developmental coordination disorder (DCD). We tested 34 children aged 6-12 years with and without DCD using the serial reaction time task, in which the standard keyboard was replaced by a touch screen in order to minimize the impact…

  17. Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly

    PubMed Central

    1983-01-01

    The principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. We show that these two isoelectric variants of alpha-tubulin are related to one another since posttranslational modification of the cell body precursor form converts it to the axonemal form. During flagellar assembly, precursor alpha-tubulin enters the flagella and is posttranslationally modified within the flagellar matrix fraction prior to or at the time of its addition to the growing axonemal microtubules. Experiments designed to identify the nature of this posttranslational modification have also been conducted. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo and, under these conditions, no other flagellar polypeptides exhibit detectable labeling. PMID:6863393

  18. Biochemical, immunological, metabolic, and molecular studies on flagellar development in Euglena gracilis

    SciTech Connect

    Levasseur, P.J.

    1989-01-01

    The emergent flagellum of Euglena gracilis arises from an anterior invagination of the organism and possesses, along with the typical eukaryotic axoneme, a glycoprotein surface layer, a complement of structurally complex mastigonemes and a paraxial rod. Nonionic detergent extraction of isolated flagella yielded a fraction containing 21% of the flagellar protein. This fraction contained at least 25 components. In vivo radiolabeling experiments indicated that Euglena possessed a pool of flagellar precursors. This was evidence by the observation that flagellar proteins radiolabeled during an initial regeneration could be mobilized to flagella of a subsequent regeneration. At least one component in the pool was present in sufficient quantity to support an entire regeneration. This protein was tentatively identified as a mastigonemal protein of M{sub r} {approximately} 220,000. A cDNA library was constructed to investigate flagellar gene expression in Euglena.

  19. Monitoring Intact Viruses Using Aptamers

    PubMed Central

    Kumar, Penmetcha K. R.

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  20. Monitoring Intact Viruses Using Aptamers.

    PubMed

    Kumar, Penmetcha K R

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  1. mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii.

    PubMed Central

    Schloss, J A; Silflow, C D; Rosenbaum, J L

    1984-01-01

    Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas. Images PMID:6546968

  2. Complex flagellar motions and swimming patterns of the flagellates Paraphysomonas vestita and Pteridomonas danica.

    PubMed

    Christensen-Dalsgaard, Karen K; Fenchel, Tom

    2004-03-01

    Most flagellates with hispid flagella, that is, flagella with rigid filamentous hairs (mastigonemes), swim in the direction of the flagellar wave propagation with an anterior position of the flagellum. Previous analysis was based on planar wave propagation showing that the mastigonemes pull fluid along the flagellar axis. In the present study, we investigate the flagellar motions and swimming patterns for two flagellates with hispid flagella: Paraphysomonas vestita and Pteridomonas danica. Studies were carried out using normal and high-speed video recording, and particles were added to visualize flow around cells generating feeding currents. When swimming or generating flow, P. vestita was able to pull fluid normal to, and not just along, the flagellum, implying the use of the mastigonemes in an as yet un-described way. When the flagellum made contact with food particles, it changed the flagellar waveform so that the particle was fanned towards the ingestion area, suggesting mechano-sensitivity of the mastigonemes. Pteridomonas danica was capable of more complex swimming than previously described for flagellated protists. This was associated with control of the flagellar beat as well as an ability to bend the plane of the flagellar waveform.

  3. Language and Williams syndrome: how intact is "intact"?

    PubMed

    Karmiloff-Smith, A; Grant, J; Berthoud, I; Davies, M; Howlin, P; Udwin, O

    1997-04-01

    It has been claimed that Williams syndrome (WS), a rare neurodevelopmental disorder, is characterized by serious cognitive deficits alongside intact language. The syndrome is often used as a prime example of the modularity of an innate faculty for morphosyntactic rules. We challenge this claim and hypothesize that morphosyntax, although surprisingly good given WS level of mental retardation, is by no means intact. We make an initial test of this hypothesis through an analysis of the receptive language of a group of English-speaking WS individuals on a standardized morphosyntactic test. We then present an experimental study of expressive language that examines grammatical gender assignment in French-speaking WS patients. Despite a Verbal Mental Age selected to be higher than the chronological age of the young control group, these people with WS continue even in adulthood to show clear-cut deficits in their production of an aspect of morphosyntax that normal children acquire effortlessly very early. The results of the 2 studies, one focusing on receptive language and the other on expressive language, challenge the notion that comprehension and use of morphosyntactic rules in WS individuals are intact. The Within-domain dissociations regarding the use of grammatical gender assignment across several sentence clements and their difficulties in understanding embedded sentences-two quintessentially linguistic skills-suggest that we must rethink the notion of spared, modular, language capacities in Williams syndrome. We conclude that WS language follows a different path to normal acquisition and may turn out to be more like second language learning. PMID:9180000

  4. Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

    SciTech Connect

    Gibbons, B.H.; Gibbons, I.R.

    1987-06-15

    Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein. The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility.

  5. Somatosensory responses in a human motor cortex.

    PubMed

    Shaikhouni, Ammar; Donoghue, John P; Hochberg, Leigh R

    2013-04-01

    Somatic sensory signals provide a major source of feedback to motor cortex. Changes in somatosensory systems after stroke or injury could profoundly influence brain computer interfaces (BCI) being developed to create new output signals from motor cortex activity patterns. We had the unique opportunity to study the responses of hand/arm area neurons in primary motor cortex to passive joint manipulation in a person with a long-standing brain stem stroke but intact sensory pathways. Neurons responded to passive manipulation of the contralateral shoulder, elbow, or wrist as predicted from prior studies of intact primates. Thus fundamental properties and organization were preserved despite arm/hand paralysis and damage to cortical outputs. The same neurons were engaged by attempted arm actions. These results indicate that intact sensory pathways retain the potential to influence primary motor cortex firing rates years after cortical outputs are interrupted and may contribute to online decoding of motor intentions for BCI applications.

  6. Somatosensory responses in a human motor cortex

    PubMed Central

    Donoghue, John P.; Hochberg, Leigh R.

    2013-01-01

    Somatic sensory signals provide a major source of feedback to motor cortex. Changes in somatosensory systems after stroke or injury could profoundly influence brain computer interfaces (BCI) being developed to create new output signals from motor cortex activity patterns. We had the unique opportunity to study the responses of hand/arm area neurons in primary motor cortex to passive joint manipulation in a person with a long-standing brain stem stroke but intact sensory pathways. Neurons responded to passive manipulation of the contralateral shoulder, elbow, or wrist as predicted from prior studies of intact primates. Thus fundamental properties and organization were preserved despite arm/hand paralysis and damage to cortical outputs. The same neurons were engaged by attempted arm actions. These results indicate that intact sensory pathways retain the potential to influence primary motor cortex firing rates years after cortical outputs are interrupted and may contribute to online decoding of motor intentions for BCI applications. PMID:23343902

  7. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions

    PubMed Central

    Juhas, Mario; Ajioka, James W

    2015-01-01

    The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. PMID:26074421

  8. A rotary motor drives Flavobacterium gliding.

    PubMed

    Shrivastava, Abhishek; Lele, Pushkar P; Berg, Howard C

    2015-02-01

    Cells of Flavobacterium johnsoniae, a rod-shaped bacterium devoid of pili or flagella, glide over glass at speeds of 2-4 μm/s [1]. Gliding is powered by a protonmotive force [2], but the machinery required for this motion is not known. Usually, cells move along straight paths, but sometimes they exhibit a reciprocal motion, attach near one pole and flip end over end, or rotate. This behavior is similar to that of a Cytophaga species described earlier [3]. Development of genetic tools for F. johnsoniae led to discovery of proteins involved in gliding [4]. These include the surface adhesin SprB that forms filaments about 160 nm long by 6 nm in diameter, which, when labeled with a fluorescent antibody [2] or a latex bead [5], are seen to move longitudinally down the length of a cell, occasionally shifting positions to the right or the left. Evidently, interaction of these filaments with a surface produces gliding. To learn more about the gliding motor, we sheared cells to reduce the number and size of SprB filaments and tethered cells to glass by adding anti-SprB antibody. Cells spun about fixed points, mostly counterclockwise, rotating at speeds of 1 Hz or more. The torques required to sustain such speeds were large, comparable to those generated by the flagellar rotary motor. However, we found that a gliding motor runs at constant speed rather than at constant torque. Now, there are three rotary motors powered by protonmotive force: the bacterial flagellar motor, the Fo ATP synthase, and the gliding motor. PMID:25619763

  9. Structural insights into bacterial flagellar hooks similarities and specificities

    PubMed Central

    Yoon, Young-Ho; Barker, Clive S.; Bulieris, Paula V.; Matsunami, Hideyuki; Samatey, Fadel A.

    2016-01-01

    Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments. PMID:27759043

  10. Real-Time Imaging of Fluorescent Flagellar Filaments

    PubMed Central

    Turner, Linda; Ryu, William S.; Berg, Howard C.

    2000-01-01

    Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form. PMID:10781548

  11. [A new type of flagellar structure. Type 9+n

    PubMed Central

    1977-01-01

    The ultrastructural study of the Eoacanthocephala sperm cell shows a variation from 0 to 5 in the number of the axial fibers in the axoneme. All the species of the order Eoacanthocephala available to us show this variation; moreover, every individual possesses simultaneously several different structural types. So, we are dealing with a new flagellar organization: 9+n, with 0 less than or equal to n less than or equal to 5. In the Quadrigyridae and the Tenuisentidae families, n varies from 0 to 4, with a maximum of 2 for most individuals, exceptionally at 1 for some individuals. In the Neoechinorhynchidae family, n varies from 0 to 5 with a conspicuous prevalence of 3 (from 84 to 99%, according to the individual). These results prompted us to reexamine the two other orders of Acanthocephala in which the structural types 9+2 or 9+0 have been considered as fixed. Indeed, we have found a few flagella the structure of which is different from the prevalent one. It seems, therefore, that the number of the central fibers of the axoneme in the Acanthocephala sperm cell is never absolutely fixed. PMID:557042

  12. Flagellar apparatus absolute orientations and the phylogeny of the green algae.

    PubMed

    O'Kelly, C J; Floyd, G L

    The absolute orientation of the flagellar apparatus in green algal motile cells is a feature of considerable value in studies of green algal systematics and phylogeny. The absolute orientation patterns found in those algae for which this feature is known or can be deduced are reviewed. Counterclockwise absolute orientation occurs in all classes except the Chlorophyceae and is considered primitive, while the clockwise absolute orientation present in most members of the Chlorophyceae is the result of progressive clockwise rotation of components during evolution. Extant intermediates documenting this rotation include Hafniomonas vegetative cells, which show counterclockwise absolute orientation, and Chaetopeltis quadriflagellate zoospores, in which the flagellar apparatus is strictly cruciate except for a slight clockwise offset of the microtubular rootlets. The V-shaped arrangement of the basal bodies in the flagellar apparatus, as well as the presence of proximal sheaths and of two layers of scales on the cell body, further identifies the Chaetopeltis zoospore as a primitive cell type within the Chlorophyceae . Trends towards the exsertion of basal bodies from a flagellar pit, either apically or laterally, the elimination of quadriflagellate cells, and, in the Chlorophyceae , an increasing amount of basal body offset, indicate advancement within the classes. Absolute orientation is conserved during flagellar apparatus replication and development. Events after flagellar apparatus division in the algae studied may be subdivided into component assembly, which is universal and preserves phylogenetically-useful features, and component reorientation, which occurs in relatively few green algae and adapts the flagellar apparatus to specialized functions. From these flagellar apparatus orientation studies, a major reevaluation of evolution within the Chlorophyceae is proposed, with weakly- thalloid algae possessing desmoschisis (e.g. Chaetopeltis ) considered primitive, and

  13. Giardia Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces

    PubMed Central

    House, Susan A.; Richter, David J.; Pham, Jonathan K.; Dawson, Scott C.

    2011-01-01

    Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is

  14. Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor

    PubMed Central

    Thomas, Dennis R.; Morgan, David Gene; DeRosier, David J.

    1999-01-01

    FliG, FliM, and FliN, key proteins for torque generation, are located in two rings. The first protein is in the M ring and the last two are in the C ring. The rotational symmetries of the C and M rings have been determined to be about 34 (this paper) and 26 (previous work), respectively. The mechanism proposed here depends on the symmetry mismatch between the rings: the C ring extends 34 levers, of which 26 can bind to the 26 equivalent sites on the M ring. The remaining 8 levers bind to proton–pore complexes (studs) to form 8 torque generators. Movement results from the swapping of stud-bound levers with M ring-bound levers. The model predicts that both the M and C rings rotate in the same direction but at different speeds. PMID:10468575

  15. Comparative analysis of the secretion capability of early and late flagellar type III secretion substrates

    PubMed Central

    Singer, Hanna M.; Erhardt, Marc; Hughes, Kelly T.

    2016-01-01

    Summary A remarkable feature of the flagellar-specific type III secretion system (T3SS) is the selective recognition of a few substrate proteins among the many thousand cytoplasmic proteins. Secretion substrates are divided into two specificity classes: early substrates secreted for hook-basal body (HBB) construction and late substrates secreted after HBB completion. Secretion was reported to require a disordered N-terminal secretion signal, mRNA secretion signals within the 5′-untranslated region (5′-UTR) and for late substrates, piloting proteins known as the T3S chaperones. Here, we utilized translational β-lactamase fusions to probe the secretion efficacy of the N-terminal secretion signal of fourteen secreted flagellar substrates in Salmonella enterica. We observed a surprising variety in secretion capability between flagellar proteins of the same secretory class. The peptide secretion signals of the early-type substrates FlgD, FlgF, FlgE and the late-type substrate FlgL were analysed in detail. Analysing the role of the 5′-UTR in secretion of flgB and flgE revealed that the native 5′-UTR substantially enhanced protein translation and secretion. Based on our data, we propose a multicomponent signal that drives secretion via the flagellar T3SS. Both mRNA and peptide signals are recognized by the export apparatus and together with substrate-specific chaperones allowing for targeted secretion of flagellar substrates. PMID:24946091

  16. Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

    PubMed Central

    Porter, Mary E.; Bower, Raqual; Knott, Julie A.; Byrd, Pamela; Dentler, William

    1999-01-01

    A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas. PMID:10069812

  17. High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms.

    PubMed

    Wilson, Laurence G; Carter, Lucy M; Reece, Sarah E

    2013-11-19

    Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the "native" axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53-72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat. PMID:24194551

  18. Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane.

    PubMed

    Emmer, Brian T; Souther, Christina; Toriello, Krista M; Olson, Cheryl L; Epting, Conrad L; Engman, David M

    2009-03-15

    Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms. PMID:19240115

  19. High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms

    PubMed Central

    Wilson, Laurence G.; Carter, Lucy M.; Reece, Sarah E.

    2013-01-01

    Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the “native” axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53–72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat. PMID:24194551

  20. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone.

    PubMed

    Wheeler, Richard J; Sunter, Jack D; Gull, Keith

    2016-02-15

    Leishmania promastigote parasites have a flagellum, which protrudes from the flagellar pocket at the cell anterior, yet, surprisingly, have homologs of many flagellum attachment zone (FAZ) proteins--proteins used in the related Trypanosoma species to laterally attach the flagellum to the cell body from the flagellar pocket to the cell posterior. Here, we use seven Leishmania mexicana cell lines that expressed eYFP fusions of FAZ protein homologs to show that the Leishmania flagellar pocket includes a FAZ structure. Electron tomography revealed a precisely defined 3D organisation for both the flagellar pocket and FAZ, with striking similarities to those of Trypanosoma brucei. Expression of two T. brucei FAZ proteins in L. mexicana showed that T. brucei FAZ proteins can assemble into the Leishmania FAZ structure. Leishmania therefore have a previously unrecognised FAZ structure, which we show undergoes major structural reorganisation in the transition from the promastigote (sandfly vector) to amastigote (in mammalian macrophages). Morphogenesis of the Leishmania flagellar pocket, a structure important for pathogenicity, is therefore intimately associated with a FAZ; a finding with implications for understanding shape changes involving component modules during evolution.

  1. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone.

    PubMed

    Wheeler, Richard J; Sunter, Jack D; Gull, Keith

    2016-02-15

    Leishmania promastigote parasites have a flagellum, which protrudes from the flagellar pocket at the cell anterior, yet, surprisingly, have homologs of many flagellum attachment zone (FAZ) proteins--proteins used in the related Trypanosoma species to laterally attach the flagellum to the cell body from the flagellar pocket to the cell posterior. Here, we use seven Leishmania mexicana cell lines that expressed eYFP fusions of FAZ protein homologs to show that the Leishmania flagellar pocket includes a FAZ structure. Electron tomography revealed a precisely defined 3D organisation for both the flagellar pocket and FAZ, with striking similarities to those of Trypanosoma brucei. Expression of two T. brucei FAZ proteins in L. mexicana showed that T. brucei FAZ proteins can assemble into the Leishmania FAZ structure. Leishmania therefore have a previously unrecognised FAZ structure, which we show undergoes major structural reorganisation in the transition from the promastigote (sandfly vector) to amastigote (in mammalian macrophages). Morphogenesis of the Leishmania flagellar pocket, a structure important for pathogenicity, is therefore intimately associated with a FAZ; a finding with implications for understanding shape changes involving component modules during evolution. PMID:26746239

  2. High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms.

    PubMed

    Wilson, Laurence G; Carter, Lucy M; Reece, Sarah E

    2013-11-19

    Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the "native" axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53-72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat.

  3. A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

    PubMed Central

    2015-01-01

    The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo- and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket. PMID:26318396

  4. Zipping and entanglement in flagellar bundle of E. coli: Role of motile cell body.

    PubMed

    Adhyapak, Tapan Chandra; Stark, Holger

    2015-01-01

    The course of a peritrichous bacterium, such as E. coli, crucially depends on the level of synchronization and self-organization of several rotating flagella. However, the rotation of each flagellum generates countermovements of the body which in turn affect the flagellar dynamics. Using a detailed numerical model of an E. coli, we demonstrate that flagellar entanglement, besides fluid flow relative to the moving body, dramatically changes the dynamics of flagella from that compared to anchored flagella. In particular, bundle formation occurs through a zipping motion in a remarkably rapid time, affected little by initial flagellar orientation. A simplified analytical model supports our observations. Finally, we illustrate how entanglement, hydrodynamic interactions, and body movement contribute to zipping and bundling. PMID:26651717

  5. Zipping and entanglement in flagellar bundle of E. coli: Role of motile cell body

    NASA Astrophysics Data System (ADS)

    Adhyapak, Tapan Chandra; Stark, Holger

    2015-11-01

    The course of a peritrichous bacterium, such as E. coli, crucially depends on the level of synchronization and self-organization of several rotating flagella. However, the rotation of each flagellum generates countermovements of the body which in turn affect the flagellar dynamics. Using a detailed numerical model of an E. coli, we demonstrate that flagellar entanglement, besides fluid flow relative to the moving body, dramatically changes the dynamics of flagella from that compared to anchored flagella. In particular, bundle formation occurs through a zipping motion in a remarkably rapid time, affected little by initial flagellar orientation. A simplified analytical model supports our observations. Finally, we illustrate how entanglement, hydrodynamic interactions, and body movement contribute to zipping and bundling.

  6. Gliding movement in Peranema trichophorum is powered by flagellar surface motility.

    PubMed

    Saito, Akira; Suetomo, Yasutaka; Arikawa, Mikihiko; Omura, Gen; Khan, S M Mostafa Kamal; Kakuta, Soichiro; Suzaki, Etsuko; Kataoka, Katsuko; Suzaki, Toshinobu

    2003-08-01

    A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.

  7. Flagellar coordination in Chlamydomonas cells held on micropipettes.

    PubMed

    Rüffer, U; Nultsch, W

    1998-01-01

    The two flagella of Chlamydomonas are known to beat synchronously: During breaststroke beating they are generally coordinated in a bilateral way while in shock responses during undulatory beating coordination is mostly parallel [Rüffer and Nultsch, 1995: Botanica Acta 108:169-276]. Analysis of a great number of shock responses revealed that in undulatory beats also periods of bilateral coordination are found and that the coordination type may change several times during a shock response, without concomitant changes of the beat envelope and the beat period. In normal wt cells no coordination changes are found during breaststroke beating, but only short temporary asynchronies: During 2 or 3 normal beats of the cis flagellum, the trans flagellum performs 3 or 4 flat beats with a reduced beat envelope and a smaller beat period, resulting in one additional trans beat. Long periods with flat beats of the same shape and beat period are found in both flagella of the non-phototactic mutant ptx1 and in defective wt 622E cells. During these periods, the coordination is parallel, the two flagella beat alternately. A correlation between normal asynchronous trans beats and the parallel-coordinated beats in the presumably cis defective cells and also the undulatory beats is discussed. In the cis defective cells, a perpetual spontaneous change between parallel beats with small beat periods (higher beat frequency) and bilateral beats with greater beat periods (lower beat frequency) are observed and render questionable the existence of two different intrinsic beat frequencies of the two flagella cis and trans. Asynchronies occur spontaneously but may also be induced by light changes, either step-up or step-down, but not by both stimuli in turn as breaststroke flagellar photoresponses (BFPRs). Asynchronies are not involved in phototaxis. They are independent of the BFPRs, which are supposed to be the basis of phototaxis. Both types of coordination must be assumed to be regulated

  8. Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579.

    PubMed

    Tagawa, Yuichi

    2014-12-01

    Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.

  9. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment

    PubMed Central

    Wilson, Corinne S.; Chang, Alex J.; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W.; Takats, Taylor A.; Zambetti, Luke J.; Springer, Amy L.

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  10. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment.

    PubMed

    Wilson, Corinne S; Chang, Alex J; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W; Takats, Taylor A; Zambetti, Luke J; Springer, Amy L

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  11. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  12. Multiplex PCR for distinguishing the most common phase-1 flagellar antigens of Salmonella spp.

    PubMed

    Herrera-León, Silvia; McQuiston, John R; Usera, Miguel A; Fields, Patricia I; Garaizar, Javier; Echeita, M Aurora

    2004-06-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z(10), H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, GA, have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz(10), H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously. This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.

  13. Flagellar regeneration in Chlamydomonas: a model system for studying organelle assembly.

    PubMed

    Johnson, K A; Rosenbaum, J L

    1993-05-01

    How do the many different components of an organelle assemble into a functional structure at an appropriate place and time? Flagellar regeneration by the biflagellate green alga Chlamydomonas is one experimental system in which genetics, biochemistry and ultrastructural analysis are being combined to investigate the assembly of a microtubule-containing organelle. Recent advances in the molecular biology of this 'green yeast' have made possible several new approaches to the problem of flagellar assembly; insights from these new approaches are the focus of this review.

  14. A protein thermometer controls temperature-dependent transcription of flagellar motility genes in Listeria monocytogenes.

    PubMed

    Kamp, Heather D; Higgins, Darren E

    2011-08-01

    Facultative bacterial pathogens must adapt to multiple stimuli to persist in the environment or establish infection within a host. Temperature is often utilized as a signal to control expression of virulence genes necessary for infection or genes required for persistence in the environment. However, very little is known about the molecular mechanisms that allow bacteria to adapt and respond to temperature fluctuations. Listeria monocytogenes (Lm) is a food-borne, facultative intracellular pathogen that uses flagellar motility to survive in the extracellular environment and to enhance initial invasion of host cells during infection. Upon entering the host, Lm represses transcription of flagellar motility genes in response to mammalian physiological temperature (37°C) with a concomitant temperature-dependent up-regulation of virulence genes. We previously determined that down-regulation of flagellar motility is required for virulence and is governed by the reciprocal activities of the MogR transcriptional repressor and the bifunctional flagellar anti-repressor/glycosyltransferase, GmaR. In this study, we determined that GmaR is also a protein thermometer that controls temperature-dependent transcription of flagellar motility genes. Two-hybrid and gel mobility shift analyses indicated that the interaction between MogR and GmaR is temperature sensitive. Using circular dichroism and limited proteolysis, we determined that GmaR undergoes a temperature-dependent conformational change as temperature is elevated. Quantitative analysis of GmaR in Lm revealed that GmaR is degraded in the absence of MogR and at 37°C (when the MogR:GmaR complex is less stable). Since MogR represses transcription of all flagellar motility genes, including transcription of gmaR, changes in the stability of the MogR:GmaR anti-repression complex, due to conformational changes in GmaR, mediates repression or de-repression of flagellar motility genes in Lm. Thus, GmaR functions as a thermo

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

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    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. PMID:26943926

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

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Hara, Noritaka; Aldridge, Phillip D.; Namba, Keiichi

    2016-01-01

    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. PMID:26943926

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

    PubMed

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    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.

  18. Integrated Control of Axonemal Dynein AAA+ Motors

    PubMed Central

    King, Stephen M.

    2012-01-01

    Axonemal dyneins are AAA+ enzymes that convert ATP hydrolysis to mechanical work. This leads to the sliding of doublet microtubules with respect to each other and ultimately the generation of ciliary/flagellar beating. However, in order for useful work to be generated, the action of individual dynein motors must be precisely controlled. In addition, cells modulate the motility of these organelles through a variety of second messenger systems and these signals too must be integrated by the dynein motors to yield an appropriate output. This review describes the current status of efforts to understand dynein control mechanisms and their connectivity focusing mainly on studies of the outer dynein arm from axonemes of the unicellular biflagellate green alga Chlamydomonas. PMID:22406539

  19. Exploratory investigations of hypervelocity intact capture spectroscopy.

    PubMed

    Tsou, P; Griffiths, D J

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  20. Exploratory investigations of hypervelocity intact capture spectroscopy

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Griffiths, D. J.

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  1. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability....

  2. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  3. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  4. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  5. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  6. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability....

  7. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  8. Understanding the efficiency of autonomous nano- and microscale motors.

    PubMed

    Wang, Wei; Chiang, Tso-Yi; Velegol, Darrell; Mallouk, Thomas E

    2013-07-17

    We analyze the power conversion efficiency of different classes of autonomous nano- and micromotors. For bimetallic catalytic motors that operate by a self-electrophoretic mechanism, there are four stages of energy loss, and together they result in a power conversion efficiency on the order of 10(-9). The results of finite element modeling agree well with experimental measurements of the efficiency of catalytic Pt-Au nanorod motors. Modifications of the composition and shape of bimetallic catalytic motors were predicted computationally and found experimentally to lead to higher efficiency. The efficiencies of bubble-propelled catalytic micromotors, magnetically driven flagellar motors, Janus micromotors driven by self-generated thermal gradients, and ultrasonically driven metallic micromotors are also analyzed and discussed.

  9. A Comparative Overview of the Flagellar Apparatus of Dinoflagellate, Perkinsids and Colpodellids

    PubMed Central

    Okamoto, Noriko; Keeling, Patrick J.

    2014-01-01

    Dinoflagellates are a member of the Alveolata, and elucidation of the early evolution of alveolates is important for our understanding of dinoflagellates, and vice versa. The ultrastructure of the flagellar apparatus has been described from several dinoflagellates in the last few decades, and the basic components appear to be well conserved. The typical dinoflagellate apparatus is composed of two basal bodies surrounded by striated collars attached to a connective fiber. The longitudinal basal body is connected to a longitudinal microtubular root (LMR; equivalent of R1) and single microtubular root (R2), whereas the transverse basal body is connected to a transverse microtubular root (TMR; R3) and transverse striated root (TSR) with a microtubule (R4). Some of these components, especially the connective fibers and collars, are dinoflagellate specific characteristics that make their flagellar apparatus relatively complex. We also compare these structures with the flagellar apparatus from a number of close relatives of dinoflagellates and their sister, the apicomplexans, including colpodellids, perkinsids, and Psammosa. Though the ultrastructural knowledge of these lineages is still relatively modest, it provides us with an interesting viewpoint of the character evolution of the flagellar apparatus among those lineages.

  10. The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

    PubMed Central

    Fowkes, Mary Elizabeth; Mitchell, David Rees

    1998-01-01

    Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr ≃ 2 × 106 dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment. PMID:9725897

  11. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

    PubMed

    Ishijima, Sumio

    2016-01-01

    It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements. PMID:26863204

  12. A Comparative Overview of the Flagellar Apparatus of Dinoflagellate, Perkinsids and Colpodellids

    PubMed Central

    Okamoto, Noriko; Keeling, Patrick J.

    2014-01-01

    Dinoflagellates are a member of the Alveolata, and elucidation of the early evolution of alveolates is important for our understanding of dinoflagellates, and vice versa. The ultrastructure of the flagellar apparatus has been described from several dinoflagellates in the last few decades, and the basic components appear to be well conserved. The typical dinoflagellate apparatus is composed of two basal bodies surrounded by striated collars attached to a connective fiber. The longitudinal basal body is connected to a longitudinal microtubular root (LMR; equivalent of R1) and single microtubular root (R2), whereas the transverse basal body is connected to a transverse microtubular root (TMR; R3) and transverse striated root (TSR) with a microtubule (R4). Some of these components, especially the connective fibers and collars, are dinoflagellate specific characteristics that make their flagellar apparatus relatively complex. We also compare these structures with the flagellar apparatus from a number of close relatives of dinoflagellates and their sister, the apicomplexans, including colpodellids, perkinsids, and Psammosa. Though the ultrastructural knowledge of these lineages is still relatively modest, it provides us with an interesting viewpoint of the character evolution of the flagellar apparatus among those lineages. PMID:27694777

  13. Organization and temporal expression of a flagellar basal body gene in Caulobacter crescentus.

    PubMed Central

    Hahnenberger, K M; Shapiro, L

    1988-01-01

    Caulobacter crescentus assembles a single polar flagellum at a defined time in the cell cycle. The protein components of the flagellar hook and filament are synthesized just prior to their assembly. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. A mutation in the flaD gene results in the assembly of a partial basal body which is missing the outermost P and L rings as well as the external hook and filament (K.M. Hahnenberger and L. Shapiro, J. Mol. Biol. 194:91-103, 1987). The flaD gene was cloned and characterized by nucleotide sequencing and S1 nuclease protection assays. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. The predicted amino acid sequence of the leader peptide of flaD is very similar to the leader peptide of the flagellar basal body P ring of Salmonella typhimurium (M. Homma, Y. Komeda, T. Iino, and R.M. Macnab, J. Bacteriol. 169:1493-1498, 1987). Images PMID:2842303

  14. Listeria monocytogenes DNA glycosylase AdiP affects flagellar motility, biofilm formation, virulence, and stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is struct...

  15. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies

    PubMed Central

    Ooi, Cher-Pheng; Rotureau, Brice; Gribaldo, Simonetta; Georgikou, Christina; Julkowska, Daria; Blisnick, Thierry; Perrot, Sylvie; Subota, Ines; Bastin, Philippe

    2015-01-01

    African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins. PMID:26218532

  16. The flagellar apparatus of Breviata anathema, a eukaryote without a clear supergroup affinity.

    PubMed

    Heiss, Aaron A; Walker, Giselle; Simpson, Alastair G B

    2013-08-01

    Breviata anathema is an anaerobic amoeboid flagellate that does not branch within any established 'supergroup'. Molecular phylogenies suggest affinities to Amoebozoa, Opisthokonta, or apusomonads. Here we describe its flagellar apparatus ultrastructure. Breviata has two basal bodies. The flagellated anterior basal body (AB) is associated with a fan of ∼18 microtubules and a short singlet microtubular root. Three microtubular roots associate with the posterior basal body. One, the right root (RR), is initially a triplet that splits into two parts. The other two are singlets: the left root (LR), and the middle root (MR), which arises on the posterior side of the basal body. The MR, LR and smaller part of RR support the left ventral side of the cell, while the larger part of RR runs down the right. Outer dynein arms were not observed on the flagellar axoneme. The mitochondrion-like organelle sometimes contains some tubular cristae. The posterior flagellar apparatus resembles that of several eukaryotic lineages, particularly apusomonads, ancyromonads, excavates, and myxogastrid amoebozoans. This comparison suggests that the complex flagellar apparatus of myxogastrids is actually plesiomorphic within Amoebozoa. The widely distributed splitting right root and posterior singlet (MR in Breviata) may be plesiomorphies in many eukaryotic lineages, and thus could be features of the last eukaryotic common ancestor.

  17. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

    PubMed Central

    Ishijima, Sumio

    2016-01-01

    It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements. PMID:26863204

  18. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems.

    PubMed

    Quelas, J Ignacio; Althabegoiti, M Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A; Marconi, Verónica I; Mongiardini, Elías J; Trejo, Sebastián A; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R

    2016-04-07

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species.

  19. S-adenosyl homocysteine hydrolase (SAHH) accelerates flagellar regeneration in Dunaliella salina.

    PubMed

    Li, Qinghua; Zhu, Liqiang; Yan, Yunmeng; Chai, Dandan; Li, Jie; Xue, Lexun

    2013-08-01

    S-adenosylhomocysteine hydrolase (SAHH) is an enzyme, which catalyzes the hydrolysis of S-adenosylhomocysteine (SAH) which is formed after the donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor in methylation reaction. As a potent regulator of methylation, SAHH plays a critical role in methylation reaction in the cells. Here we cloned the SAHH gene from unicellular green alga Dunaliella salina (dsSAHH) and investigated its effects on flagellar regeneration of D. salina, and found that dsSAHH was upregulated both at the protein and the transcription levels during pH shock-triggered flagellar regeneration of D. salina. The flagellar regeneration was accelerated when dsSAHH was overexpressed, but it was inhibited by SAHH inhibitor 3-deazaadenosine (DZA). Moreover, a receptor for activated C kinase 1 from D. salina (dsRACK1), which was identified to interact with dsSAHH, was increased when dsSAHH was overexpressed in D. salina as shown by real-time PCR. The findings of this study suggest that dsSAHH may participate in the regulation of flagellar regeneration of D. salina.

  20. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems

    PubMed Central

    Quelas, J. Ignacio; Althabegoiti, M. Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A.; Marconi, Verónica I.; Mongiardini, Elías J.; Trejo, Sebastián A.; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R.

    2016-01-01

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species. PMID:27053439

  1. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems.

    PubMed

    Quelas, J Ignacio; Althabegoiti, M Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A; Marconi, Verónica I; Mongiardini, Elías J; Trejo, Sebastián A; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R

    2016-01-01

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species. PMID:27053439

  2. Periplasmic Flagellar Export Apparatus Protein, FliH, Is Involved in Post-Transcriptional Regulation of FlaB, Motility and Virulence of the Relapsing Fever Spirochete Borrelia hermsii

    PubMed Central

    Guyard, Cyril; Raffel, Sandra J.; Schrumpf, Merry E.; Dahlstrom, Eric; Sturdevant, Daniel; Ricklefs, Stacy M.; Martens, Craig; Hayes, Stanley F.; Fischer, Elizabeth R.; Hansen, Bryan T.; Porcella, Stephen F.; Schwan, Tom G.

    2013-01-01

    Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their helical or flat-wave morphology and motility. While most other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ post-transcriptional mechanism(s) that are only partially known. In the present study, we characterize a spontaneous non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in periplasmic flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcription analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major periplasmic flagellar filament core protein FlaB. Despite the lack of FlaB, the amount of FlaA produced by the fliH mutant was similar to the wild-type level. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the post-transcriptional level by influencing the synthesis of FlaB. PMID:24009690

  3. Conditional Granger Causality Analysis of Effective Connectivity during Motor Imagery and Motor Execution in Stroke Patients.

    PubMed

    Wang, Li; Zhang, Jingna; Zhang, Ye; Yan, Rubing; Liu, Hongliang; Qiu, Mingguo

    2016-01-01

    Aims. Motor imagery has emerged as a promising technique for the improvement of motor function following stroke, but the mechanism of functional network reorganization in patients during this process remains unclear. The aim of this study is to evaluate the cortical motor network patterns of effective connectivity in stroke patients. Methods. Ten stroke patients with right hand hemiplegia and ten normal control subjects were recruited. We applied conditional Granger causality analysis (CGCA) to explore and compare the functional connectivity between motor execution and motor imagery. Results. Compared with the normal controls, the patient group showed lower effective connectivity to the primary motor cortex (M1), the premotor cortex (PMC), and the supplementary motor area (SMA) in the damaged hemisphere but stronger effective connectivity to the ipsilesional PMC and M1 in the intact hemisphere during motor execution. There were tighter connections in the cortical motor network in the patients than in the controls during motor imagery, and the patients showed more effective connectivity in the intact hemisphere. Conclusions. The increase in effective connectivity suggests that motor imagery enhances core corticocortical interactions, promotes internal interaction in damaged hemispheres in stroke patients, and may facilitate recovery of motor function. PMID:27200373

  4. Conditional Granger Causality Analysis of Effective Connectivity during Motor Imagery and Motor Execution in Stroke Patients

    PubMed Central

    Wang, Li; Zhang, Jingna; Zhang, Ye; Yan, Rubing; Liu, Hongliang; Qiu, Mingguo

    2016-01-01

    Aims. Motor imagery has emerged as a promising technique for the improvement of motor function following stroke, but the mechanism of functional network reorganization in patients during this process remains unclear. The aim of this study is to evaluate the cortical motor network patterns of effective connectivity in stroke patients. Methods. Ten stroke patients with right hand hemiplegia and ten normal control subjects were recruited. We applied conditional Granger causality analysis (CGCA) to explore and compare the functional connectivity between motor execution and motor imagery. Results. Compared with the normal controls, the patient group showed lower effective connectivity to the primary motor cortex (M1), the premotor cortex (PMC), and the supplementary motor area (SMA) in the damaged hemisphere but stronger effective connectivity to the ipsilesional PMC and M1 in the intact hemisphere during motor execution. There were tighter connections in the cortical motor network in the patients than in the controls during motor imagery, and the patients showed more effective connectivity in the intact hemisphere. Conclusions. The increase in effective connectivity suggests that motor imagery enhances core corticocortical interactions, promotes internal interaction in damaged hemispheres in stroke patients, and may facilitate recovery of motor function. PMID:27200373

  5. KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

    PubMed

    Tran, Khoa D; Rodriguez-Contreras, Dayana; Vieira, Danielle P; Yates, Phillip A; David, Larry; Beatty, Wandy; Elferich, Johannes; Landfear, Scott M

    2013-08-01

    The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

  6. KHARON1 mediates flagellar targeting of a glucose transporter in Leishmania mexicana and is critical for viability of infectious intracellular amastigotes.

    PubMed

    Tran, Khoa D; Rodriguez-Contreras, Dayana; Vieira, Danielle P; Yates, Phillip A; David, Larry; Beatty, Wandy; Elferich, Johannes; Landfear, Scott M

    2013-08-01

    The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle. PMID:23766511

  7. Membrane structure in isolated and intact myelins.

    PubMed Central

    Inouye, H; Karthigasan, J; Kirschner, D A

    1989-01-01

    The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions. PMID:2752082

  8. Centrin-mediated microtubule severing during flagellar excision in Chlamydomonas reinhardtii

    PubMed Central

    1989-01-01

    Chlamydomonas cells excise their flagella in response to a variety of experimental conditions (e.g., extremes of temperature or pH, alcohol or detergent treatment, and mechanical shear). Here, we show that flagellar excision is an active process whereby microtubules are severed at select sites within the transition zone. The transition zone is located between the flagellar axoneme and the basal body; it is characterized by a pair of central cylinders that have an H shape when viewed in longitudinal section. Both central cylinders are connected to the A tubule of each microtubule doublet of the transition zone by fibers (approximately 5 nm diam). When viewed in cross section, these fibers are seen to form a distinctive stellate pattern characteristic of the transition zone (Manton, I. 1964. J. R. Microsc. Soc. 82:279- 285; Ringo. D. L. 1967. J. Cell Biol. 33:543-571). We demonstrate that at the time of flagellar excision these fibers contract and displace the microtubule doublets of the axoneme inward. We believe that the resulting shear force and torsional load act to sever the axonemal microtubules immediately distal to the central cylinder. Structural alterations of the transition zone during flagellar excision occur both in living cells and detergent-extracted cell models, and are dependent on the presence of calcium (greater than or equal to 10(-6) M). Immunolocalization using monoclonal antibodies against the calcium- binding protein centrin demonstrate the presence of centrin in the fiber-based stellate structure of the transition zone of wild-type cells. Examination of the flagellar autotomy mutant, fa-1, which fails to excise its flagella (Lewin, R., and C. Burrascano. 1983. Experientia. 39:1397-1398), demonstrates that the fa-1 lacks the ability to completely contract the fibers of the stellate structure. We conclude that flagellar excision in Chlamydomonas involves microtubule severing that is mediated by the action of calcium-sensitive contractile fibers

  9. Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of Salmonella spp.

    PubMed Central

    Herrera-León, Silvia; McQuiston, John R.; Usera, Miguel A.; Fields, Patricia I.; Garaizar, Javier; Echeita, M. Aurora

    2004-01-01

    Most Salmonella serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the fliC and fljB genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fliC genes encoding the H:i, H:r, H:l,v, H:e,h, H:z10, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected Salmonella serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz10, H:b, and H:d first-phase antigens. These antigens are part of the most common Salmonella serotypes possessing first-phase flagellar antigens. Salmonella enterica serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161 Salmonella strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp; Salmonella serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested. PMID:15184437

  10. Singly Flagellated Pseudomonas aeruginosa Chemotaxes Efficiently by Unbiased Motor Regulation

    PubMed Central

    Cai, Qiuxian; Li, Zhaojun; Ouyang, Qi

    2016-01-01

    ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that has long been known to chemotax. More recently, it has been established that chemotaxis is an important factor in the ability of P. aeruginosa to make biofilms. Genes that allow P. aeruginosa to chemotax are homologous with genes in the paradigmatic model organism for chemotaxis, Escherichia coli. However, P. aeruginosa is singly flagellated and E. coli has multiple flagella. Therefore, the regulation of counterclockwise/clockwise flagellar motor bias that allows E. coli to efficiently chemotax by runs and tumbles would lead to inefficient chemotaxis by P. aeruginosa, as half of a randomly oriented population would respond to a chemoattractant gradient in the wrong sense. How P. aeruginosa regulates flagellar rotation to achieve chemotaxis is not known. Here, we analyze the swimming trajectories of single cells in microfluidic channels and the rotations of cells tethered by their flagella to the surface of a variable-environment flow cell. We show that P. aeruginosa chemotaxes by symmetrically increasing the durations of both counterclockwise and clockwise flagellar rotations when swimming up the chemoattractant gradient and symmetrically decreasing rotation durations when swimming down the chemoattractant gradient. Unlike the case for E. coli, the counterclockwise/clockwise bias stays constant for P. aeruginosa. We describe P. aeruginosa’s chemotaxis using an analytical model for symmetric motor regulation. We use this model to do simulations that show that, given P. aeruginosa’s physiological constraints on motility, its distinct, symmetric regulation of motor switching optimizes chemotaxis. PMID:27048795

  11. Neural activation differences in amputees during imitation of intact versus amputee movements

    PubMed Central

    Cusack, William F.; Cope, Michael; Nathanson, Sheryl; Pirouz, Nikta; Kistenberg, Robert; Wheaton, Lewis A.

    2012-01-01

    The mirror neuron system (MNS) has been attributed with increased activation in motor-related cortical areas upon viewing of another's actions. Recent work suggests that limb movements that are similar and dissimilar in appearance to that of the viewer equivalently activate the MNS. It is unclear if this result can be observed in the action encoding areas in amputees who use prosthetic devices. Intact subjects and upper extremity amputee prosthesis users were recruited to view video demonstrations of tools being used by an intact actor and a prosthetic device user. All subjects pantomimed the movements seen in the video while recording electroencephalography (EEG). Intact subjects showed equivalent left parietofrontal activity during imitation planning after watching the intact or prosthetic arm. Likewise, when prosthesis users imitated prosthesis demonstrations, typical left parietofrontal activation was observed. When prosthesis users imitated intact actors, an additional pattern was revealed which showed greater activity in right parietal and occipital regions that are associated with the mentalizing system. This change may be required for prosthesis users to plan imitation movements in which the limb states between the observed and the observer do not match. The finding that prosthesis users imitating other prosthesis users showed typical left parietofrontal activation suggests that these subjects engage normal planning related activity when they are able to imitate a limb matching their own. This result has significant implications on rehabilitation, as standard therapy involves training with an intact occupational therapist, which could necessitate atypical planning mechanisms in amputees when learning to use their prosthesis. PMID:22754516

  12. Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amoebae into flagellates

    SciTech Connect

    Lee, J.H.; Walsh, C.J.

    1988-06-01

    The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins ..cap alpha..- and BETA-tubulin and flagellar calmodulin, as well as a coordinately regulated poly(A)/sup +/ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)/sup +/ RNAs common to ameobae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in ameobae increased 3- to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flagellar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. gruberi amebae to form visible flagella.

  13. Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

    PubMed Central

    2009-01-01

    Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented. PMID:21637533

  14. Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation.

    PubMed

    Diniz, Michely C; Costa, Marcília P; Pacheco, Ana C L; Kamimura, Michel T; Silva, Samara C; Carneiro, Laura D G; Sousa, Ana P L; Soares, Carlos E A; Souza, Celeste S F; de Oliveira, Diana Magalhães

    2009-07-01

    Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented. PMID:21637533

  15. A case of mixed transcortical aphasia with intact naming.

    PubMed

    Heilman, K M; Tucker, D M; Valenstein, E

    1976-09-01

    Altholgh Lichtheim recognized that Wernicke's 'reflex arch' (primary auditory area, to Wernicke's area, to Broca's area, to primary motor area) was important for repetition, he recognized that other areas of the brain (for example, area of concepts or semantic area) must be important in comprehension and voluntary speech. He suggested that Wernicke's area (phonemic area) not only projected to Broca's area (as Wernicke suggested) but that it also projected to the area of concepts. A lesion of this latter pathway or in the area of concepts would produce a syndrome where repetition was intact but comprehension was impaired (e.g. transcortical sensory aphasia). Lichtheim also thought that the area of concepts projected directly to Broca's area and that voluntary speech was mediated by this pathway. Although Lichtheim's model could explain the mechanism underlying transcortical aphasia, his schema could not explain anomic aphasia. Unlike Lichtheim's schema, Kussmaul's schema suggested that the area of concepts projects back to Wernicke's area before projecting to Broca's area. With this schema, a patient with a hypothetical lesion which interrupted the pathway from the area of concepts to Wernicke's area (but did not interrupt the pathway from Wernicke's area to the area of concepts) should be anomic, with normal comprehension and repetition. In order for this latter schema to be plausible there should also be a lesion which interrupts the pathway from Wernicke's area to the area of concepts but does not interrupt the pathway which goes from the area of concepts to Wernicke's area. A patient with this hypothetical lesion should comprehend poorly; however, in spite of poor comprehension, naming and repetition should be intact. We report a patient who demonstrates poor comprehension with intact naming and repetition. This patient could also read aloud but could not comprehend written language. Not only could this patient name objects but he could demonstrate their use

  16. Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms

    PubMed Central

    1987-01-01

    The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha- tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules. PMID:3818788

  17. Cell-body rocking is a dominant mechanism for flagellar synchronization in a swimming alga

    PubMed Central

    Geyer, Veikko F.; Jülicher, Frank; Howard, Jonathon; Friedrich, Benjamin M.

    2013-01-01

    The unicellular green alga Chlamydomonas swims with two flagella that can synchronize their beat. Synchronized beating is required to swim both fast and straight. A long-standing hypothesis proposes that synchronization of flagella results from hydrodynamic coupling, but the details are not understood. Here, we present realistic hydrodynamic computations and high-speed tracking experiments of swimming cells that show how a perturbation from the synchronized state causes rotational motion of the cell body. This rotation feeds back on the flagellar dynamics via hydrodynamic friction forces and rapidly restores the synchronized state in our theory. We calculate that this “cell-body rocking” provides the dominant contribution to synchronization in swimming cells, whereas direct hydrodynamic interactions between the flagella contribute negligibly. We experimentally confirmed the two-way coupling between flagellar beating and cell-body rocking predicted by our theory. PMID:24145440

  18. The phylogeny of swimming kinematics: The environment controls flagellar waveforms in sperm motility

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Burton, Lisa; Zimmer, Richard; Hosoi, Anette; Stocker, Roman

    2013-11-01

    In recent years, phylogenetic and molecular analyses have dominated the study of ecology and evolution. However, physical interactions between organisms and their environment, a fundamental determinant of organism ecology and evolution, are mediated by organism form and function, highlighting the need to understand the mechanics of basic survival strategies, including locomotion. Focusing on spermatozoa, we combined high-speed video microscopy and singular value decomposition analysis to quantitatively compare the flagellar waveforms of eight species, ranging from marine invertebrates to humans. We found striking similarities in sperm swimming kinematics between genetically dissimilar organisms, which could not be uncovered by phylogenetic analysis. The emergence of dominant waveform patterns across species are suggestive of biological optimization for flagellar locomotion and point toward environmental cues as drivers of this convergence. These results reinforce the power of quantitative kinematic analysis to understand the physical drivers of evolution and as an approach to uncover new solutions for engineering applications, such as micro-robotics.

  19. Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode.

    PubMed

    Beznosov, Sergei N; Veluri, Pavan S; Pyatibratov, Mikhail G; Chatterjee, Abhijit; MacFarlane, Douglas R; Fedorov, Oleg V; Mitra, Sagar

    2015-01-13

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g(-1) after 50 cycles and with high rate capability, delivering 770 mAh g(-1) at 5 A g(-1) (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

  20. Nonlinear instability in flagellar dynamics: a novel modulation mechanism in sperm migration?

    NASA Astrophysics Data System (ADS)

    Gadelha, H.; Gaffney, E.; Smith, D.; Kirkman-Brown, J.

    2010-11-01

    Throughout biology, cells and organisms use flagella and cilia to propel fluid and achieve motility. While the mechanics of flagellum-fluid interaction has been the subject of extensive mathematical studies, these models have been restricted to being geometrically linear or weakly nonlinear. In this talk, we study the effect of geometrical nonlinearity, focusing on the spermatozoon flagellum. For a wide range of physiologically relevant parameters, the nonlinear model predicts that flagellar compression by the internal forces initiates an effective buckling behaviour, leading to a symmetry-breaking bifurcation that causes profound and complicated changes in the waveform and swimming trajectory, as well as the breakdown of the linear theory. The emergent waveform also induces curved swimming in an otherwise symmetric system, with the swimming trajectory being sensitive to head shape - no signalling or asymmetric forces are required. We conclude that non-linear models are essential in understanding the flagellar waveform in migratory human sperm.

  1. Mechanisms of flagellar motility deduced from backward-swimming bull sperm.

    PubMed

    Phillips, D M; Kalay, D

    1984-07-01

    Under certain conditions of cryopreservation, bull spermatozoa undergo an interesting structural alteration. The sperm tail becomes bent back on itself to form a hairpin shape. The bend in the tail occurs at a very precise point, 11 microns behind the neck, and it causes the tail to become kinked. Flagellar microtubules and dense fibers become broken and the ninefold symmetry of the flagellum is greatly distored. Although the portion of the flagellum between the kink and the sperm head does not propagate a wave, the distal portion of the flagellum propagates a base-to-tip wave, causing the spermatozoan to progress backward. These observations suggest that the mammalian spermatozoon does not need basal structures to propagate a flagellar wave.

  2. Porters versus rowers: a unified stochastic model of motor proteins

    PubMed Central

    1993-01-01

    We present a general phenomenological theory for chemical to mechanical energy transduction by motor enzymes which is based on the classical "tight-coupling" mechanism. The associated minimal stochastic model takes explicitly into account both ATP hydrolysis and thermal noise effects. It provides expressions for the hydrolysis rate and the sliding velocity, as functions of the ATP concentration and the number of motor enzymes. It explains in a unified way many results of recent in vitro motility assays. More importantly, the theory provides a natural classification scheme for the motors: it correlates the biochemical and mechanical differences between "porters" such as cellular kinesins or dyneins, and "rowers" such as muscular myosins or flagellar dyneins. PMID:8509455

  3. Flagellar Waveform Dynamics of Freely Swimming Algal Cells

    NASA Astrophysics Data System (ADS)

    Gollub, Jerry; Kurtuldu, Huseyin; Johnson, Karl

    2012-02-01

    We study the time-dependent conformation patterns of flagella driving freely swimming algal cells, and use this information to determine the time-dependent forces acting on the cells, the induced cell velocities, and the power injected into the surrounding fluid. Conformational waves are evident along the flagella, as are synchronization transitions. The observed dynamics relate directly to the behavior of the dynein molecular motors ``walking'' along the microtubules of the flagella. We find that the irreversibility giving rise to net propulsion is due to the hydrodynamic interactions between the flagella and cell body. We determine the time-dependent power injected into the fluid by the cell body and flagella, and show that the propulsion efficiency is only about 3%.

  4. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells

    PubMed Central

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-01-01

    Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell’s turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion. PMID:26655343

  5. Complex spatial organization and flagellin composition of flagellar propeller from marine magnetotactic ovoid strain MO-1.

    PubMed

    Zhang, Wei-Jia; Santini, Claire-Lise; Bernadac, Alain; Ruan, Juanfang; Zhang, Sheng-Da; Kato, Takayuki; Li, Ying; Namba, Keiichi; Wu, Long-Fei

    2012-03-01

    Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 μm/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an ~ 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity.

  6. The counterbend phenomenon in flagellar axonemes and cross-linked filament bundles.

    PubMed

    Gadêlha, Hermes; Gaffney, Eamonn A; Goriely, Alain

    2013-07-23

    Recent observations of flagellar counterbend in sea urchin sperm show that the mechanical induction of curvature in one part of a passive flagellum induces a compensatory countercurvature elsewhere. This apparent paradoxical effect cannot be explained using the standard elastic rod theory of Euler and Bernoulli, or even the more general Cosserat theory of rods. Here, we develop a geometrically exact mechanical model to describe the statics of microtubule bundles that is capable of predicting the curvature reversal events observed in eukaryotic flagella. This is achieved by allowing the interaction of deformations in different material directions, by accounting not only for structural bending, but also for the elastic forces originating from the internal cross-linking mechanics. Large-amplitude static configurations can be described analytically, and an excellent match between the model and the observed counterbend deformation was found. This allowed a simultaneous estimation of multiple sperm flagellum material parameters, namely the cross-linking sliding resistance, the bending stiffness, and the sperm head junction compliance ratio. We further show that small variations on the empirical conditions may induce discrepancies for the evaluation of the flagellar material quantities, so that caution is required when interpreting experiments. Finally, our analysis demonstrates that the counterbend emerges as a fundamental property of sliding resistance in cross-linked filamentous polymer bundles, which also suggests that cross-linking proteins may contribute to the regulation of the flagellar waveform in swimming sperm via counterbend mechanics. PMID:23824293

  7. Two flagellar BAR domain proteins in Trypanosoma brucei with stage-specific regulation

    PubMed Central

    Cicova, Zdenka; Dejung, Mario; Skalicky, Tomas; Eisenhuth, Nicole; Hanselmann, Steffen; Morriswood, Brooke; Figueiredo, Luisa M.; Butter, Falk; Janzen, Christian J.

    2016-01-01

    Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod. PMID:27779220

  8. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells.

    PubMed

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-12-29

    Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell's turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion. PMID:26655343

  9. Studies on the serology of flagellar antigens of Yersinia enterocolitica and related Yersinia species.

    PubMed

    Aleksić, S; Bockemühl, J; Lange, F

    1986-05-01

    A total of 1242 strains of Y. enterocolitica, 104 strains of Y. frederiksenii, 95 strains of Y. kristensenii and 85 strains of Y. intermedia were serotyped with antisera against 56 O antigens and 19 H antigens according to the extended antigenic scheme of Wauters, and with additional antisera against 4 somatic and 19 flagellar antigens not previously described. H antigens of Y. frederiksenii, Y. kristensenii, and Y. intermedia turned out to be rather homogeneous without distinct subfactors. In these species the scope of identified serovars was narrow. Flagellar antigens of Y. enterocolitica were mostly composed of several subfactors, leading to a total of 117 serovars identified in the species. A number of cross-reactions between Yersinia H antigens were observed which could be avoided by absorption without significant lowering of the titre. Flagellar antigens of Yersinia were monophasic, and species specific. The antigens remained stable after storage in agar stabs and repeated subcultures. The epidemiological value of serotyping is demonstrated by strains from three different sources. It is suggested serotyping of Yersinia strains should be performed in three steps: O typing of the prevailing enteropathogenic Y. enterocolitica serogroups in the medical routine laboratory; O and H typing of Y. enterocolitica by National Reference Centres applying a typing scheme reduced to this species; and O and H typing of Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia by specialized International Centres using an extended typing scheme. The need for international standards comparable to those established for Salmonella is emphasized.

  10. Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

    PubMed Central

    Juhas, Mario; Ajioka, James W.

    2015-01-01

    Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci. PMID:25816013

  11. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells.

    PubMed

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-12-29

    Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell's turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion.

  12. Complex spatial organization and flagellin composition of flagellar propeller from marine magnetotactic ovoid strain MO-1.

    PubMed

    Zhang, Wei-Jia; Santini, Claire-Lise; Bernadac, Alain; Ruan, Juanfang; Zhang, Sheng-Da; Kato, Takayuki; Li, Ying; Namba, Keiichi; Wu, Long-Fei

    2012-03-01

    Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 μm/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an ~ 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity. PMID:22245577

  13. Calorie restriction and susceptibility to intact pathogens

    PubMed Central

    2008-01-01

    Long-term calorie restriction (CR) causes numerous physiological changes that ultimately increase mean and maximum lifespan of most species examined to date. One physiological change that occurs with CR is enhanced immune function, as tested using antigens and mitogens to stimulate an immune response. Fewer studies have used intact pathogen exposure to test whether the enhanced capacity of the immune response during CR actually decreases susceptibility of hosts to their pathogens. So far, studies using intact bacteria, virus, and helminth worm exposure indicate that, despite similar or enhanced immune system function, CR hosts are more susceptible to infection by intact pathogens than their fully fed counterparts. Long-term CR studies that examine susceptibility to a variety of parasite taxa will help determine if direct CR or CR mimetics will be beneficial to people living in pathogen-rich environments. PMID:19424864

  14. Basic timing abilities stay intact in patients with musician's dystonia.

    PubMed

    van der Steen, M C; van Vugt, Floris T; Keller, Peter E; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument.

  15. Basic timing abilities stay intact in patients with musician's dystonia.

    PubMed

    van der Steen, M C; van Vugt, Floris T; Keller, Peter E; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument. PMID:24667273

  16. Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms

    PubMed Central

    1992-01-01

    We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229- 236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation. PMID:1387404

  17. Disentangling motor execution from motor imagery with the phantom limb.

    PubMed

    Raffin, Estelle; Mattout, Jérémie; Reilly, Karen T; Giraux, Pascal

    2012-02-01

    Amputees can move their phantom limb at will. These 'movements without movements' have generally been considered as motor imagery rather than motor execution, but amputees can in fact perform both executed and imagined movements with their phantom and they report distinct perceptions during each task. Behavioural evidence for this dual ability comes from the fact that executed movements are associated with stump muscle contractions whereas imagined movements are not, and that phantom executed movements are slower than intact hand executed movements whereas the speed of imagined movements is identical for both hands. Since neither execution nor imagination produces any visible movement, we hypothesized that the perceptual difference between these two motor tasks relies on the activation of distinct cerebral networks. Using functional magnetic resonance imaging and changes in functional connectivity (dynamic causal modelling), we examined the activity associated with imagined and executed movements of the intact and phantom hands of 14 upper-limb amputees. Distinct but partially overlapping cerebral networks were active during both executed and imagined phantom limb movements (both performed at the same speed). A region of interest analysis revealed a 'switch' between execution and imagination; during execution there was more activity in the primary somatosensory cortex, the primary motor cortex and the anterior lobe of the cerebellum, while during imagination there was more activity in the parietal and occipital lobes, and the posterior lobe of the cerebellum. In overlapping areas, task-related differences were detected in the location of activation peaks. The dynamic causal modelling analysis further confirmed the presence of a clear neurophysiological distinction between imagination and execution, as motor imagery and motor execution had opposite effects on the supplementary motor area-primary motor cortex network. This is the first imaging evidence that the

  18. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  19. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  20. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  1. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  2. 46 CFR 172.070 - Intact stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability. 172.070 Section 172.070 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO BULK CARGOES Special Rules Pertaining to a Vessel That Carries a Cargo Regulated Under 33 CFR Part...

  3. HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

    EPA Science Inventory

    The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

  4. 46 CFR 178.320 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements. 178.320 Section 178.320... TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.320 Intact stability... stability proof test in accordance with § 178.330 of this part in the presence of a Coast Guard...

  5. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements-general. 178.310 Section... (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.310 Intact stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  6. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements-general. 178.310 Section... (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.310 Intact stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  7. 46 CFR 178.310 - Intact stability requirements-general.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-general. 178.310 Section... (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178.310 Intact stability requirements—general. (a) Except as provided in paragraph (c) of this section, each vessel...

  8. Heterogeneity of dynein structure implies coordinated suppression of dynein motor activity in the axoneme.

    PubMed

    Maheshwari, Aditi; Ishikawa, Takashi

    2012-08-01

    Axonemal dyneins provide the driving force for flagellar/ciliary bending. Nucleotide-induced conformational changes of flagellar dynein have been found both in vitro and in situ by electron microscopy, and in situ studies demonstrated the coexistence of at least two conformations in axonemes in the presence of nucleotides (the apo and the nucleotide-bound forms). The distribution of the two forms suggested cooperativity between adjacent dyneins on axonemal microtubule doublets. Although the mechanism of such cooperativity is unknown it might be related to the mechanism of bending. To explore the mechanism by which structural heterogeneity of axonemal dyneins is induced by nucleotides, we used cilia from Tetrahymena thermophila to examine the structure of dyneins in a) the intact axoneme and b) microtubule doublets separated from the axoneme, both with and without additional pure microtubules. We also employed an ATPase assay on these specimens to investigate dynein activity functionally. Dyneins on separated doublets show more activation by nucleotides than those in the intact axoneme, both structurally and in the ATPase assay, and this is especially pronounced when the doublets are coupled with added microtubules, as expected. Paralleling the reduced ATPase activity in the intact axonemes, a lower proportion of these dyneins are in the nucleotide-bound form. This indicates a coordinated suppression of dynein activity in the axoneme, which could be the key for understanding the bending mechanism.

  9. Small Particles Intact Capture Experiment (SPICE)

    NASA Technical Reports Server (NTRS)

    Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

    1994-01-01

    The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

  10. Motor Starters

    NASA Astrophysics Data System (ADS)

    1986-01-01

    The power factor controller (PFC) was invented by a NASA engineer. It matches voltage with a motor's actual need by sensing shifts in the relationship between voltage and current flow. With the device, power can be trimmed as much as 65%. Intellinet adopted this technology and designed "soft start" and "load-responsive" control modes to start engines gradually and recycle voltage without reducing motor speed. Other features are lower motor heat and faster fault identification.

  11. Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

    PubMed Central

    Engel, Benjamin D.; Ludington, William B.

    2009-01-01

    The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly. PMID:19805630

  12. KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes.

    PubMed

    Sanchez, Marco A; Tran, Khoa D; Valli, Jessica; Hobbs, Sam; Johnson, Errin; Gluenz, Eva; Landfear, Scott M

    2016-09-16

    African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability. PMID:27489106

  13. Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens.

    PubMed

    Echeita, M Aurora; Herrera, Silvia; Garaizar, Javier; Usera, Miguel A

    2002-03-01

    Most Salmonella serotypes alternatively express phase 1 or phase 2 flagellar antigens encoded by fliC and fljB genes respectively. Flagellar phase reversal to identify both flagellar antigens is not necessary at the genetic level. Variable internal regions of the fljB genes encoding H:1,w, H:e,n,x and H:e,n,z15 antigens have been sequenced and the specific sites for each antigen determined in selected Salmonella serotypes. These results, together with flagellar H1 complex variable internal sequences previously published, have been used to design a multiplex-PCR to identify H:1,2, H:1,5, H:1,6, H:1,7, H:1,w, H:e,n,x and H:e,n,z15 second-phase antigens. These antigens are part of the most common Salmonella serotypes possessing second-phase flagellar antigens. This multiplex-PCR includes 10 primers. A total of 140 Salmonella strains associated with 49 different serotypes were tested. Each strain generated one second-phase-specific antigen fragment, ranging between 50 and 400 bps. Twenty-five strains associated with 17 serotypes, with no second-phase antigen or with an antigen different from those tested in this work, did not generate any fragments. The method is quick, specific and reproducible and is independent of the phase expressed by the bacteria when tested.

  14. Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion

    PubMed Central

    1976-01-01

    To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion. PMID:1245545

  15. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  16. Adenine Nucleotide Metabolism and a Role for AMP in Modulating Flagellar Waveforms in Mouse Sperm1

    PubMed Central

    Vadnais, Melissa L.; Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa; Lin, Angel M.; Al-Alao, Osama; Gerton, George L.

    2014-01-01

    ABSTRACT While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase. PMID:24740601

  17. Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli.

    PubMed

    Sampaio, Suely C F; Luiz, Wilson B; Vieira, Mônica A M; Ferreira, Rita C C; Garcia, Bruna G; Sinigaglia-Coimbra, Rita; Sampaio, Jorge L M; Ferreira, Luís C S; Gomes, Tânia A T

    2016-04-01

    The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliCa nd fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The a EPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

  18. Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli

    PubMed Central

    Sampaio, Suely C. F.; Luiz, Wilson B.; Vieira, Mônica A. M.; Ferreira, Rita C. C.; Garcia, Bruna G.; Sinigaglia-Coimbra, Rita; Sampaio, Jorge L. M.; Ferreira, Luís C. S.

    2016-01-01

    The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliC and fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of aEPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of aEPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The aEPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of aEPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

  19. Molecular motors

    NASA Astrophysics Data System (ADS)

    Allemand, Jean François Desbiolles, Pierre

    2015-10-01

    How do we move? More precisely, what are the molecular mechanisms that can explain that our muscles, made of very small components can move at a osopic scale? To answer these questions we must introduce molecular motors. Those motors are proteins, or small protein assemblies that, in our cells, transform chemical energy into mechanical work. Then, like we could do for a oscopic motor, used in a car or in a fan, we are going to study the basic behavior of these molecular machines, present what are their energy sources, calculate their power, their yield. If molecular motors are crucial for our oscopic movements, we are going to see that they are also essential to cellular transport and that considering the activity of some enzymes as molecular motors bring some interesting new insights on their activity.

  20. Silica Aerogel Captures Cosmic Dust Intact

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1994-01-01

    The mesostructure of silica aerogel resembles stings of grapes, ranging in size from 10 to 100 angstrom. This fine mesostructure transmits nearly 90 percent of incident light in the visible, while providing sufficiently gentle dissipation of the kinetric energy of hypervelocity cosmic dust particles to permit their intact capture. We introduced silica aerogel in 1987 as capture medium to take advantage of its low density, fine mesostruicture and most importantly, its transparency, allowing optical location of captured micron sized particles.

  1. The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression.

    PubMed

    Mouslim, Chakib; Hughes, Kelly T

    2014-03-01

    The flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD₄C₂ transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1(flhDC) and P5(flhDC), were functional in a wild-type background, while P6(flhDC) was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1(flhDC) initiates flagellar assembly and a negative autoregulatory loop through FlhD₄C₂-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1(flhDC) also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD₄C₂ transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.

  2. Transcriptional organization of the region encoding the synthesis of the flagellar filament in Pseudomonas fluorescens.

    PubMed

    Redondo-Nieto, Miguel; Lloret, Javier; Larenas, Javiera; Barahona, Emma; Navazo, Ana; Martínez-Granero, Francisco; Capdevila, Silvia; Rivilla, Rafael; Martín, Marta

    2008-06-01

    Pseudomonas fluorescens F113 is motile by means of type b flagella. Analysis of the region encoding the synthesis of the flagellar filament has shown a transcriptional organization different from that of type a flagella. Additionally to the promoters driving fliC, fliD, and fleQ expression, we have found promoters upstream of the flaG gene and the fliST operon. These promoters were functional in vivo. Both promoters have been mapped and appear to be dependent on the vegetative sigma factor and independent of FleQ, the master regulator of flagellum synthesis.

  3. A new automated method of image analysis: comparison of ciliary and flagellar beats.

    PubMed

    Schoevaert, D; Marano, F; Serres, C; Berrebah, H

    1990-01-01

    A new automated method of image analysis of sperm flagellar (human) and cilia (Dunaliella) bends is developed. This method permits an automatic determination of the line characterizing the flagellum. Two dynamic parameters are measured: the wave propagation velocity and the wave curvature radius. The data reveal similar patterns in the propagation of the principal and reverse waves between flagelated and ciliated cells. Conversely, differences are seen in principal wave curvature due perhaps to the presence of periaxonemal structures in the flagellum, absent in cilium. The identical patterns of reverse wave curvaturei in both systems may be linked to axonemal limitations.

  4. Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective.

    PubMed

    Pallen, Mark J; Beatson, Scott A; Bailey, Christopher M

    2005-04-01

    We review the biology of non-flagellar type-III secretion systems from a Darwinian perspective, highlighting the themes of evolution, conservation, variation and decay. The presence of these systems in environmental organisms such as Myxococcus, Desulfovibrio and Verrucomicrobium hints at roles beyond virulence. We review newly discovered sequence homologies (e.g., YopN/TyeA and SepL). We discuss synapomorphies that might be useful in formulating a taxonomy of type-III secretion. The problem of information overload is likely to be ameliorated by launch of a web site devoted to the comparative biology of type-III secretion ().

  5. Measuring mitochondrial function in intact cardiac myocytes

    PubMed Central

    Dedkova, Elena N.; Blatter, Lothar A.

    2011-01-01

    Mitochondria are involved in cellular functions that go beyond the traditional role of these organelles as the power plants of the cell. Mitochondria have been implicated in several human diseases, including cardiac dysfunction, and play a role in the aging process. Many aspects of our knowledge of mitochondria stem from studies performed on the isolated organelle. Their relative inaccessibility imposes experimental difficulties to study mitochondria in their natural environment – the cytosol of intact cells – and has hampered a comprehensive understanding of the plethora of mitochondrial functions. Here we review currently available methods to study mitochondrial function in intact cardiomyocytes. These methods primarily use different flavors of fluorescent dyes and genetically encoded fluorescent proteins in conjunction with high-resolution imaging techniques. We review methods to study mitochondrial morphology, mitochondrial membrane potential, Ca2+ and Na+ signaling, mitochondrial pH regulation, redox state and ROS production, NO signaling, oxygen consumption, ATP generation and the activity of the mitochondrial permeability transition pore. Where appropriate we complement this review on intact myocytes with seminal studies that were performed on isolated mitochondria, permeabilized cells, and in whole hearts. PMID:21964191

  6. Radioimmunoassay for intact Gross mouse leukemia virus.

    PubMed Central

    Yalow, R S; Gross, L

    1976-01-01

    A radioimmunoassay for intact Gross leukemia virus has been developed using 125I-labeled Gross virus grown in tissue culture and guinea pig antisera to Gross virus grown either in tissue culture or harvested from leukemic C3H(f) mice. Separation of bound from free labeled virus was effected using the double antibody method. The assay can detect fewer than 10(8) virus particles and has been used to measure the viral content of individual organs from inoculated leukemic C3H(f) mice and from Ak mice with spontaneous leukemia. Organs from noninoculated healthy C3H(f) mice crossreacted poorly in the system, virus generally being detectable only in the thymus and spleen and at low concentration. In some of the inoculated C3H(f) leukemic mice the viral content of as little as 0.5 mul of plasma is measurable. That this assay is for intact virus and not for soluble antigens of the viral envelope was proven by the observation that the immunoreactive material of plasma and extracts from thymus and liver of leukemic mice has a buoyant denisty in sucrose of 1.17-1.18 g/ml, corresponding to that of intact virus grown in tissue culture. With this sensitivity it may now be possible to quantitate viral concentrations in tissue and body fluids from the time of inoculation through the development of obvious pathology. PMID:1066697

  7. Multi-View Intact Space Learning.

    PubMed

    Xu, Chang; Tao, Dacheng; Xu, Chao

    2015-12-01

    It is practical to assume that an individual view is unlikely to be sufficient for effective multi-view learning. Therefore, integration of multi-view information is both valuable and necessary. In this paper, we propose the Multi-view Intact Space Learning (MISL) algorithm, which integrates the encoded complementary information in multiple views to discover a latent intact representation of the data. Even though each view on its own is insufficient, we show theoretically that by combing multiple views we can obtain abundant information for latent intact space learning. Employing the Cauchy loss (a technique used in statistical learning) as the error measurement strengthens robustness to outliers. We propose a new definition of multi-view stability and then derive the generalization error bound based on multi-view stability and Rademacher complexity, and show that the complementarity between multiple views is beneficial for the stability and generalization. MISL is efficiently optimized using a novel Iteratively Reweight Residuals (IRR) technique, whose convergence is theoretically analyzed. Experiments on synthetic data and real-world datasets demonstrate that MISL is an effective and promising algorithm for practical applications. PMID:26539856

  8. Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly.

    PubMed

    Fan, Zhen-Chuan; Behal, Robert H; Geimer, Stefan; Wang, Zhaohui; Williamson, Shana M; Zhang, Haili; Cole, Douglas G; Qin, Hongmin

    2010-08-01

    DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1-depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.

  9. Stepper motor

    NASA Technical Reports Server (NTRS)

    Dekramer, Cornelis

    1994-01-01

    The purpose of this document is to describe the more commonly used permanent magnet stepper motors for spaceflight. It will discuss the mechanical and electrical aspects of the devices, their torque behavior, those parameters which need to be controlled and measured, and test methods to be employed. It will also discuss torque margins, compare these to the existing margin requirements, and determine the applicability of these requirements. Finally it will attempt to generate a set of requirements which will be used in any stepper motor procurement and will fully characterize the stepper motor behavior in a consistent and repeatable fashion.

  10. Nonlinear instability in flagellar dynamics: a novel modulation mechanism in sperm migration?

    PubMed Central

    Gadêlha, H.; Gaffney, E. A.; Smith, D. J.; Kirkman-Brown, J. C.

    2010-01-01

    Throughout biology, cells and organisms use flagella and cilia to propel fluid and achieve motility. The beating of these organelles, and the corresponding ability to sense, respond to and modulate this beat is central to many processes in health and disease. While the mechanics of flagellum–fluid interaction has been the subject of extensive mathematical studies, these models have been restricted to being geometrically linear or weakly nonlinear, despite the high curvatures observed physiologically. We study the effect of geometrical nonlinearity, focusing on the spermatozoon flagellum. For a wide range of physiologically relevant parameters, the nonlinear model predicts that flagellar compression by the internal forces initiates an effective buckling behaviour, leading to a symmetry-breaking bifurcation that causes profound and complicated changes in the waveform and swimming trajectory, as well as the breakdown of the linear theory. The emergent waveform also induces curved swimming in an otherwise symmetric system, with the swimming trajectory being sensitive to head shape—no signalling or asymmetric forces are required. We conclude that nonlinear models are essential in understanding the flagellar waveform in migratory human sperm; these models will also be invaluable in understanding motile flagella and cilia in other systems. PMID:20462879

  11. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein-TbBILBO1.

    PubMed

    Perdomo, Doranda; Bonhivers, Mélanie; Robinson, Derrick R

    2016-03-02

    Sub-species of Trypanosoma brucei are the causal agents of human African sleeping sickness and Nagana in domesticated livestock. These pathogens have developed an organelle-like compartment called the flagellar pocket (FP). The FP carries out endo- and exocytosis and is the only structure this parasite has evolved to do so. The FP is essential for parasite viability, making it an interesting structure to evaluate as a drug target, especially since it has an indispensible cytoskeleton component called the flagellar pocket collar (FPC). The FPC is located at the neck of the FP where the flagellum exits the cell. The FPC has a complex architecture and division cycle, but little is known concerning its organization. Recent work has focused on understanding how the FP and the FPC are formed and as a result of these studies an important calcium-binding, polymer-forming protein named TbBILBO1 was identified. Cellular biology analysis of TbBILBO1 has demonstrated its uniqueness as a FPC component and until recently, it was unknown what structural role it played in forming the FPC. This review summarizes the recent data on the polymer forming properties of TbBILBO1 and how these are correlated to the FP cytoskeleton.

  12. Role of flgA for Flagellar Biosynthesis and Biofilm Formation of Campylobacter jejuni NCTC11168.

    PubMed

    Kim, Joo-Sung; Park, Changwon; Kim, Yun-Ji

    2015-11-01

    The complex roles of flagella in the pathogenesis of Campylobacter jejuni, a major cause of worldwide foodborne diarrheal disease, are important. Compared with the wild-type, an insertional mutation of the flgA gene (cj0769c) demonstrated significant decrease in the biofilm formation of C. jejuni NCTC11168 on major food contact surfaces, such as polystyrene, stainless steel, and borosilicate glass. The flgA mutant was completely devoid of flagella and non-motile whereas the wild-type displayed the full-length flagella and motility. In addition, the biofilm formation of the wild-type was inversely dependent on the viscosity of the media. These results support that flagellar-mediated motility plays a significant role in the biofilm formation of C. jejuni NCTC11168. Moreover, our adhesion assay suggests that it plays an important role during biofilm maturation after initial attachment. Furthermore, C. jejuni NCTC11168 wild-type formed biofilm with a net-like structure of extracellular fiber-like material, but such a structure was significantly reduced in the biofilm of the flgA mutant. It supports that the extracellular fiber-like material may play a significant role in the biofilm formation of C. jejuni. This study demonstrated that flgA is essential for flagellar biosynthesis and motility, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

  13. Common Evolutionary Origin for the Rotor Domain of Rotary Atpases and Flagellar Protein Export Apparatus

    PubMed Central

    Kishikawa, Jun-ichi; Ibuki, Tatsuya; Nakamura, Shuichi; Nakanishi, Astuko; Minamino, Tohru; Miyata, Tomoko; Namba, Keiichi; Konno, Hiroki; Ueno, Hiroshi; Imada, Katsumi; Yokoyama, Ken

    2013-01-01

    The V1- and F1- rotary ATPases contain a rotor that rotates against a catalytic A3B3 or α3β3 stator. The rotor F1-γ or V1-DF is composed of both anti-parallel coiled coil and globular-loop parts. The bacterial flagellar type III export apparatus contains a V1/F1-like ATPase ring structure composed of FliI6 homo-hexamer and FliJ which adopts an anti-parallel coiled coil structure without the globular-loop part. Here we report that FliJ of Salmonella enterica serovar Typhimurium shows a rotor like function in Thermus thermophilus A3B3 based on both biochemical and structural analysis. Single molecular analysis indicates that an anti-parallel coiled-coil structure protein (FliJ structure protein) functions as a rotor in A3B3. A rotary ATPase possessing an F1-γ-like protein generated by fusion of the D and F subunits of V1 rotates, suggesting F1-γ could be the result of a fusion of the genes encoding two separate rotor subunits. Together with sequence comparison among the globular part proteins, the data strongly suggest that the rotor domains of the rotary ATPases and the flagellar export apparatus share a common evolutionary origin. PMID:23724081

  14. Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas

    PubMed Central

    Tam, Lai-Wa; Dentler, William L.; Lefebvre, Paul A.

    2003-01-01

    Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435–442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly. PMID:14610061

  15. Building a radial spoke: flagellar radial spoke protein 3 (RSP3) is a dimer.

    PubMed

    Wirschell, Maureen; Zhao, Feifei; Yang, Chun; Yang, Pinfen; Diener, Dennis; Gaillard, Anne; Rosenbaum, Joel L; Sale, Winfield S

    2008-03-01

    Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility. PMID:18157907

  16. Calcium ion-mediated assembly and function of glycosylated flagellar sheath of marine magnetotactic bacterium.

    PubMed

    Lefèvre, Christopher T; Santini, Claire-Lise; Bernadac, Alain; Zhang, Wei-Jia; Li, Ying; Wu, Long-Fei

    2010-12-01

    Flagella of some pathogens or marine microbes are sheathed by an apparent extension of the outer cell membrane. Although flagellar sheath has been reported for almost 60 years, little is known about its function and the mechanism of its assembly. Recently, we have observed a novel type of sheath that encloses a flagellar bundle, instead of a single flagellum, in a marine magnetotactic bacterium MO-1. Here, we reported isolation and characterization of the sheath which can be described as a six-start, right-handed helical tubular structure with a diameter of about 100 nm, and a pitch of helix of about 260 nm. By proteomic, microscopic and immunolabelling analyses, we showed that the sheath of MO-1 consists of glycoprotein with an apparent molecular mass > 350 kDa. This protein, named sheath-associated protein (Sap), shows homology with bacterial adhesins and eukaryotic calcium-dependent adherent proteins (cadherin). Most importantly, we showed that calcium ions mediate the assembly of the tubular-shaped sheath and disintegration of the sheath was deleterious for smooth swimming of MO-1 cells. The disintegrated sheath was efficiently reconstituted in vitro by adding calcium ions. Altogether, these results demonstrate a novel bacterial Ca(2+) -dependent surface architecture, which is essential for bacterial swimming. PMID:21091512

  17. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin

    PubMed Central

    Brumley, Douglas R.; Polin, Marco; Pedley, Timothy J.; Goldstein, Raymond E.

    2015-01-01

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behaviour—possibly influenced by both mechanical interactions and direct biological regulation—is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves. PMID:26040592

  18. Serotyping of Serratia marcescens: current status of seven recently described flagellar (H) antigens.

    PubMed

    Traub, W H; Fukushima, P I

    1979-07-01

    The slightly revised, current scheme of 20 flagellar (H) antigens of Serratia marcesens was examined. The seven new H antigens were demonstrated to be antigenically distinct as determined with Le Minor's H-immobilization test. The H-immobilization antibodies of rabbit anti-H immune sera proved resistant to treatment with 2-mercaptoethanol and dithiothreitol, respectively. On the other hand, dual absorptions of rabbit anti-H immune sera with killed cells of Staphylococcus aureus strain Cowan I, i.e., protein A, failed to reduce significantly H-immobilization titers of rabbit sera, although human immunoglobulins G and M were bound by protein A. It was tentatively concluded that the 2-mercaptoethanol- and dithiothreitol-refractory H-immobilizing rabbit antibodies belonged to the immunoglublin M class. H-antigen (phase) variation was not demonstrable in several extramural, clinical isolates of S. marcescens for which this phenomenon had been claimed. Rather, four of these six isolates were found to consist of cell populations of two distinct serotypes, as also borne out by bacteriocin typing; the flagellar H-antigens of the remaining two isolates were stable, with minor, hterologous H-antigen cross-reactivity.

  19. Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania.

    PubMed

    Katta, Santharam S; Sahasrabuddhe, Amogh A; Gupta, Chhitar M

    2009-04-01

    Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids. PMID:19121339

  20. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin.

    PubMed

    Brumley, Douglas R; Polin, Marco; Pedley, Timothy J; Goldstein, Raymond E

    2015-07-01

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behavior--possibly influenced by both mechanical interactions and direct biological regulation--is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves. PMID:26040592

  1. A “Mechanistic” Explanation of the Multiple Helical Forms Adopted by Bacterial Flagellar Filaments

    PubMed Central

    Calladine, C.R.; Luisi, B.F.; Pratap, J.V.

    2013-01-01

    The corkscrew-like flagellar filaments emerging from the surface of bacteria such as Salmonella typhimurium propel the cells toward nutrient and away from repellents. This kind of motility depends upon the ability of the flagellar filaments to adopt a range of distinct helical forms. A filament is typically constructed from ~ 30,000 identical flagellin molecules, which self-assemble into a tubular structure containing 11 near-longitudinal protofilaments. A “mechanical” model, in which the flagellin building block has the capacity to switch between two principal interfacial states, predicts that the filament can assemble into a “canonical” family of 12 distinct helical forms, each having unique curvature and twist: these include two “extreme” straight forms having left- and right-handed twists, respectively, and 10 intermediate helical forms. Measured shapes of the filaments correspond well with predictions of the model. This report is concerned with two unanswered questions. First, what properties of the flagellin determine which of the 12 discrete forms is preferred? Second, how does the interfacial “switch” work, at a molecular level? Our proposed solution of these problems is based mainly on a detailed examination of differences between the available electron cryo-microscopy structures of the straight L and R filaments, respectively. PMID:23274110

  2. Differentiation of the major flagellar antigens of Pseudomonas aeruginosa by the slide coagglutination technique.

    PubMed

    Ansorg, R A; Knoche, M E; Spies, A F; Kraus, C J

    1984-07-01

    Antisera against the two major flagellar antigens of Pseudomonas aeruginosa were obtained by immunization of rabbits with isolated flagella and absorption of contaminating antisomatic antibodies. In the conventional slide agglutination test, the pure H antisera did not agglutinate the flagellated cells of the homologous strains. The addition of protein A-bearing staphylococci to H antiserum and homologous flagellated cells, the so-called slide coagglutination, results in a rapid development of flaky clumps. H coagglutination tests of reference strains, which formerly have been H typed by long-term tube agglutination and by the indirect fluorescent-antibody technique, yielded exactly the same subdivision of the strains in H type a and H type b as the more laborious and time-consuming methods. O grouping and H typing of 181 isolates from clinical specimens revealed a free combination of the somatic and flagellar antigens. 25 OH serovars were found. The simple and rapid coagglutination technique can promote the serovar determination of P. aeruginosa, particularly for the purpose of hospital infection control. PMID:6430957

  3. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein—TbBILBO1

    PubMed Central

    Perdomo, Doranda; Bonhivers, Mélanie; Robinson, Derrick R.

    2016-01-01

    Sub-species of Trypanosoma brucei are the causal agents of human African sleeping sickness and Nagana in domesticated livestock. These pathogens have developed an organelle-like compartment called the flagellar pocket (FP). The FP carries out endo- and exocytosis and is the only structure this parasite has evolved to do so. The FP is essential for parasite viability, making it an interesting structure to evaluate as a drug target, especially since it has an indispensible cytoskeleton component called the flagellar pocket collar (FPC). The FPC is located at the neck of the FP where the flagellum exits the cell. The FPC has a complex architecture and division cycle, but little is known concerning its organization. Recent work has focused on understanding how the FP and the FPC are formed and as a result of these studies an important calcium-binding, polymer-forming protein named TbBILBO1 was identified. Cellular biology analysis of TbBILBO1 has demonstrated its uniqueness as a FPC component and until recently, it was unknown what structural role it played in forming the FPC. This review summarizes the recent data on the polymer forming properties of TbBILBO1 and how these are correlated to the FP cytoskeleton. PMID:26950156

  4. DksA and ppGpp Directly Regulate Transcription of the Escherichia coli Flagellar Cascade

    PubMed Central

    Lemke, Justin J.; Durfee, Tim; Gourse, Richard L.

    2009-01-01

    The components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade coordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the σ70-dependent fliA promoter transcribing the gene for σ28. However, DksA and ppGpp do not affect the σ28-dependent fliA promoter or the σ28-dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and σ28 expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby coordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell’s largest macromolecular complexes. PMID:19889089

  5. Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits

    PubMed Central

    Juhas, Mario; Evans, Lewis D. B.; Frost, Joe; Davenport, Peter W.; Yarkoni, Orr; Fraser, Gillian M.; Ajioka, James W.

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

  6. NgUNC-119, Naegleria homologue of UNC-119, localizes to the flagellar rootlet.

    PubMed

    Chung, Sunglan; Kang, Seungmin; Paik, Soonyoung; Lee, Joohun

    2007-03-01

    The UNC-119 family of proteins is ubiquitous in animals. The expression of UNC-119 is prominent in neural tissues including photoreceptor cells. Homologues of UNC-119 are also found in ciliated (or flagellated) single-celled organisms; however, the cellular distribution of this protein in protists is unknown. We cloned and characterized a homologue of unc-119 from the ameboflagellate Naegleria gruberi (Ngunc-119) and identified the cellular distribution of the protein. The Ngunc-119 open reading frame contained 570 nucleotides encoding a protein of 189 amino acids with a predicted molecular weight of 22.1 kDa, which is similar to that of Paramecium UNC-119 and Trypanosoma UNC-119. These three proteins are 46-48% identical in their amino acid sequences. The smaller NgUNC-119 corresponds to the conserved C-terminal 3/4 of the UNC-119 from multi-cellular organisms. The amino acid sequence of NgUNC-119 is 43-50% identical to that of the conserved C-terminal regions. NgUNC-119 was not found in growing amoebae but accumulated rapidly after the initiation of differentiation into flagellates. Indirect immunofluorescence staining of differentiating N. gruberi showed that NgUNC-119 begins to concentrate at a spot near the nucleus of differentiating cells and then elongates into a filamentous structure. Purification and indirect immunofluorescence staining of the Naegleria flagellar rootlet suggested that NgUNC-119 is a component of the flagellar rootlet.

  7. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin.

    PubMed

    Brumley, Douglas R; Polin, Marco; Pedley, Timothy J; Goldstein, Raymond E

    2015-07-01

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behavior--possibly influenced by both mechanical interactions and direct biological regulation--is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves.

  8. Calcium Signaling in Intact Dorsal Root Ganglia

    PubMed Central

    Gemes, Geza; Rigaud, Marcel; Koopmeiners, Andrew S.; Poroli, Mark J.; Zoga, Vasiliki; Hogan, Quinn H.

    2013-01-01

    Background Ca2+ is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca2+ signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca2+ recording techniques use high K+ or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. Methods Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. Results Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca2+ transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Aβ-type neurons. Unitary Ca2+ transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca2+ transients in C-type neurons, but increased transients in Aβ-type neurons. Conclusions Refined observation of Ca2+ signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state. PMID:20526180

  9. Quantitative proteomic analysis of intact plastids.

    PubMed

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described. PMID:24136541

  10. Quantitative proteomic analysis of intact plastids.

    PubMed

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

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

    PubMed

    Terauchi, Takashi; Terashima, Hiroyuki; Kojima, Seiji; Homma, Michio

    2011-08-01

    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.

  12. 50 CFR 622.276 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated,...

  13. 50 CFR 622.381 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.381 Section 622... Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia... head and fins intact. Such fish may be eviscerated, gilled, and scaled, but must otherwise...

  14. 50 CFR 622.276 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated,...

  15. 7 CFR 160.29 - Containers to remain intact.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Containers to remain intact. 160.29 Section 160.29... STANDARDS FOR NAVAL STORES Analysis, Inspection, and Grading on Request § 160.29 Containers to remain intact... the containers holding such naval stores remain intact as sampled until the analysis,...

  16. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  17. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  18. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  19. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  20. 33 CFR 157.22 - Intact stability requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Intact stability requirements... OIL IN BULK Design, Equipment, and Installation § 157.22 Intact stability requirements. All tank ships of 5,000 DWT and above contracted after December 3, 2001 must comply with the intact...

  1. Seroprevalence in Chickens against Campylobacter jejuni Flagellar Capping Protein (FliD) in Selected Areas of the U.S.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis. Poultry products are regarded as a major source for human infection with this microorganism. We have demonstrated that the flagellar capping protein (FliD) of C. jejuni is highl...

  2. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

    2005-01-01

    The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

  3. The Structure of the Salmonella typhimurium Type III Secretion System Needle Shows Divergence from the Flagellar System

    PubMed Central

    Galkin, Vitold E.; Schmied, Wolfgang H.; Schraidt, Oliver; Marlovits, Thomas C.; Egelman, Edward H.

    2010-01-01

    The Type III Secretion System (T3SS) is essential for the infectivity of many pathogenic Gram-negative bacteria. The T3SS contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. The homology between the T3SS and the bacterial flagellar system has been firmly established, based upon both sequence similarities between respective proteins in the two systems and the structural homology of the higher-order assemblies. It has previously been shown that the Shigella flexneri needle has a helical symmetry of ~ 5.6 subunits per turn, which is quite similar to that of the most intensively studied flagellar filament, from Salmonella typhimurium, which has ~ 5.5 subunits per turn. We now show that the S. typhimurium needle, expected by homology arguments to be more similar to the S. typhimurium flagellar filament than is the needle from Shigella, actually has ~ 6.3 subunits per turn. It is not currently understood how host cell contact, made at the tip of the needle, is communicated to the secretory system at the base. In contrast to the S. typhimurium flagellar filament, which shows a nearly crystalline order, the S. typhimurium needle has a highly variable symmetry, which could be used to transmit information about host cell contact. PMID:20060835

  4. Induction of Overt Menstruation in Intact Mice

    PubMed Central

    Rudolph, Marion; Döcke, Wolf-Dietrich; Müller, Andrea; Menning, Astrid; Röse, Lars; Zollner, Thomas Matthias; Gashaw, Isabella

    2012-01-01

    The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply. PMID:22412950

  5. An Empirical Failure Criterion for Intact Rocks

    NASA Astrophysics Data System (ADS)

    Peng, Jun; Rong, Guan; Cai, Ming; Wang, Xiaojiang; Zhou, Chuangbing

    2014-03-01

    The parameter m i is an important rock property parameter required for use of the Hoek-Brown failure criterion. The conventional method for determining m i is to fit a series of triaxial compression test data. In the absence of laboratory test data, guideline charts have been provided by Hoek to estimate the m i value. In the conventional Hoek-Brown failure criterion, the m i value is a constant for a given rock. It is observed that using a constant m i may not fit the triaxial compression test data well for some rocks. In this paper, a negative exponent empirical model is proposed to express m i as a function of confinement, and this exercise leads us to a new empirical failure criterion for intact rocks. Triaxial compression test data of various rocks are used to fit parameters of this model. It is seen that the new empirical failure criterion fits the test data better than the conventional Hoek-Brown failure criterion for intact rocks. The conventional Hoek-Brown criterion fits the test data well in the high-confinement region but fails to match data well in the low-confinement and tension regions. In particular, it overestimates the uniaxial compressive strength (UCS) and the uniaxial tensile strength of rocks. On the other hand, curves fitted by the proposed empirical failure criterion match test data very well, and the estimated UCS and tensile strength agree well with test data.

  6. Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor

    PubMed Central

    Mizuno, Katsutoshi; Shiba, Kogiku; Okai, Masahiko; Takahashi, Yusuke; Shitaka, Yuji; Oiwa, Kazuhiro; Tanokura, Masaru; Inaba, Kazuo

    2012-01-01

    Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca2+ influx through specific Ca2+ channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca2+ is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca2+-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca2+ concentrations. This study describes the missing link between chemoattractant-mediated Ca2+ signaling and motor-driven microtubule sliding during sperm chemotaxis. PMID:23169663

  7. Recollections of Parent Characteristics and Attachment Patterns for College Women of Intact vs. Non-Intact Families

    ERIC Educational Resources Information Center

    Kilmann, Peter R.; Carranza, Laura V.; Vendemia, Jennifer M. C.

    2006-01-01

    This study contrasted offsprings' attachment patterns and recollections of parent characteristics in two college samples: 147 females from intact biological parents and 157 females of parental divorce. Secure females from intact or non-intact families rated parents positively, while insecure females rated parents as absent, distant, and demanding.…

  8. Advanced Motors

    SciTech Connect

    Knoth, Edward A; Chelluri, Bhanumathi; Schumaker, Edward J

    2012-12-14

    vProject Summary Transportation energy usage is predicted to increase substantially by 2020. Hybrid vehicles and fuel cell powered vehicles are destined to become more prominent as fuel prices rise with the demand. Hybrid and fuel cell vehicle platforms are both dependent on high performance electric motors. Electric motors for transportation duty will require sizeable low-speed torque to accelerate the vehicle. As motor speed increases, the torque requirement decreases which results in a nearly constant power motor output. Interior permanent magnet synchronous motors (IPMSM) are well suited for this duty. , , These rotor geometries are configured in straight lines and semi circular arc shapes. These designs are of limited configurations because of the lack of availability of permanent magnets of any other shapes at present. We propose to fabricate rotors via a novel processing approach where we start with magnet powders and compact them into a net shape rotor in a single step. Using this approach, widely different rotor designs can be implemented for efficiency. The current limitation on magnet shape and thickness will be eliminated. This is accomplished by co-filling magnet and soft iron powders at specified locations in intricate shapes using specially designed dies and automatic powder filling station. The process fundamentals for accomplishing occurred under a previous Applied Technology Program titled, Motors and Generators for the 21st Century. New efficient motor designs that are not currently possible (or cost prohibitive) can be accomplished by this approach. Such an approach to motor fabrication opens up a new dimension in motor design. Feasibility Results We were able to optimize a IPMSM rotor to take advantage of the powder co-filling and DMC compaction processing methods. The minimum low speed torque requirement of 5 N-m can be met through an optimized design with magnet material having a Br capability of 0.2 T. This level of magnetic performance can

  9. Motor activity under weightless conditions

    NASA Technical Reports Server (NTRS)

    Kasyan, I. I.; Kopanev, V. I.; Cherepakhin, M. A.; Yuganov, Y. M.

    1975-01-01

    The material presented on the motor activity under weightless conditions (brief and long) leads to the conclusion that it is not significantly disrupted, if those being examined are secured at the workplaces. Some discoordination of movement, moderately expressed disruption of the precision of reproduction of assigned muscular forces, etc., were observed. Motor disorders decrease significantly in proportion to the length of stay under weightless conditions. This apparently takes place, as a consequence of formation of a new functional system, adequate to the conditions of weightlessness. Tests on intact and labyrinthectomized animals have demonstrated that signaling from the inner ear receptors is superfluous in weightlessness, since it promotes the onset of disruptions in the combined work of the position analyzers.

  10. Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis.

    PubMed Central

    Limberger, R J; Slivienski, L L; Samsonoff, W A

    1994-01-01

    The periplasmic flagellum of Treponema phagedenis consists of the flagellar filament and hook-basal body. We report here a characterization of the hook gene and flagellar hook of T. phagedenis, and in the process of this analysis we found evidence that the hook polypeptide is likely cross-linked in situ. A T. phagedenis genomic library was screened with a Treponema pallidum antiserum, and the DNA segments from several positive plaques were subcloned and sequenced. DNA sequencing of two overlapping segments revealed a 1,389-nucleotide (nt) open reading frame (ORF) with a deduced amino acid sequence that was 36% identical to that of FlgE, the hook polypeptide of Salmonella typhimurium. This gene was designated T. phagedenis flgE. Beginning at 312 nt downstream from flgE was a partial ORF of 486 nt with a deduced amino acid sequence that was 33% identical to that of MotA of Bacillus subtilis, a polypeptide that enables flagellar rotation. Upstream of flgE, separated by 39 nt, was a partial (291-nt) ORF with a deduced amino acid sequence that was homologous to that of ORF8, a polypeptide of unknown function located in an operon encoding polypeptides involved in motility of B. subtilis. The T. phagedenis flgE gene was cloned into an Escherichia coli protein expression plasmid, and the purified recombinant protein was used to prepare a FlgE antiserum. Western blots (immunoblots) of whole-cell lysates probed with this antiserum revealed a 55-kDa polypeptide and a ladder of polypeptide bands with increasing molecular masses. T. phagedenis hooks were then isolated and purified, and electron microscopic analysis revealed that the morphology of the hooks resembled that in other bacteria. The hooks were slightly curved and had an average length of 69 +/- 8 nm and a diameter of 23 +/- 1 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots of purified hook preparations using the FlgE antiserum also revealed a polypeptide ladder, suggesting that the

  11. Oligodeoxynucleotide Probes for Detecting Intact Cells

    NASA Technical Reports Server (NTRS)

    Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

    2004-01-01

    A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

  12. Motor neglect.

    PubMed Central

    Laplane, D; Degos, J D

    1983-01-01

    Motor neglect is characterised by an underutilisation of one side, without defects of strength, reflexes or sensibility. Twenty cases of frontal, parietal and thalamic lesions causing motor neglect, but all without sensory neglect, are reported. It is proposed that the cerebral structures involved in motor neglect are the same as those for sensory neglect and for the preparation of movement. As in sensory neglect, the multiplicity of the structures concerned suggests that this interconnection is necessary to maintain a sufficient level of activity. Predominance of left sided neglect by right sided lesions suggests that the left hemisphere is dominant for deliberate activity; hemispheric dominance could be applied to sensory neglect where conscious awareness would play the role of deliberate activity. PMID:6842219

  13. Heartbeat control in leeches. II. Fictive motor pattern.

    PubMed

    Wenning, Angela; Hill, Andrew A V; Calabrese, Ronald L

    2004-01-01

    The rhythmic beating of the tube-like hearts in the medicinal leech is driven and coordinated by rhythmic activity in segmental heart motor neurons. The motor neurons are controlled by rhythmic inhibitory input from a network of heart interneurons that compose the heartbeat central pattern generator. In the preceding paper, we described the constriction pattern of the hearts in quiescent intact animals and showed that one heart constricts in a rear-to-front wave (peristaltic coordination mode), while the other heart constricts in near unison over its length (synchronous coordination mode) and that they regularly switch coordination modes. Here we analyze intersegmental and side-to-side-coordination of the fictive motor pattern for heartbeat in denervated nerve cords. We show that the intersegmental phase relations among heart motor neurons in both coordination modes are independent of heartbeat period. This finding enables us to combine data from different experiments to form a detailed analysis of the relative phases, duty cycle, and intraburst spike frequency of the bursts of the segmental heart motor neurons. The fictive motor pattern and the constriction pattern seen in intact leeches closely match in their intersegmental and side-to-side coordination, indicating that sensory feedback is not necessary for properly phased intersegmental coordination. Moreover, the regular switches in coordination mode of the fictive motor pattern mimic those seen in intact animals indicating that these switches likely arise by a central mechanism.

  14. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  15. Protein methylation reactions in intact pea chloroplasts

    SciTech Connect

    Niemi, K.J. )

    1989-04-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.

  16. Drilling to gabbro in intact ocean crust.

    PubMed

    Wilson, Douglas S; Teagle, Damon A H; Alt, Jeffrey C; Banerjee, Neil R; Umino, Susumu; Miyashita, Sumio; Acton, Gary D; Anma, Ryo; Barr, Samantha R; Belghoul, Akram; Carlut, Julie; Christie, David M; Coggon, Rosalind M; Cooper, Kari M; Cordier, Carole; Crispini, Laura; Durand, Sedelia Rodriguez; Einaudi, Florence; Galli, Laura; Gao, Yongjun; Geldmacher, Jörg; Gilbert, Lisa A; Hayman, Nicholas W; Herrero-Bervera, Emilio; Hirano, Nobuo; Holter, Sara; Ingle, Stephanie; Jiang, Shijun; Kalberkamp, Ulrich; Kerneklian, Marcie; Koepke, Jürgen; Laverne, Christine; Vasquez, Haroldo L Lledo; Maclennan, John; Morgan, Sally; Neo, Natsuki; Nichols, Holly J; Park, Sung-Hyun; Reichow, Marc K; Sakuyama, Tetsuya; Sano, Takashi; Sandwell, Rachel; Scheibner, Birgit; Smith-Duque, Chris E; Swift, Stephen A; Tartarotti, Paola; Tikku, Anahita A; Tominaga, Masako; Veloso, Eugenio A; Yamasaki, Toru; Yamazaki, Shusaku; Ziegler, Christa

    2006-05-19

    Sampling an intact sequence of oceanic crust through lavas, dikes, and gabbros is necessary to advance the understanding of the formation and evolution of crust formed at mid-ocean ridges, but it has been an elusive goal of scientific ocean drilling for decades. Recent drilling in the eastern Pacific Ocean in Hole 1256D reached gabbro within seismic layer 2, 1157 meters into crust formed at a superfast spreading rate. The gabbros are the crystallized melt lenses that formed beneath a mid-ocean ridge. The depth at which gabbro was reached confirms predictions extrapolated from seismic experiments at modern mid-ocean ridges: Melt lenses occur at shallower depths at faster spreading rates. The gabbros intrude metamorphosed sheeted dikes and have compositions similar to the overlying lavas, precluding formation of the cumulate lower oceanic crust from melt lenses so far penetrated by Hole 1256D.

  17. Drilling to gabbro in intact ocean crust.

    PubMed

    Wilson, Douglas S; Teagle, Damon A H; Alt, Jeffrey C; Banerjee, Neil R; Umino, Susumu; Miyashita, Sumio; Acton, Gary D; Anma, Ryo; Barr, Samantha R; Belghoul, Akram; Carlut, Julie; Christie, David M; Coggon, Rosalind M; Cooper, Kari M; Cordier, Carole; Crispini, Laura; Durand, Sedelia Rodriguez; Einaudi, Florence; Galli, Laura; Gao, Yongjun; Geldmacher, Jörg; Gilbert, Lisa A; Hayman, Nicholas W; Herrero-Bervera, Emilio; Hirano, Nobuo; Holter, Sara; Ingle, Stephanie; Jiang, Shijun; Kalberkamp, Ulrich; Kerneklian, Marcie; Koepke, Jürgen; Laverne, Christine; Vasquez, Haroldo L Lledo; Maclennan, John; Morgan, Sally; Neo, Natsuki; Nichols, Holly J; Park, Sung-Hyun; Reichow, Marc K; Sakuyama, Tetsuya; Sano, Takashi; Sandwell, Rachel; Scheibner, Birgit; Smith-Duque, Chris E; Swift, Stephen A; Tartarotti, Paola; Tikku, Anahita A; Tominaga, Masako; Veloso, Eugenio A; Yamasaki, Toru; Yamazaki, Shusaku; Ziegler, Christa

    2006-05-19

    Sampling an intact sequence of oceanic crust through lavas, dikes, and gabbros is necessary to advance the understanding of the formation and evolution of crust formed at mid-ocean ridges, but it has been an elusive goal of scientific ocean drilling for decades. Recent drilling in the eastern Pacific Ocean in Hole 1256D reached gabbro within seismic layer 2, 1157 meters into crust formed at a superfast spreading rate. The gabbros are the crystallized melt lenses that formed beneath a mid-ocean ridge. The depth at which gabbro was reached confirms predictions extrapolated from seismic experiments at modern mid-ocean ridges: Melt lenses occur at shallower depths at faster spreading rates. The gabbros intrude metamorphosed sheeted dikes and have compositions similar to the overlying lavas, precluding formation of the cumulate lower oceanic crust from melt lenses so far penetrated by Hole 1256D. PMID:16627698

  18. Flagellar Kinematics and Swimming Behavior of Algal Cells in Viscoelastic Fluids

    NASA Astrophysics Data System (ADS)

    Arratia, Paulo; Yang, Jing; Gollub, Jerry

    2013-11-01

    The motility behavior of microorganisms can be significantly affected by the rheology of their fluidic environment. In this talk, we experimentally investigate the effects of fluid elasticity on both the flagella kinematics and swimming dynamics of the microscopic alga Chlamydomonas reinhardtii. We find that the flagellar beating frequency and wave speed are both enhanced by fluid elasticity. Interestingly, the swimming speeds during the alga power and recovery strokes are enhanced by fluid elasticity for De>1. Despite such enhancements, however, the alga net forward speed is hindered by fluid elasticity by as much as 30% compared to Newtonian fluids of similar shear viscosities. The motility enhancements could be explained by the mechanism of stress accumulation in the viscoelastic fluid. This work was supported by the National Science Foundation - DMR-1104705.

  19. Anatomical and Molecular Design of the Drosophila Antenna as a Flagellar Auditory Organ

    PubMed Central

    TODI, SOKOL V.; SHARMA, YASHODA; EBERL, DANIEL F.

    2007-01-01

    The molecular basis of hearing is less well understood than many other senses. However, recent studies in Drosophila have provided some important steps towards a molecular understanding of hearing. In this report, we summarize these findings and their implications on the relationship between hearing and touch. In Drosophila, hearing is accomplished by Johnston’s Organ, a chordotonal organ containing over 150 scolopidia within the second antennal segment. We will discuss anatomical features of the antenna and how they contribute to the function of this flagellar auditory receptor. The effects of several mutants, identified through mutagenesis screens or as homologues of vertebrate auditory genes, will be summarized. Based on evidence gathered from these studies, we propose a speculative model for how the chordotonal organ might function. PMID:15252880

  20. From Organelle to Protein Gel: A 6-Wk Laboratory Project on Flagellar Proteins

    PubMed Central

    Graziano, Mary R.

    2006-01-01

    Research suggests that undergraduate students learn more from lab experiences that involve longer-term projects. We have developed a one-semester laboratory sequence aimed at sophomore-level undergraduates. In designing this curriculum, we focused on several educational objectives: 1) giving students a feel for the scientific research process, 2) introducing them to commonly used lab techniques, and 3) building skills in both data analysis and scientific writing. Over the course of the semester, students carry out two project-based lab experiences and write two substantial lab reports modeled on primary literature. Student assessment data indicate that this lab curriculum achieved these objectives. This article describes the first of these projects, which uses the biflagellate alga Chlamydomonas reinhardtii to introduce students to the study of flagellar motility, protein synthesis, microtubule polymerization, organelle assembly, and protein isolation and characterization. PMID:17012215

  1. Ciliary and flagellar structure and function--their regulations by posttranslational modifications of axonemal tubulin.

    PubMed

    Konno, Alu; Setou, Mitsutoshi; Ikegami, Koji

    2012-01-01

    Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, α- and β-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, Δ2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge. PMID:22364873

  2. Ciliary and flagellar structure and function--their regulations by posttranslational modifications of axonemal tubulin.

    PubMed

    Konno, Alu; Setou, Mitsutoshi; Ikegami, Koji

    2012-01-01

    Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, α- and β-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, Δ2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge.

  3. Restriction fragment length polymorphism of the periplasmic flagellar flaA1 gene of Serpulina species.

    PubMed Central

    Fisher, L N; Mathiesen, M R; Duhamel, G E

    1997-01-01

    Forty-one reference and field isolates of intestinal spirochetes representing Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Brachyspira aalborgi, and nonclassified weakly beta-hemolytic intestinal spirochetes were compared by restriction fragment length polymorphism (RFLP) of the periplasmic flagellar (PF) flaA1 gene. Six genetically distinct groups (I through VI), each with a unique RFLP fingerprint pattern, were identified by Southern blotting analysis of EcoRV chromosomal DNA digests with a PCR-amplified digoxigenin-labeled 1-kb fragment of the S. hyodysenteriae isolate B78 PF flaA1 gene. The RFLP fingerprint patterns corresponded to known DNA homology differences between Serpulina species and to provisionally designated species described previously by using phenotypic and genotypic classification schemes. RFLP fingerprinting of the PF flaA1 gene provides a relatively simple genotypic method for identification of intestinal spirochetes without the use of radioisotopes. PMID:9384289

  4. Flagellar hydrodynamics. A comparison between resistive-force theory and slender-body theory.

    PubMed

    Johnson, R E; Brokaw, C J

    1979-01-01

    This paper investigates the accuracy of the resistive-force theory (Gray and Hancock method) which is commonly used for hydrodynamic analysis of swimming flagella. We made a comparison between the forces, bending moments, and shear moments calculated by resistive-force theory and by the more accurate slender-body theory for large-amplitude, planar wave forms computed for a flagellar model. By making an upward empirical adjustment, by about 35%, of the classical drag coefficient values used in the resistive-force theory calculations, we obtained good agreement between the distributions of the forces and moments along the length of the flagellum predicted by the two methods when the flagellum has no cell body attached. After this adjustment, we found the rate of energy expenditure calculated by the two methods for the few typical test cases to be almost identical. The resistive-force theory is thus completely satisfactory for use in analysis of mechanisms for the control of flagellar bending, at the current level of sophistication of this analysis. We also examined the effects of the presence of a cell body attached to one end of the flagellum, which modifies the flow field experienced by the flagellum. This interaction, which is not considered in resistive-force theory, is probably insignificant for small cell bodies, such as the heads of simple spermatozoa, but for larger cell bodies, or cell bodies that have large-amplitude motions transverse to the swimming direction, use of slender-body theory is required for accurate analysis. PMID:262381

  5. Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

    PubMed

    Brown, Robert W B; Collingridge, Peter W; Gull, Keith; Rigden, Daniel J; Ginger, Michael L

    2014-01-01

    Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed. PMID:25050549

  6. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

  7. BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

    PubMed Central

    Florimond, Célia; Sahin, Annelise; Vidilaseris, Keni; Dong, Gang; Landrein, Nicolas; Dacheux, Denis; Albisetti, Anna; Byard, Edward H.; Bonhivers, Mélanie; Robinson, Derrick R.

    2015-01-01

    The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC. PMID:25822645

  8. Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

    PubMed

    Brown, Robert W B; Collingridge, Peter W; Gull, Keith; Rigden, Daniel J; Ginger, Michael L

    2014-01-01

    Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed.

  9. Evidence for Loss of a Partial Flagellar Glycolytic Pathway during Trypanosomatid Evolution

    PubMed Central

    Brown, Robert W. B.; Collingridge, Peter W.; Gull, Keith; Rigden, Daniel J.; Ginger, Michael L.

    2014-01-01

    Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed. PMID:25050549

  10. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  11. Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ

    PubMed Central

    1985-01-01

    Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination. PMID:4030899

  12. Motor learning.

    PubMed

    Wolpert, Daniel M; Flanagan, J Randall

    2010-06-01

    Although learning a motor skill, such as a tennis stroke, feels like a unitary experience, researchers who study motor control and learning break the processes involved into a number of interacting components. These components can be organized into four main groups. First, skilled performance requires the effective and efficient gathering of sensory information, such as deciding where and when to direct one's gaze around the court, and thus an important component of skill acquisition involves learning how best to extract task-relevant information. Second, the performer must learn key features of the task such as the geometry and mechanics of the tennis racket and ball, the properties of the court surface, and how the wind affects the ball's flight. Third, the player needs to set up different classes of control that include predictive and reactive control mechanisms that generate appropriate motor commands to achieve the task goals, as well as compliance control that specifies, for example, the stiffness with which the arm holds the racket. Finally, the successful performer can learn higher-level skills such as anticipating and countering the opponent's strategy and making effective decisions about shot selection. In this Primer we shall consider these components of motor learning using as an example how we learn to play tennis. PMID:20541489

  13. Motor Controllers

    NASA Technical Reports Server (NTRS)

    1984-01-01

    Kollmorgen Corporation's Mermaid II two person submersible is propeller-driven by a system of five DC brushless motors with new electronic controllers that originated in work performed in a NASA/DOE project managed by Lewis Research Center. A key feature of the system is electric commutation rather than mechanical commutation for converting AC current to DC.

  14. Motor learning.

    PubMed

    Wolpert, Daniel M; Flanagan, J Randall

    2010-06-01

    Although learning a motor skill, such as a tennis stroke, feels like a unitary experience, researchers who study motor control and learning break the processes involved into a number of interacting components. These components can be organized into four main groups. First, skilled performance requires the effective and efficient gathering of sensory information, such as deciding where and when to direct one's gaze around the court, and thus an important component of skill acquisition involves learning how best to extract task-relevant information. Second, the performer must learn key features of the task such as the geometry and mechanics of the tennis racket and ball, the properties of the court surface, and how the wind affects the ball's flight. Third, the player needs to set up different classes of control that include predictive and reactive control mechanisms that generate appropriate motor commands to achieve the task goals, as well as compliance control that specifies, for example, the stiffness with which the arm holds the racket. Finally, the successful performer can learn higher-level skills such as anticipating and countering the opponent's strategy and making effective decisions about shot selection. In this Primer we shall consider these components of motor learning using as an example how we learn to play tennis.

  15. Therma motor

    DOEpatents

    Kandarian, R.

    The disclosure is directed to a thermal motor utilizing two tapered prestressed parallel adjacent cylinders lengthwise disposed about one third in a coolant. Heat is applied to contacting portions of the cylinders outside the coolant to cause them to deform and turn. Heat sources such as industrial waste heat, geothermal hot water, solar radiation, etc. can be used.

  16. Auditory-Motor Interactions in Pediatric Motor Speech Disorders: Neurocomputational Modeling of Disordered Development

    PubMed Central

    Terband, H.; Maassen, B.; Guenther, F.H.; Brumberg, J.

    2014-01-01

    Background/Purpose Differentiating the symptom complex due to phonological-level disorders, speech delay and pediatric motor speech disorders is a controversial issue in the field of pediatric speech and language pathology. The present study investigated the developmental interaction between neurological deficits in auditory and motor processes using computational modeling with the DIVA model. Method In a series of computer simulations, we investigated the effect of a motor processing deficit alone (MPD), and the effect of a motor processing deficit in combination with an auditory processing deficit (MPD+APD) on the trajectory and endpoint of speech motor development in the DIVA model. Results Simulation results showed that a motor programming deficit predominantly leads to deterioration on the phonological level (phonemic mappings) when auditory self-monitoring is intact, and on the systemic level (systemic mapping) if auditory self-monitoring is impaired. Conclusions These findings suggest a close relation between quality of auditory self-monitoring and the involvement of phonological vs. motor processes in children with pediatric motor speech disorders. It is suggested that MPD+APD might be involved in typically apraxic speech output disorders and MPD in pediatric motor speech disorders that also have a phonological component. Possibilities to verify these hypotheses using empirical data collected from human subjects are discussed. PMID:24491630

  17. Motor Neuron Diseases

    MedlinePlus

    ... called upper motor neurons ) are transmitted to nerve cells in the brain stem and spinal cord (called lower motor neurons ) and from them to particular muscles. Upper motor neurons direct the lower motor neurons ...

  18. Motor Neuron Diseases

    MedlinePlus

    ... Enhancing Diversity Find People About NINDS NINDS Motor Neuron Diseases Information Page Condensed from Motor Neuron Diseases ... and Information Publicaciones en Español What are Motor Neuron Diseases? The motor neuron diseases (MNDs) are a ...

  19. Teaching basic neurophysiology using intact earthworms.

    PubMed

    Kladt, Nikolay; Hanslik, Ulrike; Heinzel, Hans-Georg

    2010-01-01

    Introductory neurobiology courses face the problem that practical exercises often require expensive equipment, dissections, and a favorable student-instructor ratio. Furthermore, the duration of an experiment might exceed available time or the level of required expertise is too high to successfully complete the experiment. As a result, neurobiological experiments are commonly replaced by models and simulations, or provide only very basic experiments, such as the frog sciatic nerve preparation, which are often time consuming and tedious. Action potential recordings in giant fibers of intact earthworms (Lumbricus terrestris) circumvent many of these problems and result in a nearly 100% success rate. Originally, these experiments were introduced as classroom exercises by Charles Drewes in 1978 using awake, moving earthworms. In 1990, Hans-Georg Heinzel described further experiments using anesthetized earthworms. In this article, we focus on the application of these experiments as teaching tools for basic neurobiology courses. We describe and extend selected experiments, focusing on specific neurobiological principles with experimental protocols optimized for classroom application. Furthermore, we discuss our experience using these experiments in animal physiology and various neurobiology courses at the University of Bonn. PMID:23494516

  20. Non-genetic individuality in Escherichia coli motor switching

    NASA Astrophysics Data System (ADS)

    Mora, Thierry; Bai, Fan; Che, Yong-Suk; Minamino, Tohru; Namba, Keiichi; Wingreen, Ned S.

    2011-04-01

    By analyzing 30 min, high-resolution recordings of single Escherichia coli flagellar motors in the physiological regime, we show that two main properties of motor switching—the mean clockwise and mean counter-clockwise interval durations—vary significantly. When we represent these quantities on a two-dimensional plot for several cells, the data do not fall on a one-dimensional curve, as expected with a single control parameter, but instead spread in two dimensions, pointing to motor individuality. The largest variations are in the mean counter-clockwise interval, and are attributable to variations in the concentration of the internal signaling molecule CheY-P. In contrast, variations in the mean clockwise interval are interpreted in terms of motor individuality. We argue that the sensitivity of the mean counter-clockwise interval to fluctuations in CheY-P is consistent with an optimal strategy of run and tumble. The concomittent variability in mean run length may allow populations of cells to better survive in rapidly changing environments by 'hedging their bets'.

  1. An intact action-perception coupling depends on the integrity of the cerebellum.

    PubMed

    Christensen, Andrea; Giese, Martin A; Sultan, Fahad; Mueller, Oliver M; Goericke, Sophia L; Ilg, Winfried; Timmann, Dagmar

    2014-05-01

    It is widely accepted that action and perception in humans functionally interact on multiple levels. Moreover, areas originally suggested to be predominantly motor-related, as the cerebellum, are also involved in action observation. However, as yet, few studies provided unequivocal evidence that the cerebellum is involved in the action perception coupling (APC), specifically in the integration of motor and multisensory information for perception. We addressed this question studying patients with focal cerebellar lesions in a virtual-reality paradigm measuring the effect of action execution on action perception presenting self-generated movements as point lights. We measured the visual sensitivity to the point light stimuli based on signal detection theory. Compared with healthy controls cerebellar patients showed no beneficial influence of action execution on perception indicating deficits in APC. Applying lesion symptom mapping, we identified distinct areas in the dentate nucleus and the lateral cerebellum of both hemispheres that are causally involved in APC. Lesions of the right ventral dentate, the ipsilateral motor representations (lobules V/VI), and most interestingly the contralateral posterior cerebellum (lobule VII) impede the benefits of motor execution on perception. We conclude that the cerebellum establishes time-dependent multisensory representations on different levels, relevant for motor control as well as supporting action perception. Ipsilateral cerebellar motor representations are thought to support the somatosensory state estimate of ongoing movements, whereas the ventral dentate and the contralateral posterior cerebellum likely support sensorimotor integration in the cerebellar-parietal loops. Both the correct somatosensory as well as the multisensory state representations are vital for an intact APC.

  2. CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

    PubMed Central

    Krähling, Anke Miriam; Alvarez, Luis; Debowski, Katharina; Van, Qui; Gunkel, Monika; Irsen, Stephan; Al-Amoudi, Ashraf; Strünker, Timo; Kremmer, Elisabeth; Krause, Eberhard; Voigt, Ingo; Wörtge, Simone; Waisman, Ari; Weyand, Ingo; Seifert, Reinhard; Kaupp, Ulrich Benjamin; Wachten, Dagmar

    2013-01-01

    The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that—in mature sperm—are involved in flagellar bending. PMID:24339785

  3. Positive youth development, life satisfaction, and problem behaviors of adolescents in intact and non-intact families in Hong Kong.

    PubMed

    Shek, Daniel T L; Leung, Hildie

    2013-01-01

    This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families. PMID:24400264

  4. Positive Youth Development, Life Satisfaction, and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

    PubMed Central

    Shek, Daniel T. L.; Leung, Hildie

    2013-01-01

    This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families. PMID:24400264

  5. Coordination and Fine Motor Control Depends on Drosophila TRPγ

    PubMed Central

    Akitake, Bradley; Ren, Qiuting; Boiko, Nina; Ni, Jinfei; Sokabe, Takaaki; Stockand, James D.; Eaton, Benjamin A.; Montell, Craig

    2015-01-01

    Motor coordination is broadly divided into gross and fine motor control, both of which depend on proprioceptive organs. However, the channels that function specifically in fine motor control are unknown. Here, we show that mutations in trpγ disrupt fine motor control while leaving gross motor proficiency intact. The mutants are unable to coordinate precise leg movements during walking, and are ineffective in traversing large gaps due to an inability in making subtle postural adaptations that are requisite for this task. TRPγ is expressed in proprioceptive organs, and is required in both neurons and glia for gap crossing. We expressed TRPγ in vitro, and found that its activity is promoted by membrane stretch. A mutation eliminating the Na+/Ca2+ exchanger suppresses the gap crossing phenotype of trpγ flies. Our findings indicate that TRPγ contributes to fine motor control through mechanical activation in proprioceptive organs, thereby promoting Ca2+ influx, which is required for function. PMID:26028119

  6. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis.

    PubMed

    Jiang, Lingyan; Ni, Zhiwei; Wang, Lei; Feng, Lu; Liu, Bin

    2015-03-01

    Salmonella, a genus that is closely related to Escherichia coli, includes many pathogens of humans and other animals. A notable feature that distinguishes Salmonella from E. coli is lactose negativity, because the lac operon is lost in most Salmonella genomes. Here, we expressed the lac operon in Salmonella enterica serovar Typhimurium and compared the virulence of the Lac(+) strain to that of the wild-type strain in a murine model, invasion assays, and macrophage replication assays. We showed that the Lac(+) strain is attenuated in vivo and the attenuation of virulence is caused by its defect in epithelial cell invasion. However, the invasion-defective phenotype is unrelated to lactose utilization. Through sequencing and the comparison of the transcriptome profile between the Lac(+) and wild-type strains during invasion, we found that most flagellar genes were markedly downregulated in the Lac(+) strain, while other genes associated with invasion, such as the majority of genes encoded in Salmonella pathogenicity island 1, were not differentially expressed. Moreover, we discovered that lacA is the major repressor of flagellar gene expression in the lac operon. In conclusion, these data demonstrate that the lac operon decreases Salmonella invasion of epithelial cells through repression of flagellar biosynthesis. As the ability to invade epithelial cells is a critical virulence determinant of Salmonella, our results provide important evidence that the loss of the lac operon contributes to the evolution of Salmonella pathogenicity. PMID:25362512

  7. Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

    PubMed Central

    Rompolas, Panteleimon; Patel-King, Ramila S.; King, Stephen M.

    2012-01-01

    The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters. PMID:22855525

  8. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis.

    PubMed

    Jiang, Lingyan; Ni, Zhiwei; Wang, Lei; Feng, Lu; Liu, Bin

    2015-03-01

    Salmonella, a genus that is closely related to Escherichia coli, includes many pathogens of humans and other animals. A notable feature that distinguishes Salmonella from E. coli is lactose negativity, because the lac operon is lost in most Salmonella genomes. Here, we expressed the lac operon in Salmonella enterica serovar Typhimurium and compared the virulence of the Lac(+) strain to that of the wild-type strain in a murine model, invasion assays, and macrophage replication assays. We showed that the Lac(+) strain is attenuated in vivo and the attenuation of virulence is caused by its defect in epithelial cell invasion. However, the invasion-defective phenotype is unrelated to lactose utilization. Through sequencing and the comparison of the transcriptome profile between the Lac(+) and wild-type strains during invasion, we found that most flagellar genes were markedly downregulated in the Lac(+) strain, while other genes associated with invasion, such as the majority of genes encoded in Salmonella pathogenicity island 1, were not differentially expressed. Moreover, we discovered that lacA is the major repressor of flagellar gene expression in the lac operon. In conclusion, these data demonstrate that the lac operon decreases Salmonella invasion of epithelial cells through repression of flagellar biosynthesis. As the ability to invade epithelial cells is a critical virulence determinant of Salmonella, our results provide important evidence that the loss of the lac operon contributes to the evolution of Salmonella pathogenicity.

  9. Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface.

    PubMed

    Yoshihara, Akinori; Nobuhira, Noritaka; Narahara, Hisaya; Toyoda, Syunsuke; Tokumoto, Hayato; Konishi, Yasuhiro; Nomura, Toshiyuki

    2015-07-01

    The effects of the presence or absence of microbial flagella and the microbial motility on the colloidal behaviors of microbial cells were quantitatively evaluated. The microbial cell attachment and detachment processes on a glass surface were observed directly using a parallel-plate flow chamber. Wild-type, flagellar paralyzed, and nonflagellated Escherichia coli strains were used as model microbial cells. In the cell attachment tests, the microbial adhesion rate in a 160mM NaCl solution was approximately 10 times higher than that in a 10mM solution, for all E. coli strains. The colloidal behavior of the microbial cells agreed well with the predictions of the DLVO theory. In addition, the microbial flagella and motility did not significantly affect the cell attachment, regardless of the existence of a potential barrier between the cell and the glass substratum. In the cell detachment tests, the cumulative number of microbial cells detached from the glass substratum with increasing flow rate was fit well with the Weibull distribution function. The list of strains arranged in order of increasing median drag force required to remove them was nonflagellated strain, flagellar paralyzed strain, and wild-type strain. These results indicated that the flagella and the flagellar motility inhibited the cell detachment from the glass substratum. Furthermore, a large external force would likely be required to inhibit the microbial adhesion in the early stage of the biofilm formation.

  10. Tubulin polyglutamylation regulates flagellar motility by controlling a specific inner-arm dynein that interacts with the dynein regulatory complex.

    PubMed

    Kubo, Tomohiro; Yagi, Toshiki; Kamiya, Ritsu

    2012-12-01

    The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.

  11. Experimental serpentinization of intact dunite cores

    NASA Astrophysics Data System (ADS)

    Luhmann, A. J.; Tutolo, B. M.; Kong, X. Z.; Bagley, B. C.; Schaen, A. T.; Saar, M. O.; Seyfried, W. E., Jr.

    2014-12-01

    Serpentinization in ultramafic-hosted hydrothermal systems, such as Lost City, produces relatively cool and alkaline fluids that support diverse ecosystems. To simulate serpentinization in such systems, we conducted single-pass, flow-through experiments on dunite cores cut out of a sample from Jackson County, North Carolina. Experimental seawater prepared using laboratory-grade reagents and standards was pumped through a core at 150ºC and 150 bar pore-fluid outlet pressure at a flow rate of 0.01 ml/min. An additional experiment will be conducted at 200ºC. At 150ºC, permeability decreased by 2.3 times with reaction progress over the course of the 36 day experiment. Fluid-rock reaction generally produced CO2, H2, CH4, and CO concentrations of 100 μmol/kg, up to 40 μmol/kg, 2 μmol/kg, and less than 1 μmol/kg, respectively. Outlet fluid chemistry was relatively stable, except for initial peaks in Al, Ba, Fe, Mn, and Si. pH of outlet fluids increased with reaction progress, but it was always lower (6.9-7.4) than the initial seawater (7.8). X-ray computed tomography scans were/will be collected for both pre- and post-experimental cores. The combination of flow-through experiments on solid, intact rock cores cut out of natural samples and X-ray tomography permits visualization and quantification of mineralogical changes and flow path evolution during serpentinization. This approach further permits physical and chemical processes to be documented on a fine scale to better understand feedbacks between chemical reactions and flow fields, with implications for ultramafic-hosted hydrothermal systems.

  12. 50 CFR 622.186 - Landing fish intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but...

  13. 50 CFR 622.186 - Landing fish intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but...

  14. 50 CFR 622.247 - Landing golden crab intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing golden crab intact. 622.247... ATLANTIC Golden Crab Fishery of the South Atlantic Region § 622.247 Landing golden crab intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that golden crab on that vessel...

  15. 50 CFR 622.247 - Landing golden crab intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing golden crab intact. 622.247... ATLANTIC Golden Crab Fishery of the South Atlantic Region § 622.247 Landing golden crab intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that golden crab on that vessel...

  16. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  17. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  18. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  19. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  20. 46 CFR 28.570 - Intact righting energy.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of...

  1. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  2. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  3. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  4. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  5. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations...

  6. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  7. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  8. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  9. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  10. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  11. 46 CFR 172.165 - Intact stability calculations.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability calculations. 172.165 Section 172.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.165 Intact stability calculations. (a) Design calculations...

  12. 46 CFR 174.020 - Alternate intact stability criterion.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Alternate intact stability criterion. 174.020 Section 174.020 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY... Alternate intact stability criterion. A barge need not comply with § 174.015 and subparts C and E of...

  13. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  14. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact longitudinal stability. 172.095 Section 172.095 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must...

  15. 46 CFR 170.165 - International Code on Intact Stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false International Code on Intact Stability. 170.165 Section 170.165 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.165 International Code...

  16. 50 CFR 622.455 - Landing spiny lobster intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing spiny lobster intact. 622.455... ATLANTIC Spiny Lobster Fishery of Puerto Rico and the U.S. Virgin Islands § 622.455 Landing spiny lobster intact. (a) A Caribbean spiny lobster in or from the Caribbean EEZ must be maintained with head...

  17. 50 CFR 622.455 - Landing spiny lobster intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing spiny lobster intact. 622.455... ATLANTIC Spiny Lobster Fishery of Puerto Rico and the U.S. Virgin Islands § 622.455 Landing spiny lobster intact. (a) A Caribbean spiny lobster in or from the Caribbean EEZ must be maintained with head...

  18. 50 CFR 622.10 - Landing fish intact--general.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH... landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified... intact. (a) Finfish in or from the Gulf EEZ or Caribbean EEZ, except as specified in paragraphs (b)...

  19. 50 CFR 622.10 - Landing fish intact--general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE FISHERIES OF THE CARIBBEAN, GULF OF MEXICO, AND SOUTH... landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified... intact. (a) Finfish in or from the Gulf EEZ or Caribbean EEZ, except as specified in paragraphs (b)...

  20. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  1. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  2. 46 CFR 172.095 - Intact longitudinal stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact longitudinal stability. 172.095 Section 172.095... Under Subchapter O of This Chapter § 172.095 Intact longitudinal stability. Each tank barge must be shown by design calculations to have a longitudinal metacentric height (GM) in feet (meters) in...

  3. Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm.

    PubMed

    Lesich, Kathleen A; Pelle, Dominic W; Lindemann, Charles B

    2008-07-01

    Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 muM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 +/- 0.29 nN/microm in 0.1 mM ATP and increasing to 2.9 +/- 1.2 nN/microm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed. PMID:18375503

  4. Insights into the Mechanism of ADP Action on Flagellar Motility Derived from Studies on Bull Sperm

    PubMed Central

    Lesich, Kathleen A.; Pelle, Dominic W.; Lindemann, Charles B.

    2008-01-01

    Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1–4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 μM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 ± 0.29 nN/μm in 0.1 mM ATP and increasing to 2.9 ± 1.2 nN/μm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed. PMID:18375503

  5. Motor Switching Rates in Caulobacter Crescentus Follow First Passage Time Distribution

    NASA Astrophysics Data System (ADS)

    Tang, Jay; Morse, Michael; Bell, Jordan; Li, Guanglai

    2015-03-01

    The flagellar motor of uni-flagellated bacterium Caulobacter crescentus switches stochastically between clockwise (CW) and counterclockwise (CCW) rotation. We performed measurements of the time intervals between switches in order to gain insight on motor dynamics and regulation. Our measurements were performed both on free swimming cells and tethered cells with their flagella attached to a glass slide. A peak time of approximately one second was observed in both motor directions with counterclockwise intervals more sharply peaked. The distributions of switching times can be fitted using biased first passage time statistics. We present a model of motor switching dynamics, which is controlled by the binding of CheY-P to motor subunits FliM. A lower threshold number of FliM with CheY-P bound triggers a switch in motor rotation from CW to CCW, whereas a higher threshold triggers an opposing switch from CCW to CW. The time intervals between alternating switches may be increased or decreased by regulating CheY-P concentration, resulting in biased directional motion in the cells swimming trajectory over many motor cycles under external spatial or temporal gradients. Work funded by the United States National Science Foundation.

  6. Siphon motor

    SciTech Connect

    Bunn, C.H.

    1980-01-01

    A siphon motor comprises the combination of siphon means and generating means for generating electrical energy from a water source located below the generating means and a water discharge at a lower level than the water source. Water rises by siphonic action upward from the water source to a sealed working region maintained under partial vacuum, and descends to the water discharge. The working region contains the generating means. The system has particular utility as a source of power generation in remote locations having a water table within about 30 feet of the ground.

  7. Tests for intact and collapsed magnetofossil chains

    NASA Astrophysics Data System (ADS)

    Egli, R.

    2012-04-01

    first time. These results match measurements obtained previously on magnetosome-rich sediments in smallest details, showing that the identification of distinct intact and collapsed chain structures is possible. On the other hand, these results show that caution should be used when interpreting sediment hysteresis properties as mixtures of single domain (SD), multidomain (MD), and superparamagnetic (SP) particles; because some collapsed chain structures closely mimic SD-MD-SP mixing trends in a Day plot, although being made only of SD particles.

  8. A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components.

    PubMed

    de Leon, Jessica Cruz; Scheumann, Nicole; Beatty, Wandy; Beck, Josh R; Tran, Johnson Q; Yau, Candace; Bradley, Peter J; Gull, Keith; Wickstead, Bill; Morrissette, Naomi S

    2013-07-01

    SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

  9. Narrow-spectrum inhibitors of Campylobacter jejuni flagellar expression and growth.

    PubMed

    Johnson, Jeremiah G; Yuhas, Caroline; McQuade, Thomas J; Larsen, Martha J; DiRita, Victor J

    2015-07-01

    Campylobacter jejuni is a major cause of food-borne illness due to its ability to reside within the gastrointestinal tracts of chickens. Multiple studies have identified the flagella of C. jejuni as a major determinant of chicken colonization. An inhibitor screen of approximately 147,000 small molecules was performed to identify compounds that are able to inhibit flagellar expression in a reporter strain of C. jejuni. Several compounds that modestly inhibited motility of wild-type C. jejuni in standard assays were identified, as were a number of small molecules that robustly inhibited C. jejuni growth, in vitro. Examination of similar bacterial screens found that many of these small molecules inhibited only the growth of C. jejuni. Follow-up assays demonstrated inhibition of other strains of C. jejuni and Campylobacter coli but no inhibition of the closely related Helicobacter pylori. The compounds were determined to be bacteriostatic and nontoxic to eukaryotic cells. Preliminary results from a day-of-hatch chick model of colonization suggest that at least one of the compounds demonstrates promise for reducing Campylobacter colonization loads in vivo, although further medicinal chemistry may be required to enhance bioavailability.

  10. Stochastic and Deterministic Flagellar Dynamics Provide a Mechanism for Eukaryotic Swimming Reorientation

    NASA Astrophysics Data System (ADS)

    Polin, Marco; Tuval, Idan; Drescher, Knut; Goldstein, Raymond

    2009-03-01

    The biflagellated alga Chlamydomonas reinhardtii is a good model organism to study the origin of flagellar synchronization. Here we employ high-speed imaging to study the beating of the two flagella of Chlamydomonas, and show that a single cell can alternate between two distinct dynamical regimes: asynchronous and synchronous. The asynchronous state is characterized by a large interflagellar frequency difference. In the synchronous state, the flagella beat in phase for lengthy periods, interrupted episodically by an extra beat of either flagellum. The statistics of these events are consistent with a model of hydrodynamically coupled noisy oscillators. Previous observations have suggested that the two flagella have well separated intrinsic beat frequencies, and are synchronized by their mutual coupling. Our analysis shows instead that the synchronized state is incompatible with coupling-induced synchronization of flagella with those intrinsic frequencies. This suggests that the beat frequencies themselves are under the control of the cell. Moreover, high-resolution three-dimensional tracking of swimming cells provides strong evidence that these dynamical states are related to non-phototactic reorientation events in the trajectories, yielding a eukaryotic equivalent of the ``run and tumble'' motion of peritrichously flagellated bacteria.

  11. Investigation of the Swimming and Flagellar Dynamics of Shear guided Motile Alga

    NASA Astrophysics Data System (ADS)

    Chengala, Anwar; Hondzo, Miki; Sheng, Jian

    2011-11-01

    We examine the behavior of force-free swimming cell in a shear flow having spheroidal bodies with two flagella located at the anterior part of the cell that uses breast-like motion for its propulsion. The cell, Dunaliella primolecta, displays a unique behavior of propelling along the local vorticity direction in a linear shear flow. The cell rotation is absent during this display and the flagella beating is observed to be asynchronous. The forces and moments generated by the flagella are estimated numerically. Based on the Resistive Force Theory approach, we attempt to demonstrate that the moments generated by beating flagella and their alignment to flow are necessary to counter-act the vorticity of the flow. In addition, we explore the various mechanisms that cell may use to re-orient while in a shear flow as well as how critical the variations in the flagellar beating pattern are to a cell's swimming dynamics. NSF (No: CBET-0844647), Minnesota Futures Grant.

  12. Flagellar antigen based CI-ELISA for sero-surveillance of surra.

    PubMed

    Ligi, M; Sengupta, P P; Rudramurthy, G R; Rahman, H

    2016-03-30

    Trypanosoma evansi causes a disease known as 'surra' in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive-negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen's Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra. PMID:26921034

  13. Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

    PubMed

    Lesich, Kathleen A; dePinho, Tania G; Pelle, Dominic W; Lindemann, Charles B

    2016-05-01

    The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend. This phenomenon was shown to be caused by the induction of inter-doublet shear and is dependent on the presence of an inter-doublet shear resistance. Here we report that in sea urchin flagella there is systematically less shear induced in the distal flagellum than is predicted by the sliding doublet mechanism, if we follow the assumption that the diameter of the flagellum is uniform. To account for the reduced shear that is observed, the likeliest and most direct interpretation is that the portion of the axoneme that is forced to bend undergoes substantial compression of the axoneme in the bending plane. A compression of 30-50 nm would be sufficient to account for the shear reduction from a bend of 2 radians. A compression of this magnitude would require considerable flexibility in the axoneme structure. This would necessitate that the radial spokes and/or the central pair apparatus are easily compressed by transverse stress. © 2016 Wiley Periodicals, Inc. PMID:27001352

  14. Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids.

    PubMed

    Casanova, Magali; Crobu, Lucien; Blaineau, Christine; Bourgeois, Nathalie; Bastien, Patrick; Pagès, Michel

    2009-03-01

    Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubule-severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as < or = 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubule-severing proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models. PMID:19183280

  15. Bacterial flagellar motility on hydrated rough surfaces controlled by aqueous film thickness and connectedness.

    PubMed

    Tecon, Robin; Or, Dani

    2016-01-01

    Recent studies have shown that rates of bacterial dispersion in soils are controlled by hydration conditions that define size and connectivity of the retained aqueous phase. Despite the ecological implications of such constraints, microscale observations of this phenomenon remain scarce. Here, we quantified aqueous film characteristics and bacterial flagellated motility in response to systematic variations in microhydrological conditions on porous ceramic surfaces that mimic unsaturated soils. We directly measured aqueous film thickness and documented its microscale heterogeneity. Flagellar motility was controlled by surface hydration conditions, as cell velocity decreased and dispersion practically ceased at water potentials exceeding -2 kPa (resulting in thinner and disconnected liquid films). The fragmentation of aquatic habitats was delineated indirectly through bacterial dispersal distances within connected aqueous clusters. We documented bacterial dispersal radii ranging from 100 to 10 μm as the water potential varied from 0 to -7 kPa, respectively. The observed decrease of flagellated velocity and dispersal ranges at lower matric potentials were in good agreement with mechanistic model predictions. Hydration-restricted habitats thus play significant role in bacterial motility and dispersal, which has potentially important impact on soil microbial ecology and diversity. PMID:26757676

  16. Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

    PubMed

    Lesich, Kathleen A; dePinho, Tania G; Pelle, Dominic W; Lindemann, Charles B

    2016-05-01

    The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend. This phenomenon was shown to be caused by the induction of inter-doublet shear and is dependent on the presence of an inter-doublet shear resistance. Here we report that in sea urchin flagella there is systematically less shear induced in the distal flagellum than is predicted by the sliding doublet mechanism, if we follow the assumption that the diameter of the flagellum is uniform. To account for the reduced shear that is observed, the likeliest and most direct interpretation is that the portion of the axoneme that is forced to bend undergoes substantial compression of the axoneme in the bending plane. A compression of 30-50 nm would be sufficient to account for the shear reduction from a bend of 2 radians. A compression of this magnitude would require considerable flexibility in the axoneme structure. This would necessitate that the radial spokes and/or the central pair apparatus are easily compressed by transverse stress. © 2016 Wiley Periodicals, Inc.

  17. Bacterial flagellar motility on hydrated rough surfaces controlled by aqueous film thickness and connectedness

    PubMed Central

    Tecon, Robin; Or, Dani

    2016-01-01

    Recent studies have shown that rates of bacterial dispersion in soils are controlled by hydration conditions that define size and connectivity of the retained aqueous phase. Despite the ecological implications of such constraints, microscale observations of this phenomenon remain scarce. Here, we quantified aqueous film characteristics and bacterial flagellated motility in response to systematic variations in microhydrological conditions on porous ceramic surfaces that mimic unsaturated soils. We directly measured aqueous film thickness and documented its microscale heterogeneity. Flagellar motility was controlled by surface hydration conditions, as cell velocity decreased and dispersion practically ceased at water potentials exceeding –2 kPa (resulting in thinner and disconnected liquid films). The fragmentation of aquatic habitats was delineated indirectly through bacterial dispersal distances within connected aqueous clusters. We documented bacterial dispersal radii ranging from 100 to 10 μm as the water potential varied from 0 to –7 kPa, respectively. The observed decrease of flagellated velocity and dispersal ranges at lower matric potentials were in good agreement with mechanistic model predictions. Hydration-restricted habitats thus play significant role in bacterial motility and dispersal, which has potentially important impact on soil microbial ecology and diversity. PMID:26757676

  18. Structure and protein composition of the striated flagellar rootlets of some protists.

    PubMed

    Dingle, A D; Larson, D E

    1981-01-01

    The striated rootlets of different protists are extremely diverse and, on the basis of structural organization, can be assigned to no fewer than four major types. In light of this extreme variation in fine-structure is it reasonable to expect that all striated rootlets may share common protein species? Using the flagellar rootlet of Naegleria gruberi strain NB-1 as reference, we compared rootlet size, structure, and protein composition to another Naegleria strain, another amoebo-flagellate, Tetramitus rostratus, and to the ciliate Tetrahymena pyriformis. Although differing in size and distribution in the cells, the rootlets of all three amoebo-flagellates appeared very similar in structure, periodicity, and in the presence of a common 170 000 Dalton subunit. Kinetodesmal fibres of Tetrahymena differed markedly in detailed fine-structure, in periodicity, and in the apparent absence of the 170 000 Dalton subunit as tested either by SDS gel electrophoresis or by indirect immunofluorescence staining using a specific antiserum directed against the NB-1 major rootlet protein. Consideration of literature describing striated rootlet structure in a wide variety of ciliated and flagellated cells led to the speculation that striated rootlets arose subsequent to primitive flagella and likely evolved along two major pathways: a narrow-period rootlet similar to those discussed above, which developed from the microtubule rootlets of algal flagellates, and a wide-band, contractile rootlet which originated from the primitive interbasal body connector prominent in both algal and protozoan species.

  19. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    NASA Technical Reports Server (NTRS)

    Barker, Ed; Abercromby, Kira J.; Abell, Paul

    2009-01-01

    A key objective of NASA s Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the 0.6 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectra is flat and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar panels with a strong band gap feature near 1 micron. The two spacecraft are spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but with fewer

  20. Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

    NASA Astrophysics Data System (ADS)

    Albercromby, Kira J.; Abell, Paul; Barker, Ed

    2009-03-01

    A key objective of NASA's Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the ~ 0.65 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectrum has a slight slope and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar cells with a strong band gap feature near 1 micron. The two spacecraft were spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but

  1. RflM functions as a transcriptional repressor in the autogenous control of the Salmonella Flagellar master operon flhDC.

    PubMed

    Singer, Hanna M; Erhardt, Marc; Hughes, Kelly T

    2013-09-01

    Motility of bacteria like Salmonella enterica is a highly regulated process that responds to a variety of internal and external stimuli. A hierarchy of three promoter classes characterizes the Salmonella flagellar system, and the onset of flagellar gene expression depends on the oligomeric regulatory complex and class 1 gene product FlhD(4)C(2). The flhDC promoter is a target for a broad range of transcriptional regulators that bind within the flhDC promoter region and either negatively or positively regulate flhDC operon transcription. In this work, we demonstrate that the RflM protein is a key component of flhDC regulation. Transposon mutagenesis was performed to investigate a previously described autoinhibitory effect of the flagellar master regulatory complex FlhD(4)C(2). RflM is a LuxR homolog that functions as a flagellar class 1 transcriptional repressor. RflM was found to be the negative regulator of flhDC expression that is responsible for the formerly described autoinhibitory effect of the FlhD(4)C(2) complex on flhDC operon transcription (K. Kutsukake, Mol. Gen. Genet. 254:440-448, 1997). We conclude that upon commencement of flagellar gene expression, the FlhD(4)C(2) complex initiates a regulatory feedback loop by activating rflM gene expression. rflM encodes a transcriptional repressor, RflM, which fine-tunes flhDC expression levels.

  2. T-POP array identifies EcnR and PefI-SrgD as novel regulators of flagellar gene expression.

    PubMed

    Wozniak, Christopher E; Lee, Changhan; Hughes, Kelly T

    2009-03-01

    The T-POP transposon was employed in a general screen for tetracycline (Tet)-induced chromosomal loci that exhibited Tet-activated or Tet-repressed expression of a fliC-lac transcriptional fusion. Insertions that activated flagellar transcription were located in flagellar genes. T-POP insertions that exhibited Tet-dependent fliC-lac inhibition were isolated upstream of the ecnR, fimZ, pefI-srgD, rcsB, and ydiV genes and in the flagellar gene flgA, which is located upstream of the anti-sigma(28) factor gene flgM. When expressed from the chromosomal P(araBAD) promoter, EcnR, FimZ, PefI-SrgD, and RcsB inhibited the transcription of the flagellar class 1 flhDC operon. YdiV, which is weakly homologous to EAL domain proteins involved in cyclic-di-GMP regulation, appears to act at a step after class 1 transcription. By using a series of deletions of the regulatory genes to try to disrupt each pathway, these regulators were found to act largely independently of one another. These results identify EcnR and PefI-SrgD as additional components of the complex regulatory network controlling flagellar expression. PMID:19114490

  3. Crystallization and preliminary X-ray analysis of Salmonella FliI, the ATPase component of the type III flagellar protein-export apparatus

    SciTech Connect

    Minamino, Tohru; Imada, Katsumi; Tahara, Aiko; Kihara, May; Macnab, Robert M.; Namba, Keiichi

    2006-10-01

    Crystals of an N-terminally truncated variant of the Salmonella flagellar ATPase FliI, which exports substrate proteins into the central channel of the growing flagellar structure by utilizing the energy of ATP hydrolysis, have been obtained and characterized by X-ray diffraction. Most of the structural components making up the bacterial flagellum are translocated through the central channel of the growing flagellar structure by the type III flagellar protein-export apparatus in an ATPase-driven manner and are assembled at the growing end. FliI is the ATPase that drives flagellar protein export using the energy of ATP hydrolysis. FliI forms an oligomeric ring structure in order to attain maximum ATPase activity. In this study, FliI(Δ1–18), an N-terminally truncated variant of FliI lacking the first 18 residues, was purified and crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 8000 as a precipitant. FliI(Δ1–18) crystals grew in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 48, b = 73, c = 126 Å, β = 94°, and diffracted to 2.4 Å resolution. Anomalous difference Patterson maps of Os-derivative and Pt-derivative crystals showed significant peaks in their Harker sections, indicating that both derivatives are suitable for structure determination.

  4. An Evolutionarily Conserved Coiled-Coil Protein Implicated in Polycystic Kidney Disease Is Involved in Basal Body Duplication and Flagellar Biogenesis in Trypanosoma brucei†

    PubMed Central

    Morgan, Gareth W.; Denny, Paul W.; Vaughan, Sue; Goulding, David; Jeffries, Tim R.; Smith, Deborah F.; Gull, Keith; Field, Mark C.

    2005-01-01

    Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism. PMID:15831481

  5. Multifocal Motor Neuropathy

    MedlinePlus

    ... Diversity Find People About NINDS NINDS Multifocal Motor Neuropathy Information Page Table of Contents (click to jump ... done? Clinical Trials Organizations What is Multifocal Motor Neuropathy? Multifocal motor neuropathy is a progressive muscle disorder ...

  6. Myosin VI: a multifunctional motor.

    PubMed

    Lister, I; Roberts, R; Schmitz, S; Walker, M; Trinick, J; Veigel, C; Buss, F; Kendrick-Jones, J

    2004-11-01

    Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed. PMID:15493988

  7. The steady-state phase distribution of the motor switch complex model of Halobacterium salinarum.

    PubMed

    del Rosario, Ricardo C H; Diener, Francine; Diener, Marc; Oesterhelt, Dieter

    2009-12-01

    Steady-state analysis is performed on the kinetic model for the switch complex of the flagellar motor of Halobacterium salinarum (Nutsch et al.). The existence and uniqueness of a positive steady-state of the system is established and it is demonstrated why the steady-state is centered around the competent phase, a state of the motor in which it is able to respond to light stimuli. It is also demonstrated why the steady-state shifts to the refractory phase when the steady-state value of the response regulator CheYP increases. This work is one aspect of modeling in systems biology wherein the mathematical properties of a model are established. PMID:19857501

  8. Motor control for a brushless DC motor

    NASA Technical Reports Server (NTRS)

    Peterson, William J. (Inventor); Faulkner, Dennis T. (Inventor)

    1985-01-01

    This invention relates to a motor control system for a brushless DC motor having an inverter responsively coupled to the motor control system and in power transmitting relationship to the motor. The motor control system includes a motor rotor speed detecting unit that provides a pulsed waveform signal proportional to rotor speed. This pulsed waveform signal is delivered to the inverter to thereby cause an inverter fundamental current waveform output to the motor to be switched at a rate proportional to said rotor speed. In addition, the fundamental current waveform is also pulse width modulated at a rate proportional to the rotor speed. A fundamental current waveform phase advance circuit is controllingly coupled to the inverter. The phase advance circuit is coupled to receive the pulsed waveform signal from the motor rotor speed detecting unit and phase advance the pulsed waveform signal as a predetermined function of motor speed to thereby cause the fundamental current waveform to be advanced and thereby compensate for fundamental current waveform lag due to motor winding reactance which allows the motor to operate at higher speeds than the motor is rated while providing optimal torque and therefore increased efficiency.

  9. Vesta is not an intact protoplanet

    NASA Astrophysics Data System (ADS)

    Consolmagno, G.; Turrini, D.; Golabek, G.; Svetsov, V.; Sirono, S.; Tsiganis, K.

    2014-07-01

    The Dawn mission was designed to explore ''remnant intact protoplanets from the earliest epoch of solar system formation'' [1]. However, models of Vesta composed of an iron core, olivine mantle, and HED crust in chondritic proportions cannot match the joint constraints from Dawn [1] of Vesta's density, core size, and the extremely limited presence of exposed olivine on its surface. Vesta has a mean density of 3456 kg/m3 and its surface composition is well matched by howardites. The Dawn gravity data suggest a nickel-iron core of radius 110 km and density 7500--7800 kg/m3. The Rheasilvia impact basin, formed within a pre-existing large basin, Veneneia, should have excavated material from a depth of 50 km to 80 km or more below Vesta's surface [2]. If the howardite crust were thinner than 50--80 km, a significant amount of olivine-rich material, derived from depth, would have been exposed within this basin; models suggest that olivine would also be distributed both on Vesta's surface and in space as meteorite-source Vestoids. Such olivine is rare on Vesta, among the Vestoids, or in our meteorite collection. Vesta's density is similar to an L chondrite, but the Na and K abundances in Vesta are strongly depleted compared to chondrites and the average metal content of an L chondrite, 8.4% by mass, would give a core radius less than 90 km. A 110 km radius metallic core, via the Dawn data, represents 15% of Vesta's mass. The Mg/Al ratio in cosmic abundances is about 10:1, but roughly 1:1 within the eucrites; thus if Vesta started with cosmic abundances, the eucrites can only represent 10% of the parent body total mass. Likewise the 10 x chondritic rare earth trace elements (REE) abundance seen in most eucrites demands that, regardless of formation mechanism, these basalts were crystallized from a melt representing 10% of the mass of the source region [3]. Thus the howardite crust of a chondritic HED parent body, mixing all the available eucritic and diogenitic material

  10. 56. POWDER MAGAZINE, VIEW OF INTACT WOOD SHEATHING ON THE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    56. POWDER MAGAZINE, VIEW OF INTACT WOOD SHEATHING ON THE SOUTHWEST REAR VENTILATION PASSAGE. (SHEATHING HELP CONTROL HUMIDITY AND DECREASE DANGER OF MAETAL STRIKING STONE AND SPARKING.) - Fort Monroe, Fortress, Hampton, Hampton, VA

  11. A Novel Trypanosoma cruzi Protein Associated to the Flagellar Pocket of Replicative Stages and Involved in Parasite Growth.

    PubMed

    Durante, Ignacio M; Cámara, María de Los Milagros; Buscaglia, Carlos A

    2015-01-01

    The flagellar pocket constitutes an active and strategic site in the body of trypanosomatids (i.e. parasitic protozoa that cause important human and/or livestock diseases), which participates in several important processes such as cell polarity, morphogenesis and replication. Most importantly, the flagellar pocket is the unique site of surface protein export and nutrient uptake in trypanosomatids, and thus constitutes a key portal for the interaction with the host. In this work, we identified and characterized a novel Trypanosoma cruzi protein, termed TCLP 1, that accumulates at the flagellar pocket area of parasite replicative forms, as revealed by biochemical, immuno-cytochemistry and electron microscopy techniques. Different in silico analyses revealed that TCLP 1 is the founding member of a family of chimeric molecules restricted to trypanosomatids bearing, in addition to eukaryotic ubiquitin-like and protein-protein interacting domains, a motif displaying significant structural homology to bacterial multi-cargo chaperones involved in the secretion of virulence factors. Using the fidelity of an homologous expression system we confirmed TCLP 1 sub-cellular distribution and showed that TCLP 1-over-expressing parasites display impaired survival and accelerated progression to late stationary phase under starvation conditions. The reduced endocytic capacity of TCLP 1-over-expressors likely underlies (at least in part) this growth phenotype. TCLP 1 is involved in the uptake of extracellular macromolecules required for nutrition and hence in T. cruzi growth. Due to the bacterial origin, sub-cellular distribution and putative function(s), we propose TCLP 1 and related orthologs in trypanosomatids as appealing therapeutic targets for intervention against these health-threatening parasites. PMID:26086767

  12. A Novel Trypanosoma cruzi Protein Associated to the Flagellar Pocket of Replicative Stages and Involved in Parasite Growth.

    PubMed

    Durante, Ignacio M; Cámara, María de Los Milagros; Buscaglia, Carlos A

    2015-01-01

    The flagellar pocket constitutes an active and strategic site in the body of trypanosomatids (i.e. parasitic protozoa that cause important human and/or livestock diseases), which participates in several important processes such as cell polarity, morphogenesis and replication. Most importantly, the flagellar pocket is the unique site of surface protein export and nutrient uptake in trypanosomatids, and thus constitutes a key portal for the interaction with the host. In this work, we identified and characterized a novel Trypanosoma cruzi protein, termed TCLP 1, that accumulates at the flagellar pocket area of parasite replicative forms, as revealed by biochemical, immuno-cytochemistry and electron microscopy techniques. Different in silico analyses revealed that TCLP 1 is the founding member of a family of chimeric molecules restricted to trypanosomatids bearing, in addition to eukaryotic ubiquitin-like and protein-protein interacting domains, a motif displaying significant structural homology to bacterial multi-cargo chaperones involved in the secretion of virulence factors. Using the fidelity of an homologous expression system we confirmed TCLP 1 sub-cellular distribution and showed that TCLP 1-over-expressing parasites display impaired survival and accelerated progression to late stationary phase under starvation conditions. The reduced endocytic capacity of TCLP 1-over-expressors likely underlies (at least in part) this growth phenotype. TCLP 1 is involved in the uptake of extracellular macromolecules required for nutrition and hence in T. cruzi growth. Due to the bacterial origin, sub-cellular distribution and putative function(s), we propose TCLP 1 and related orthologs in trypanosomatids as appealing therapeutic targets for intervention against these health-threatening parasites.

  13. A Novel Trypanosoma cruzi Protein Associated to the Flagellar Pocket of Replicative Stages and Involved in Parasite Growth

    PubMed Central

    Durante, Ignacio M.; Cámara, María de los Milagros; Buscaglia, Carlos A.

    2015-01-01

    The flagellar pocket constitutes an active and strategic site in the body of trypanosomatids (i.e. parasitic protozoa that cause important human and/or livestock diseases), which participates in several important processes such as cell polarity, morphogenesis and replication. Most importantly, the flagellar pocket is the unique site of surface protein export and nutrient uptake in trypanosomatids, and thus constitutes a key portal for the interaction with the host. In this work, we identified and characterized a novel Trypanosoma cruzi protein, termed TCLP 1, that accumulates at the flagellar pocket area of parasite replicative forms, as revealed by biochemical, immuno-cytochemistry and electron microscopy techniques. Different in silico analyses revealed that TCLP 1 is the founding member of a family of chimeric molecules restricted to trypanosomatids bearing, in addition to eukaryotic ubiquitin-like and protein-protein interacting domains, a motif displaying significant structural homology to bacterial multi-cargo chaperones involved in the secretion of virulence factors. Using the fidelity of an homologous expression system we confirmed TCLP 1 sub-cellular distribution and showed that TCLP 1-over-expressing parasites display impaired survival and accelerated progression to late stationary phase under starvation conditions. The reduced endocytic capacity of TCLP 1-over-expressors likely underlies (at least in part) this growth phenotype. TCLP 1 is involved in the uptake of extracellular macromolecules required for nutrition and hence in T. cruzi growth. Due to the bacterial origin, sub-cellular distribution and putative function(s), we propose TCLP 1 and related orthologs in trypanosomatids as appealing therapeutic targets for intervention against these health-threatening parasites. PMID:26086767

  14. Effect of iacP Mutation on Flagellar Phase Variation in Salmonella enterica Serovar Typhimurium Strain UK-1

    PubMed Central

    Eom, Jeong Seon; Kim, Jin Seok; Jang, Jung Im; Kim, Hyeon Guk; Bang, Iel-Soo

    2012-01-01

    Flagella are surface appendages that are important for bacterial motility and invasion of host cells. Two flagellin subunits in Salmonella enterica serovar Typhimurium, FliC and FljB, are alternatively expressed by a site-specific DNA inversion mechanism called flagellar phase variation. Although this inversion mechanism is understood at the molecular level, the key factor controlling the expression of the two flagellin subunits has not been determined. In this study, we found that a putative acyl carrier protein, IacP, affects flagellar phase variation in S. Typhimurium strain UK-1 under Salmonella pathogenicity island 1 (SPI1)-inducing conditions. Liquid chromatography-mass spectrometry analysis of the secreted proteins from S. Typhimurium determined that the amount of FljB secreted was significantly higher in the iacP mutant strain, a finding confirmed by Western blot analysis. Northern blotting, quantitative PCR, and microarray data showed that the level of FljB in the iacP mutant strain was regulated at the transcriptional level, although the transcription and expression of the fliC gene were independent of IacP. FljB production was abolished by the deletion of the Hin DNA invertase but could be restored by the introduction of a plasmid carrying the hin gene. We also found that in the iacP mutant strain, the orientation of the invertible H segment is in the FljB-expressing phase. Furthermore, electron microscopy observations indicated that the iacP mutant strain had more flagella per cell than the wild-type strain. These results suggest that IacP is associated with flagellar phase switching under SPI1-inducing conditions. PMID:22685287

  15. Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

    PubMed Central

    Bastin, Philippe; MacRae, Thomas H.; Francis, Susan B.; Matthews, Keith R.; Gull, Keith

    1999-01-01

    The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii. PMID:10567544

  16. Entropy and information in flagellar axoneme cybernetics: a radial spokes integrative function.

    PubMed

    Cibert, Christian

    2003-04-01

    Radial spokes and the consequences of their relationships with the central apparatus seem to play a very important role in the regulation of axonemal activity. We modeled their behavior and observed that it appears to differ in the cilium and the flagellum with respect to the development of bending as a function of time. Specifically, our calculation raises the question of the real function of the radial spokes in the regulation of the axoneme, because a given curvature of the flagellar axoneme may correspond to two opposite of their tilts. The stable nil/low amplitude shear points that we had characterized along the flagellum allowed us to describe their axoneme as a series of modules [Cibert, 2002: Cell Motil. Cytoskeleton 51:89-111]. We observed that a nil/low shearing point moves along each module during beating when a new bend is created at the base of the flagellum [Cibert, 2001: Cell Motil. Cytoskeleton 49:161-175]. We propose that the structural gradients of isoforms of tubulin could be basic verniers that act as structural references for the axonemal machinery during the beating. This allowed us to interpret the axonemal organization as a segmented structure, that could be analyzed according to the complexion(1) theory and Shannon's information theory, which associate entropy and probability in the creation of information. The important consequence of this interpretation is that regulation of the axonemal machinery appears to be due to the upstream and downstream cross-talk between the axonemal segments that do not involve any dedicated integrative structure but depend on the energy level of the entire length of each module.

  17. Plasticity of Intact Rubral Projections Mediates Spontaneous Recovery of Function after Corticospinal Tract Injury

    PubMed Central

    Siegel, Chad S.; Fink, Kathren L.; Strittmatter, Stephen M.

    2015-01-01

    Axons in the adult CNS fail to regenerate after injury, and therefore recovery from spinal cord injury (SCI) is limited. Although full recovery is rare, a modest degree of spontaneous recovery is observed consistently in a broad range of clinical and nonclinical situations. To define the mechanisms mediating spontaneous recovery of function after incomplete SCI, we created bilaterally complete medullary corticospinal tract lesions in adult mice, eliminating a crucial pathway for voluntary skilled movement. Anatomic and pharmacogenetic tools were used to identify the pathways driving spontaneous functional recovery in wild-type and plasticity-sensitized mice lacking Nogo receptor 1. We found that plasticity-sensitized mice recovered 50% of normal skilled locomotor function within 5 weeks of lesion. This significant, yet incomplete, spontaneous recovery was accompanied by extensive sprouting of intact rubrofugal and rubrospinal projections with the emergence of a de novo circuit between the red nucleus and the nucleus raphe magnus. Transient silencing of this rubro–raphe circuit in vivo via activation of the inhibitory DREADD (designer receptor exclusively activated by designer drugs) receptor hM4di abrogated spontaneous functional recovery. These data highlight the pivotal role of uninjured motor circuit plasticity in supporting functional recovery after trauma, and support a focus of experimental strategies on enhancing intact circuit rearrangement to promote functional recovery after SCI. PMID:25632122

  18. Disruption of the A-Kinase Anchoring Domain in Flagellar Radial Spoke Protein 3 Results in Unregulated Axonemal cAMP-dependent Protein Kinase Activity and Abnormal Flagellar Motility

    PubMed Central

    Gaillard, Anne R.; Fox, Laura A.; Rhea, Jeanne M.; Craige, Branch

    2006-01-01

    Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility. PMID:16571668

  19. Vulnerability of ecosystems to climate change moderated by habitat intactness.

    PubMed

    Eigenbrod, Felix; Gonzalez, Patrick; Dash, Jadunandan; Steyl, Ilse

    2015-01-01

    The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network.

  20. Vulnerability of ecosystems to climate change moderated by habitat intactness.

    PubMed

    Eigenbrod, Felix; Gonzalez, Patrick; Dash, Jadunandan; Steyl, Ilse

    2015-01-01

    The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network. PMID:25059822