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, 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-nanometerdiameter 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 (Ϸ300 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).colloidal gold ͉ molecular motor ͉ motility In Escherichia coli, a motor torque-generating unit is composed of four copies of MotA and two copies of MotB, enclosing two proton-conducting transmembrane channels (1). Protonation and subsequent de-protonation of Asp-32 at the cytoplasmic end of either channel is thought to drive conformational changes that exert forces on the periphery of the rotor, via electrostatic interactions between MotA and the rotor protein FliG (2). Estimates of the number of torque-generating units per motor range from 8 to 11 (3, 4). At high loads and low speeds, motors operate near thermodynamic equilibrium, where rates of ion movement or conformational change do not matter (5). One can heat cells up or cool them down or change the solvent from H 2 O to D 2 O without changing motor torque; however, at low loads and high speeds, rates do matter, and motors run more slowly at lower temperatures or in D 2 O (6, 7).Because power output is the product of torque and speed, it is of interest to measure the torque generated at different speeds. The torque-speed relationship is useful for testing specific models of motor rotation (1, 5). In E. coli, motor torque is maximum at stall, it falls Ϸ10% between 0 and Ϸ170 Hz (at room temperature), and then it drops rapidly, reaching 0 at Ϸ330 Hz (6, 8). We have measured torque-speed curves for motors with different numbers of torque-generating units by linking latex spheres of different sizes to f lagellar filament stubs on motA cells and then inducing expression of wild-type MotA (9). This work suggested that the family of torque-speed curves might converge to the same zero-torque value, as expected if torque generators move independently and ...

Individuals with Down syndrome (DS) will inevitably developAlzheimer disease (AD) neuropathology sometime after middle age, which may be attributable to genes triplicated in individuals with DS. The characteristics of AD neuropathology include neuritic plaques, neurofibrillary tangles, and neuronal loss in various brain regions. The mechanism underlying neurodegeneration in AD and DS remains elusive. Regulator of calcineurin 1 (RCAN1) has been implicated in the pathogenesis of DS. Our data show that RCAN1 expression is elevated in the cortex of DS and AD patients. RCAN1 expression can be activated by the stress hormone dexamethasone. A functional glucocorticoid response element was identified in the RCAN1 isoform 1 (RCAN1-1) promoter region, which is able to mediate the upregulation of RCAN1 expression. Here we show that overexpression of RCAN1-1 in primary neurons activates caspase-9 and caspase-3 and subsequently induces neuronal apoptosis. Furthermore, we found that the neurotoxicity of RCAN1-1 is inhibited by knock-out of caspase-3 in caspase-3 ؊/؊ neurons. Our study provides a novel mechanism by which RCAN1 functions as a mediator of stress-and A␤-induced neuronal death, and overexpression of RCAN1 due to an extra copy of the RCAN1 gene on chromosome 21 contributes to AD pathogenesis in DS. Down syndrome (DS)4 is the most common genetic cause of mental retardation, affecting approximately one in 800 -1,000 babies (1). Individuals with DS inevitably develop characteristic Alzheimer disease (AD) neuropathology, which includes neuritic plaques, neurofibrillary tangles, and neuronal cell death following middle age (2-5). DS is a valuable model system for understanding AD pathogenesis. Both DS and AD feature prominent neuronal losses in the hippocampus and cortex, resulting in progressive cognitive deficits in AD patients (5). Apoptosis has been implicated in playing a major role in neuronal cell death in both AD and DS (6 -9). Increased immunoreactivity of activated caspase-3 is observed in neurons of AD and DS (7); however, the underlying mechanism leading to neuronal apoptosis in AD and DS remains elusive.The development of DS is caused by the presence of an extra copy of human chromosome 21 (10, 11). The DSCR1 (Down syndrome critical region 1) gene was identified and located on chromosome 21 (12, 13). Dysregulation of a regulatory circuit involving DSCR1-calcineurin-nuclear factor of activated T cells (NFAT) plays an important role in DS development (14). DSCR1 proteins physically interact with calcineurin subunit A and inhibit calcineurin activity in vitro and in vivo (15-19). DSCR1 was accordingly renamed as RCAN1 (regulator of calcineurin 1) (20). RCAN1 is phosphorylated at Ser 112 by BMK1 (big MAP kinase 1), which is the priming site for the subsequent phosphorylation at Ser 108 by GSK-3 (21-24). The phosphorylation form of RCAN1 can release the inhibition effect on calcineurin. Furthermore, phosphorylated RCAN1 is a substrate for calcineurin (79). RCAN1 FLISPP motif phosphorylation could increase its ab...

Cells of 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). Rotation in either direction has been thought to be symmetric and exhibit the same torques and speeds. The relationship between torque and speed is one of the most important measurable characteristics of the motor, used to distinguish specific mechanisms of motor rotation. Previous measurements of the torque-speed relationship have been made with cells lacking the response regulator CheY that spin their motors exclusively CCW. In this case, the torque declines slightly up to an intermediate speed called the "knee speed" after which it falls rapidly to zero. This result is consistent with a "power-stroke" mechanism for torque generation. Here, we measure the torque-speed relationship for cells that express large amounts of CheY and only spin their motors CW. We find that the torque decreases linearly with speed, a result remarkably different from that for CCW rotation. We obtain similar results for wild-type cells by reexamining data collected in previous work. We speculate that CCW rotation might be optimized for runs, with higher speeds increasing the ability of cells to sense spatial gradients, whereas CW rotation might be optimized for tumbles, where the object is to change cell trajectories. But why a linear torque-speed relationship might be optimum for the latter purpose we do not know. molecular motor | motility | nanogold | switch M easurements of the torque-speed relationship of the bacterial flagellar motor provide a crucial test of models for motor rotation (1, 2). Previous measurements of this relationship have been made with smooth-swimming [counterclockwise (CCW) rotating] mutants of a variety of species: with the proton-driven motor of Escherichia coli (3, 4), with the sodiumdriven motor of Vibrio alginolyticus (5), and with a sodium-driven chimeric motor in E. coli (6). In all cases, motor torque is approximately constant up to a knee speed, after which it drops rapidly to zero. In E. coli at room temperature, the knee speed is about 170 Hz, and the zero-torque speed is about 300 Hz. It has been assumed that CCW and clockwise (CW) rotation are symmetric and exhibit the same torques and speeds (7).Here, we measured the torque-speed relationship for an E. coli strain locked in CW rotation. This strain is deleted for the genes that encode the response regulator, CheY, and its phosphatase, CheZ, as well as the adaptation enzymes CheR and CheB. Introduction of a plasmid that encodes wild-type CheY that can be induced to high levels with isopropyl β-D-thiogalactoside (IPTG) enables CW rotation. For comparison, we also measured the torque-speed relationship with the same strain lacking the plasmid, which is locked in CCW rotation. The measurements were made by adsorbing 0.356 μm diameter latex spheres to sticky-filament stubs (8) and monitoring rotation rates in motility medium containing diffe...

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.

The flagellar motor of Escherichia coli has been shown to adapt to changes in the steady-state level of the chemotaxis response regulator, CheY-P, by adjusting the number of molecules to which CheY-P binds, FliM. Previous measurements of motor ultrasensitivity have been made on cells containing different amounts of CheY-P, and thus different amounts of FliM. Here, we designed an experiment to measure the sensitivity of motors containing fixed amounts of FliM, finding Hill coefficients about twice as large as those observed before. This ultrasensitivity provides further insights into the motor switching mechanism and plays important roles in chemotaxis signal amplification and coordination of multiple motors. The Hill coefficients observed here appear to be the highest known for allosteric protein complexes, either biological or synthetic. Extreme motor ultrasensitivity broadens our understanding of mechanisms of allostery, and serves as an inspiration for future design of synthetic protein switches.

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