Mechanosensing by flagella is thought to trigger bacterial swarmercell 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.Escherichia coli | mechanical load | stator remodeling M echanical forces play an important role in triggering changes in gene expression and biochemical signaling in many biological systems (1, 2). Mechanical inhibition of the rotation of flagellar motors (due to higher viscosities or proximity to surfaces) is known to trigger swarmer-cell differentiation, a key step in pathogenesis in some bacterial species (3-5). Previous investigations have measured the long-term effects of mechanical stimuli on gene expression and bacterial swarming, e.g., by varying the mechanical properties of the growth medium (6). However, little is known about how the flagellar motor senses mechanical stimuli at the molecular level. Here, we apply direct mechanical stimuli and report short-time adaptation in motor response that has not been measured before. These results reveal details of the immediate response to mechanical signals in flagellar motors.Escherichia coli swim by rotating helical filaments driven by motors embedded in the cell wall. The motors switch between clockwise (CW) and counterclockwise (CCW) directions of rotation to bias cell movements in response to chemical gradients.Motor outputs, such as speed and the probability of CW rotation (CW bias), can be experimentally measured and reveal the immediate effects of stimuli on protein interactions associated with the motor...

In the bacterial chemotaxis network, receptor clusters process input 1 – 3 , and flagellar motors generate output 4 . Receptor and motor complexes are coupled by the diffusible protein CheY-P. Receptor output (the steady-state concentration of CheY-P) varies from cell to cell 5 . However, the motor is ultrasensitive, with a narrow [CheY-P] operating range 6 . How might the match between receptor output and motor input be optimized? Here we show that the motor can shift its operating range by changing its composition. The number of FliM subunits in the C-ring increases in response to a decrement in the concentration of CheY-P, increasing motor sensitivity. This shift in sensitivity explains the slow partial adaptation observed in mutants that lack the receptor methyltransferase and methylesterase 7 – 8 and why motors exhibit signal-dependent FliM turnover 9 . Adaptive remodelling is likely to be a common feature in the operation of many molecular machines.

Flagellated bacteria can swim within a thin film of fluid that coats a solid surface, such as agar; this is a means for colony expansion known as swarming. We found that micrometer-sized bubbles make excellent tracers for the motion of this fluid. The microbubbles form explosively when small aliquots of an aqueous suspension of droplets of a water-insoluble surfactant (Span 83) are placed on the agar ahead of a swarm, as the water is absorbed by the agar and the droplets are exposed to air. Using these bubbles, we discovered an extensive stream (or river) of swarm fluid flowing clockwise along the leading edge of an Escherichia coli swarm, at speeds of order 10 μm/s, about three times faster than the swarm expansion. The flow is generated by the action of counterclockwise rotating flagella of cells stuck to the substratum, which drives fluid clockwise around isolated cells (when viewed from above), counterclockwise between cells in dilute arrays, and clockwise in front of cells at the swarm edge. The river provides an avenue for long-range communication in the swarming colony, ideally suited for secretory vesicles that diffuse poorly. These findings broaden our understanding of swarming dynamics and have implications for the engineering of bacterial-driven microfluidic devices.bacterial motility | flagellar rotation | microscopic bubbles W hen grown on a moist nutrient-rich medium, many flagellated bacteria elongate, produce wetting agents, and colonize the surface by swimming outward in densely packed groups within a thin layer of fluid, a process known as swarming (1-3). Swarming promotes invasiveness and virulence of infectious pathogens (2) and is regulated by pathways that enable cells to choose between swimming or forming sessile biofilms (4). A model system that we study is the bacterium Escherichia coli, which was shown to swarm by Harshey and Matsuyama (5). Our focus is on swarm mechanics, how cells move and how this motion is promoted by flagellar rotation (6-8, but see also refs. 9-11).However, little is known about the swarm fluid, despite its significance to the expansion and physiology of swarms. The swarm fluid is important for swarm expansion, not only because it supports the operation of flagella (12), but also because it carries nutrients that support growth or molecules that regulate cellular functions, such as signals involved in quorum-sensing (13,14). Quorum-sensing has been found to regulate the synthesis of biosurfactants that facilitate swarming in Bacillus subtilis (15, 16), Serratia liquefaciens (17), and Pseudomonas aeruginosa (18). The quorum-sensing molecules involved in these systems are released from cells and diffuse in the swarm fluid before entering cells again.The layer of swarm fluid can be as thin as the width of a cell (∼1 μm) and even thinner near the swarm edge (8), which makes it difficult to probe the hydrodynamics of swarm fluid with conventional microfluid markers, such as polystyrene microspheres (19). Large markers tend to be entrained by cells within the body of th...

We have designed and constructed a versatile magnetic tweezer primarily for intracellular investigations. The micromanipulator uses only two coils to simultaneously magnetize to saturation micron-size superparamagnetic particles and generate high magnitude constant field gradients over cellular dimensions. The apparatus resembles a miniaturized Faraday balance, an industrial device used to measure magnetic susceptibility. The device operates in both continuous and pulse modes. Due to its compact size, the tweezers can conveniently be mounted on the stage of an inverted microscope and used for intracellular manipulations. A built-in temperature control unit maintains the sample at physiological temperatures. The operation of the tweezers was tested by moving 1.28 m diameter magnetic beads inside macrophages with forces near 500 pN.

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ∼ 30 μm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ∼ 120 μm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (∼ 30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.

Effective cryopreservation of oocytes is critically needed in many areas of human reproductive medicine and basic science, such as stem cell research. Currently, oocyte cryopreservation has a low success rate. The goal of this study was to understand the mechanisms associated with oocyte cryopreservation through biophysical means using a mouse model. Specifically, we experimentally investigated the biomechanical properties of the ooplasm prior and after cryopreservation as well as the consequences of reversible dismantling of the F-actin network in mouse oocytes prior to freezing. The study was complemented with the evaluation of post-thaw developmental competence of oocytes after in vitro fertilization. Our results show that the freezing-thawing process markedly alters the physiological viscoelastic properties of the actin cytoskeleton. The reversible depolymerization of the F-actin network prior to freezing preserves normal ooplasm viscoelastic properties, results in high post-thaw survival and significantly improves developmental competence. These findings provide new information on the biophysical characteristics of mammalian oocytes, identify a pathophysiological mechanism underlying cryodamage and suggest a novel cryopreservation method.

The molecular cascade that controls switching of the direction of rotation of Escherichia coli flagellar motors is well known, but the conformational changes that allow the rotor to switch are still unclear. The signaling molecule CheY, when phosphorylated, binds to the C-ring at the base of the rotor, raising the probability that the motor spins clockwise. When the concentration of CheY-P is so low that the motor rotates exclusively counterclockwise (CCW), the C-ring recruits more monomers of FliM and tetramers of FliN, the proteins to which CheY-P binds, thus increasing the motor's sensitivity to CheY-P and allowing it to switch once again. Motors that rotate exclusively CCW have more FliM and FliN subunits in their C-rings than motors that rotate exclusively clockwise. How are the new subunits accommodated? Does the diameter of the C-ring increase, or do FliM and FliN get packed in a different pattern, keeping the overall diameter of the C-ring constant? Here, by measuring fluorescence anisotropy of yellow fluorescent protein-labeled motors, we show that the CCW C-rings accommodate more FliM monomers without changing the spacing between them, and more FliN monomers at the same time as increasing their effective spacing and/or changing their orientation within the tetrameric structure.