Oxford Molecular Motors

Abstract:

Purpose

The investigation of cortical porosity is an important aspect of understanding biological, pathoetiological, and biomechanical processes occurring within the skeleton. With the emergence of HR-pQCT as a noninvasive tool suitable for clinical use, cortical porosity at appendicular sites can be directly visualized in vivo. The aim of this study was to introduce a novel topological analysis of the cortical pore network for HR-pQCT data and determine the influence of resolution on measures of cortical pore network microstructure and topology.

Methods

Cadaveric radii were scanned using HR-pQCT at two different voxel sizes (41 and 82 μm) and also using μCT at a voxel size of 18 μm. HR-pQCT and μCT image sets were spatially coregistered. Segmentation and quantification of cortical porosity (Ct.Po) and mean pore diameter (Ct.Po.Dm) were achieved using an established extended cortical analysis technique. Topological classification of individual pores was performed using topology-preserving skeletonization and multicolor dilation algorithms. Based on the pore skeleton topological classification, the following parameters were quantified: total number of planar surface-skeleton canals (N.Slabs), tubular curve-skeleton canals (N.Tubes), and junction elements (N.Junctions), mean slab volume (Slab.Vol), mean tube volume (Tube.Vol), mean slab orientation (Slab.θ), mean tube orientation (Tube.θ), N.Slabs/N.Tubes, and integral (total) slab volume/integral tube volume (iSlab.Vol/iTube.Vol). An in vivo reproducibility study was also conducted to assess short-term precision of the topology parameters. Precision error was characterized using root mean square coefficient of variation (RMSCV%).

Results

Correlations to μCT values for Ct.Po were significant for both the 41 and 82 μm HR-pQCT data (41: r(2) = 0.82, p < 0.001, 82: r(2) = 0.75, p < 0.001). For Ct.Po.Dm, only the 41 μm data were significantly predictive of μCT values (r(2) = 0.72, p < 0.01) Data at both HR-pQCT voxel sizes were strongly predictive of the μCT values for N.Slabs (41: r(2) = 0.93, p < 0.001; 82: r(2) = 0.84, p < 0.001), N.Tubes (41: r(2) = 0.94, p < 0.001; 82: r(2) = 0.84, p < 0.001), and N.Junctions (41: r(2) = 0.93, p < 0.001; 82: r(2) = 0.78, p < 0.001), though proportional bias was evident in these correlations. Weak correlations were seen for iSlab.Vol/iTube.Vol at both voxel sizes (41: r(2) = 0.52, p < 0.01; 82: r(2) = 0.39, p < 0.05). Slab.Vol was significantly correlated to μCT data at 41 μm (r(2) = 0.60, p < 0.01) but not at 82 μm, while Tube.Vol was significantly correlated at both voxel sizes (41: r(2) = 0.79, p < 0.001; 82: r(2) = 0.68, p < 0.01). In vivo precision error for these parameters ranged from 2.31 to 9.68 RMSCV%.

Conclusions

Strong correlations between μCT- and HR-pQCT-derived measurements were found, particularly in HR-pQCT images obtained at 41 μm. These data are in agreement with our previous study investigating the effect of voxel size on standard HR-pQCT metrics of trabecular and cortical microstructure, and extend our previous findings to include topological descriptors of the cortical pore network.

Abstract:

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na(+)-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H(+)-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1-2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.

Abstract:

UNLABELLED: It is becoming clear that the bacterial flagellar motor output is important not only for bacterial locomotion but also for mediating the transition from liquid to surface living. The output of the flagellar motor changes with the mechanical load placed on it by the external environment: at a higher load, the motor runs more slowly and produces higher torque. Here we show that the number of torque-generating units bound to the flagellar motor also depends on the external mechanical load, with fewer stators at lower loads. Stalled motors contained at least as many stators as rotating motors at high load, indicating that rotation is unnecessary for stator binding. Mutant stators incapable of generating torque could not be detected around the motor. We speculate that a component of the bacterial flagellar motor senses external load and mediates the strength of stator binding to the rest of the motor. IMPORTANCE: The transition between liquid living and surface living is important in the life cycles of many bacteria. In this paper, we describe how the flagellar motor, used by bacteria for locomotion through liquid media and across solid surfaces, is capable of adjusting the number of bound stator units to better suit the external load conditions. By stalling motors using external magnetic fields, we also show that rotation is not required for maintenance of stators around the motor; instead, torque production is the essential factor for motor stability. These new results, in addition to previous data, lead us to hypothesize that the motor stators function as mechanosensors as well as functioning as torque-generating units.

Abstract:

The rotary motor F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) is one of the best-studied of all molecular machines. F(1)-ATPase is the part of the enzyme F(1)F(O)-ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F(1)-ATPase from Escherichia coli (EF(1)) is governed by the same mechanism as TF(1) under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF(1) molecules, we characterized the ATP-binding, catalytic and inhibited states of EF(1). We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF(1) than in TF(1), and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.