Spiral growth in the radially-expanding piloboloid mutants ofPhycomyces blakesleeanus

The growth zone of the sporangiophore of a piloboloid mutant,pil, ofPhycomyces expands radially at an increased rate until the growth zone becomes nearly spherical, in sharp contrast to that of the wild-type sporangiophore which exhibits longitudinal elongation only and is conical. The rotation of thepil sporangiophore reverses its direction from clockwise (CW) to counterclockwise (CCW) during the period of increased radial expansion, and the CCW rotation continues as long as does the radial expansion. The direction of rotation and the time of reversal are correlated with the relative rates of cell-wall expansion in the longitudinal and transverse directions. The CCW rotation of the sporangiophore of this mutant can be explained by the behavior of the microfibrils, as previously proposed to explain the rotation of the wild-type sporangiophore.Abbreviations CW clockwise - CCW counterclockwise — both as viewed from above     

Noise underlies switching behavior of the bacterial flagellum

We report the switching behavior of the full bacterial flagellum system that includes the filament and the motor in wild-type Escherichia coli cells. In sorting the motor behavior by the clockwise bias, we find that the distributions of the clockwise (CW) and counterclockwise (CCW) intervals are either exponential or nonexponential with long tails. At low bias, CW intervals are exponentially distributed and CCW intervals exhibit long tails. At intermediate CW bias (0.5) both CW and CCW intervals are mainly exponentially distributed. A simple model suggests that these two distinct switching behaviors are governed by the presence of signaling noise within the chemotaxis network. Low noise yields exponentially distributed intervals, whereas large noise yields nonexponential behavior with long tails. These drastically different motor statistics may play a role in optimizing bacterial behavior for a wide range of environmental conditions.     

Switching of the Bacterial Flagellar Motor Near Zero Load

Flagellated bacteria, such as Escherichia coli, are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counterclockwise (CCW). Chemotactic behavior has been studied under a variety of conditions, mostly at high loads (at large motor torques). Here, we examine motor switching at low loads. Nano-gold spheres of various sizes were attached to hooks (the flexible coupling at the base of the flagellar filament) of cells lacking flagellar filaments in media containing different concentrations of the viscous agent Ficoll. The speeds and directions of rotation of the spheres were measured. Contrary to the case at high loads, motor switching rates increased appreciably with load. Both the CW → CCW and CCW → CW switching rates increased linearly with motor torque. Evidently, the switch senses stator-rotor interactions as well as the CheY-P concentration.     

Temperature-induced switching of the bacterial flagellar motor.

Chemotaxis signaling proteins normally control the direction of rotation of the flagellar motor of Escherichia coli. In their absence, a wild-type motor spins exclusively counterclockwise. Although the signaling pathway is well defined, relatively little is known about switching, the mechanism that enables the motor to change direction. We found that switching occurs in the absence of signaling proteins when cells are cooled to temperatures below about 10 degrees C. The forward rate constant (for counterclockwise to clockwise, CCW to CW, switching) increases and the reverse rate constant (for CW to CCW switching) decreases as the temperature is lowered. At about -2 degrees C, most motors spin exclusively CW. At temperatures for which reversals are frequent enough to generate a sizable data set, both CCW and CW interval distributions appear to be exponential. From the rate constants we computed equilibrium constants and standard free energy changes, and from the temperature dependence of the standard free energy changes we determined standard enthalpy and entropy changes. Using transition-state theory, we also calculated the activation free energy, enthalpy, and entropy. We conclude that the CW state is preferred at very low temperatures and that it is relatively more highly bonded and restricted than the CCW state.     

Changes in the short-term deflector loft effect are linked to the sun compass of homing pigeons

Despite being the most studied of all avian orientation systems, important questions still remain about the sun compass of homing pigeons, Columba livia. White it is well-documented that the sun compass is usually learned by young pigeons during the first 10–12 weeks of life, the mechanism by which it is calibrated to adjust for seasonal changes in the sun's azimuth is not known with certainty. Previous experiments using short-term deflector loft pigeons indicated that the sun compass may be calibrated by referencing celestial polarization patterns. The present paper describes important measurable changes in the previously reported orientation behaviour of short-term deflector loft birds, and suggests a correlation between these changes and the presence of a massive upper-atmospheric dust cloud of volcanic origin which significantly altered natural skylight polarization patterns in 1982 and 1983. Moreover, it is shown that when the short-term effect was absent (at times when data from previous years suggested it should be present), the birds were also not using sun compass orientation, as demonstrated by their failure to show the standard ‘clockshift’ response to a 6-h fast shift of their internal clocks. These results support the hypothesis that reflected light cues, rather than odours, are the basis of the deflector loft effect in pigeon homing.     

Direction of flagellar rotation in bacterial cell envelopes

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable.     

Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor.

Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro. Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.     

Influence of vision and motor imagery styles on equilibrium control during whole-body rotations

To investigate the influence of vision and motor imagery styles on equilibrium control, displacements of the supporting foot during spontaneous whole-body rotations (“pirouette”) by expert female ballet dancers were analyzed using three-dimensional kinematics. Four turn types were defined according to direction (clockwise, CW vs. counterclockwise, CCW) and supporting foot (SF, left vs. right). Visual influences were examined by including two visual conditions (blindfolded vs. full-vision). Motor imagery styles were determined using the Vividness of Movement Imagery Questionnaire (VMIQ) (Kinesthetic, n = 4 vs. Visual/Kinesthetic, n = 6). Turning direction preference was assessed by a closed-response questionnaire in which all dancers indicated that they preferred CW turn direction. Kinesthetic dancers showed more SF displacement during CCW (non-preferred direction) than CW (preferred direction) pirouettes. However, Visual/Kinesthetic dancers showed no significant effect of turn direction. Furthermore, Kinesthetic dancers showed no significant effect of vision on SF displacement whereas Visual/Kinesthetic dancers showed significantly higher SF displacement when vision was occluded. Thus there appears to be a selective effect of vision on Visual/Kinesthetic dancers, and a selective effect of turn direction on Kinesthetic dancers. These results suggest that perceptual styles should be taken into consideration when training tasks that require fine equilibrium control because the factors that perturb balance differ depending on perceptual style.     

Coordinated reversal of flagellar motors on a single Escherichia coli cell

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.     

Temperature dependence of switching of the bacterial flagellar motor by the protein CheY(13DK106YW).

The behavior of the bacterium Escherichia coli is controlled by switching of the flagellar rotary motor between the two rotational states, clockwise (CW) and counterclockwise (CCW). The molecular mechanism for switching remains unknown, but binding of the response regulator CheY-P to the motor component FliM enhances CW rotation. This effect is mimicked by the unphosphorylated double mutant CheY13DK106YW (CheY**). To learn more about switching, we measured the fraction of time that a motor spends in the CW state (the CW bias) at different concentrations of CheY** and at different temperatures. From the CW bias, we computed the standard free energy change of switching. In the absence of CheY, this free energy change is a linear function of temperature (. Biophys. J. 71:2227-2233). In the presence of CheY**, it is nonlinear. However, the data can be fit by models in which binding of each molecule of CheY** shifts the difference in free energy between CW and CCW states by a fixed amount. The shift increases linearly from approximately 0.3kT per molecule at 5 degrees C to approximately 0.9kT at 25 degrees C, where k is Boltzmann's constant and T is 289 Kelvin (= 16 degrees C). The entropy and enthalpy contributions to this shift are about -0. 031kT/ degrees C and 0.10kT, respectively.     

The effect of rectal gas on dose distribution during prostate cancer treatment using full arc and partial arc Volumetric Modulated Arc Therapy (VMAT) treatment plans

Background/AimIn this study, we investigated the effect of rectal gas on the dose distribution of prostate cancer using a volumetric modulated arc therapy (VMAT) treatment planning.Materials and MethodsThe first is the original structure set, clinical target volume (CTV), the rectum, and the bladder used clinically. The second is a structure set (simulated gas structure set) in which the overlapping part of the rectum and PTV is overwritten with Hounsfield Unit −950 as gas. Full arc and limited gantry rotation angle with VMAT were the two arcs. The VMAT of the full arc was 181°–179° in the clockwise (CW) direction and 179°–181° in the counterclockwise (CCW) direction. Three partial arcs with a limited gantry rotation angle were created: 200°–160 °CW and 160°–200 °CCW; 220°–140 °CW and 140°–220 °CCW; and finally, 240°–120 °CW and 120°–240 °CCW. The evaluation items were dose difference, distance to agreement, and gamma analysis.ResultIn the CTV, the full arc was the treatment planning technique with the least effect of rectal gas. In the rectum, when the gantry rotation angle range was short, the pass rate tended to reduce for all evaluation indices. The bladder showed no characteristic change between the treatment planning techniques in any of the evaluation indices.ConclusionsThe VMAT treatment planning with the least effect on dose distribution caused by rectal gas was shown to be a full arc.     

Chemotactic response regulator mutant CheY95IV exhibits enhanced binding to the flagellar switch and phosphorylation-dependent constitutive signalling

CheY, a response regulator protein in bacterial chemotaxis, mediates swimming behaviour through interaction with the flagellar switch protein, FliM. In its active, phosphorylated state, CheY binds to the motor switch complex and induces a change from counterclockwise (CCW) to clockwise (CW) flagellar rotation. The conformation of a conserved aromatic residue, tyrosine 106, has been proposed to play an important role in this signalling process. Here, we show that an isoleucine to valine substitution in CheY at position 95 — in close proximity to residue 106 — results in an extremely CW, hyperactive phenotype that is dependent on phosphorylation. Further biochemical characterization of this mutant protein revealed phosphorylation and dephosphorylation rates that were indistinguishable from those of wild-type CheY. CheY95IV, however, exhibited an increased binding affinity to FliM. Taken together, these results show for the first time a correlation between enhanced switch binding and constitutive signalling in bacterial chemotaxis. Considering present structural information, we also propose possible models for the role of residue 95 in the mechanism of CheY signal transduction.     

Handwriting: Hand–pen contact force synergies in circle drawing tasks

This study investigated synergistic actions of hand–pen contact forces during circle drawing tasks in three-dimensional (3D) space. Twenty-four right-handed participants drew thirty concentric circles in the counterclockwise (CCW) and clockwise (CW) directions. Three-dimensional forces acting on an instrumented pen as well as 3D linear and angular positions of the pen were recorded. These contact forces were then transformed into the 3D radial, tangential, and normal force components specific to circle drawing. Uncontrolled manifold (UCM) analysis was employed to calculate the magnitude of the hand–pen contact force synergy. Three hypotheses were tested. First, hand–pen contact force synergies during circle drawing are dependent on the angular position of the pen tip. Second, hand–pen contact force synergies are dependent on force components in circle drawing. Third, hand–pen contact force synergies are greater in CCW direction than CW direction. The results showed that the strength of the hand–pen contact force synergy increased during the initial phase of circle drawing and decreased during the final phase. The synergy strength was greater for the radial and tangential components as compared to the normal component. Also, the circle drawing in CW direction was associated with greater hand–pen contact force synergy than the CCW direction. The results of this study suggest that the central nervous system (CNS) prioritizes hand–pen contact force synergies for the force components (i.e., radial and tangential) that are critical for circle drawing. The CNS modulates hand–pen contact force synergies for preparation and conclusion of circle drawing, respectively.     

Phylogeny of the quadriflagellate Volvocales (Chlorophyceae) based on chloroplast multigene sequences

Since the phylogenetic relationships of the green plants (green algae and land plants) have been extensively studied using 18S ribosomal RNA sequences, change in the arrangement of basal bodies in flagellate cells is considered to be one of the major evolutionary events in the green plants. However, the phylogenetic relationships between biflagellate and quadriflagellate species within the Volvocales remain uncertain. This study examined the phylogeny of three genera of quadriflagellate Volvocales (Carteria, Pseudocarteria, and Hafniomonas) using concatenated sequences from three chloroplast genes. Using these multigene sequences, all three quadriflagellate genera were basal to other members (biflagellates) of the CW (clockwise) group (the Volvocales and their relatives, the Chlorophyceae) and formed three robust clades. Since the flagellar apparatuses of these three quadriflagellate lineages are diverse, including counter clockwise (CCW) and CW orientation of the basal bodies, the CW orientation of the basal bodies might have evolved from the CCW orientation in the ancestral quadriflagellate volvocalean algae, giving rise to the biflagellates, major members of the CW group.     

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

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

The phylogenetic relationship between the Chlamydomonadales and Chlorococcales inferred from 18SrDNA sequence data

Nuclear-encoded small subunit ribosomal RNA gene (185rDNA) sequences were determined for Chlamydomonas moewusii Gerloff and five chlorococcalean algae (Chlorococcum hypnosporum Starr; Chlorococcum oleofaciens Trainor et Bold; Chlorococcum sp.; Tetracystis aeria Brown et Bold; Protosiphon botryoides (Kützingl Klebs). All these algae are characterized by a clockwise CCW) flagellar apparatus. Phylogenetic trees were constructed from sequences from these algae together with 20 green algae. All algae with a CW flagellar apparatus form a monophyletic clade (CW group). Three principal clades can be recognized in the CW group, although no morphological character supports monophyly of any of these three clades. The 185rDNA trees clearly demonstrate the non-monophyly of the Chlamydomonadales and Chlorococcales, suggesting that vegetative morphology does not reflect phylogenetic relationships in the CW group. The paraphyly or polyphyly of the genus Chlamydomonas and Chlorococcum are also revealed. Present analysis suggests that the presence or absence of a zoospore's cell wall and the multinucleate condition have limited taxonomic values at higher taxonomic ranks.     

Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor.

FliG, FliM, and FliN are three proteins of Salmonella typhimurium that affect the rotation and switching of direction of the flagellar motor. An analysis of mutant alleles of FliM has been described recently (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992). We have now analyzed a large number of mutations in the fliG and fliN genes that are responsible for four different types of defects: failure to assembly flagella (nonflagellate phenotype), failure to rotate flagella (paralyzed phenotype), and failure to display normal chemotaxis as a result of an abnormally high bias to clockwise (CW) or counterclockwise (CCW) rotation (CW-bias and CCW-bias phenotypes, respectively). The null phenotype for fliG, caused by nonsense or frameshift mutations, was nonflagellate. However, a considerable part of the FliG amino acid sequence was not needed for flagellation, with several substantial in-frame deletions preventing motor rotation but not flagellar assembly. Missense mutations in fliG causing paralysis or abnormal switching occurred at a number of positions, almost all within the middle one-third of the gene. CW-bias and CCW-bias mutations tended to segregate into separate subclusters. The null phenotype of fliN is uncertain, since frameshift and nonsense mutations gave in some cases the nonflagellate phenotype and in other cases the paralyzed phenotype; in none of these cases was the phenotype a consequence of polar effects on downstream flagellar genes. Few positions in FliN were found to affect switching: only one gave rise to the CW mutant bias and only four gave rise to the CCW mutant bias. The different properties of the FliM, FliG, and FliN proteins with respect to the processes of assembly, rotation, and switching are discussed.     

Development of the navigational map in homing pigeons: effects of flight experience on orientation performance

Experiments have shown that pigeons, Columba livia, are able to develop navigational abilities even if reared and kept confined in an aviary, provided that they are exposed to the natural winds. In contrast, pigeons reared in a wind-screened aviary do not learn to navigate. Pigeons kept confined in a screened aviary when young do not learn to navigate even if, once they become adult, the screens are removed and the birds are exposed to natural winds for a period sufficiently long for map learning. In all of these experiments, pigeons were kept confined until the day of test release. In the present study we found that pigeons reared in a wind-screened aviary developed navigational abilities if, once adult, they were allowed to perform spontaneous flights around the loft. Nevertheless, their navigational performance never reached the level of controls.Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.      

Pigeon Navigation: Different Routes Lead to Frankfurt

Background

Tracks of pigeons homing to the Frankfurt loft revealed an odd phenomenon: whereas birds returning from the North approach their loft more or less directly in a broad front, pigeons returning from the South choose, from 25 km from home onward, either of two corridors, a direct one and one with a considerable detour to the West. This implies differences in the navigational process.

Methodology/Principle Findings

Pigeons released at sites at the beginning of the westerly corridor and in this corridor behave just like pigeons returning from farther south, deviating to the west before turning towards their loft. Birds released at sites within the straight corridors, in contrast, take more or less straight routes. The analysis of the short-term correlation dimension, a quantity reflecting the complexity of the system and with it, the number of factors involved in the navigational process, reveals that it is significantly larger in pigeons choosing the westerly corridor than in the birds flying straight - 3.03 vs. 2.85. The difference is small, however, suggesting a different interpretation of the same factors, with some birds apparently preferring particular factors over others.

Conclusions

The specific regional distribution of the factors which pigeons use to determine their home course seems to provide ambiguous information in the area 25 km south of the loft, resulting in the two corridors. Pigeons appear to navigate by deriving their routes directly from the locally available navigational factors which they interpret in an individual way. The fractal nature of the correlation dimensions indicates that the navigation process of pigeons is chaotic-deterministic; published tracks of migratory birds suggest that this may apply to avian navigation in general.