Pushkar P. Lele

Dynamics of mechanosensing in the bacterial flagellar motor

Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP–MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6–11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene fliL or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.

Polarization and interactions of colloidal particles in ac electric fields

Micrometer-sized polystyrene particles form two-dimensional crystals in alternating current (ac) electric fields. The induced dipole-dipole interaction is the dominant force that drives this assembly. We report measurements of forces between colloidal particles in ac electric fields using optical tweezers and find good agreement with the point dipole model. The magnitude of the pair interaction forces depends strongly on the bulk solution conductivity and decreases as the ionic strength increases. The forces also decrease with increasing field frequency. The salt and frequency dependences are consistent with double layer polarization with a characteristic relaxation frequency omega(CD) approximately a(2)/D, where a is the particle radius and D is the ion diffusivity. This enables us to reinterpret the order-disorder transition reported for micrometer-sized polystyrene particles [Lumsdon et al., Langmuir 20, 2108 (2004)], including the dependence on particle size, frequency, and ionic strength. These results provide a rational framework for identifying assembly conditions of colloidal particles in ac fields over a wide range of parameters.

Mechanism for adaptive remodeling of the bacterial flagellar switch

The bacterial flagellar motor has been shown in previous work to adapt to changes in the steady-state concentration of the chemotaxis signaling molecule, CheY-P, by changing the FliM content. We show here that the number of FliM molecules in the motor and the fraction of FliM molecules that exchange depend on the direction of flagellar rotation, not on CheY-P binding per se. Our results are consistent with a model in which the structural differences associated with the direction of rotation modulate the strength of FliM binding. When the motor spins counterclockwise, FliM binding strengthens, the fraction of FliM molecules that exchanges decreases, and the ring content increases. The larger number of CheY-P binding sites enhances the motor’s sensitivity, i.e., the motor adapts. An interesting unresolved question is how additional copies of FliM might be accommodated.

Assemble-and-stretch method for creating two- and three-dimensional structures of anisotropic particles.

We fabricate two-dimensional ordered arrays of anisotropic particles by embedding hexagonally close-packed monolayers of spherical latex particles in polymer films and subjecting them to an extensional strain. Furthermore, by stacking several films, we create three-dimensional structures. An important advantage of this approach is the ability to independently control the particle orientation in each layer of the structure. Additionally, the symmetry of a layer is controlled by the orientation of the original array of spherical particles versus the stretch direction. This technique may be readily used to generate structures for photonic applications, as well as to aid fundamental studies of packing of anisotropic particles.

A Rotary Motor Drives Flavobacterium Gliding

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.

Anomalous particle rotation and resulting microstructure of colloids in AC electric fields.

We study the transition of ordered structures to disordered bands and vortices in colloidal suspensions subjected to AC electric fields. We map the critical frequencies and field biases at which particles form disordered bands and vortices. These results are interpreted based on the trajectory dynamics of particle pairs using blinking optical tweezers. Under conditions that vortices are observed, individual particle pairs rotate out of alignment with the field. The direction and magnitude of these interactions determine the orientation and average angular velocity of the band revolution. Increasing the frequency of the electric field reduces the anomalous rotation of the particles pairs, consistent with the frequency dependence of the suspension order-to-disorder transition. This anomalous rotation is consistent with a torque on doublets generated by the mutual polarization of particles and phase lag of the induced dipoles.

Colloidal diffusion and hydrodynamic screening near boundaries

The hydrodynamic interactions between colloidal particles in small ensembles are measured at varying distances from a no-slip surface over a range of inter-particle separations. The diffusion tensor for motion parallel to the wall of each ensemble is calculated by analyzing thousands of particle trajectories generated by blinking holographic optical tweezers and by dynamic simulation. The Stokesian Dynamics simulations predict similar particle dynamics. By separating the dynamics into three classes of modes: self, relative and collective diffusion, we observe qualitatively different behavior depending on the relative magnitudes of the distance of the ensemble from the wall and the inter-particle separation. A simple picture of the pair-hydrodynamic interactions is developed, while many-body-hydrodynamic interactions give rise to more complicated behavior. The results demonstrate that the effect of many-body hydrodynamic interactions in the presence of a wall is much richer than the single particle behavior and that the multiple-particle behavior cannot be simply predicted by a superposition of pair interactions.

FRAP Analysis: Accounting for Bleaching during Image Capture

The analysis of Fluorescence Recovery After Photobleaching (FRAP) experiments involves mathematical modeling of the fluorescence recovery process. An important feature of FRAP experiments that tends to be ignored in the modeling is that there can be a significant loss of fluorescence due to bleaching during image capture. In this paper, we explicitly include the effects of bleaching during image capture in the model for the recovery process, instead of correcting for the effects of bleaching using reference measurements. Using experimental examples, we demonstrate the usefulness of such an approach in FRAP analysis.

Response thresholds in bacterial chemotaxis

The absence of responses to shallow temporal ramps of chemicals does not appear to be accounted for by motor remodeling. Stimulation of Escherichia coli by exponential ramps of chemoattractants generates step changes in the concentration of the response regulator, CheY-P. Because flagellar motors are ultrasensitive, this should change the fraction of time that motors spin clockwise, the CWbias. However, early work failed to show changes in CWbias when ramps were shallow. This was explained by a model for motor remodeling that predicted plateaus in plots of CWbias versus [CheY-P]. We looked for these plateaus by examining distributions of CWbias in populations of cells with different mean [CheY-P]. We did not find such plateaus. Hence, we repeated the work on shallow ramps and found that motors did indeed respond. These responses were quantitatively described by combining motor remodeling with ultrasensitivity in a model that exhibited high sensitivities over a wide dynamic range.

Switching of Bacterial Flagellar Motors Triggered by Mutant FliG

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 (FliG(CW)). 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 FliM(CW) 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.