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An approaching predator and self-motion toward an object can generate similar looming patterns on the retina, but these situations demand different rapid responses. How central circuits flexibly process visual cues to activate appropriate, fast motor pathways remains unclear. Here we identify two descending neuron (DN) types that control landing and contribute to visuomotor flexibility in Drosophila. For each, silencing impairs visually evoked landing, activation drives landing, and spike rate determines leg extension amplitude. Critically, visual responses of both DNs are severely attenuated during non-flight periods, effectively decoupling visual stimuli from the landing motor pathway when landing is inappropriate. The flight-dependence mechanism differs between DN types. Octopamine exposure mimics flight effects in one, whereas the other probably receives neuronal feedback from flight motor circuits. Thus, this sensorimotor flexibility arises from distinct mechanisms for gating action-specific descending pathways, such that sensory and motor networks are coupled or decoupled according to the behavioral state.

NmMetQ is a substrate binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To address the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the absence and presence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function/topology, playing a role in NmMetNI-mediated transport at the inner-membrane in addition to moonlighting functions on the bacterial surface.

Significance The high-affinity methionine importer MetNI belongs to the ATP Binding Cassette (ABC) family of transporters that carry out the ATP-dependent uptake of substrates into cells. As with other ABC importers, MetNI requires a soluble binding protein (MetQ) that in the canonical mechanistic model delivers substrates to the transporter. We made the unexpected observation that a MetQ variant with significantly impaired ligand-binding properties supports d-selenomethionine uptake at a higher rate than wild-type MetQ. A crystal structure of MetNIQ in the outward-facing conformation reveals access channels through the binding protein to the transmembrane translocation pathway. These studies support a noncanonical role for the binding protein in facilitating the uptake of certain substrates directly through the transporter–binding protein complex., The Escherichia coli methionine ABC transporter MetNI exhibits both high-affinity transport toward l-methionine and broad specificity toward methionine derivatives, including d-methionine. In this work, we characterize the transport of d-methionine derivatives by the MetNI transporter. Unexpectedly, the N229A substrate-binding deficient variant of the cognate binding protein MetQ was found to support high MetNI transport activity toward d-selenomethionine. We determined the crystal structure at 2.95 Å resolution of the ATPγS-bound MetNIQ complex in the outward-facing conformation with the N229A apo MetQ variant. This structure revealed conformational changes in MetQ providing substrate access through the binding protein to the transmembrane translocation pathway. MetQ likely mediates uptake of methionine derivatives through two mechanisms: in the methionine-bound form delivering substrate from the periplasm to the transporter (the canonical mechanism) and in the apo form by facilitating ligand binding when complexed to the transporter (the noncanonical mechanism). This dual role for substrate-binding proteins is proposed to provide a kinetic strategy for ABC transporters to transport both high- and low-affinity substrates present in a physiological concentration range.

Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. Here, we map the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, we quantified the behavioral effects of activating 2,204 genetically targeted populations of neurons. We combined a novel quantification of anatomy with our behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, we generated hypotheses of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. Our maps directly specify genetic tools to target these regions, which we used to identify a small population of neurons with a role in the control of walking.

Mammalian cerebral cortex is accepted as being critical for voluntary motor control, but what functions depend on cortex is still unclear. Here we used rapid, reversible optogenetic inhibition to test the role of cortex during a head-fixed task in which mice reach, grab, and eat a food pellet. Sudden cortical inhibition blocked initiation or froze execution of this skilled prehension behavior, but left untrained forelimb movements unaffected. Unexpectedly, kinematically normal prehension occurred immediately after cortical inhibition, even during rest periods lacking cue and pellet. This 'rebound' prehension was only evoked in trained and food-deprived animals, suggesting that a motivation-gated motor engram sufficient to evoke prehension is activated at inhibition's end. These results demonstrate the necessity and sufficiency of cortical activity for enacting a learned skill.

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a diverse array of substrates across cell membranes. We present here the dynamics of complex formation of three structurally characterized ABC transporters-the BtuCD vitamin B(12) importer and MetNI d/l-methionine importer from Escherichia coli and the Hi1470/1 metal-chelate importer from Haemophilus influenzae-in complex with their cognate binding proteins. Similarly to other ABC importers, MetNI interacts with its binding protein with low affinity (K(d) approximately 10(-4) M). In contrast, BtuCD-BtuF and Hi1470/1-Hi1472 form stable, high-affinity complexes (K(d) approximately 10(-13) and 10(-9) M, respectively). In BtuCD-BtuF, vitamin B(12) accelerates the complex dissociation rate approximately 10(7)-fold, with ATP having an additional destabilizing effect. The findings presented here highlight substantial mechanistic differences between BtuCD-BtuF, and likely Hi1470/1-Hi1472, and the better-characterized maltose and related ABC transport systems, indicating that there is considerable mechanistic diversity within this large protein super-family.

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)–binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.

Mammalian cerebral cortex is accepted as being critical for voluntary motor control, but what functions depend on cortex is still unclear. Here we used rapid, reversible optogenetic inhibition to test the role of cortex during a head-fixed task in which mice reach, grab, and eat a food pellet. Sudden cortical inhibition blocked initiation or froze execution of this skilled prehension behavior, but left untrained forelimb movements unaffected. Unexpectedly, kinematically normal prehension occurred immediately after cortical inhibition, even during rest periods lacking cue and pellet. This ‘rebound’ prehension was only evoked in trained and food-deprived animals, suggesting that a motivation-gated motor engram sufficient to evoke prehension is activated at inhibition’s end. These results demonstrate the necessity and sufficiency of cortical activity for enacting a learned skill. DOI: http://dx.doi.org/10.7554/eLife.10774.001, eLife digest Many of the movements that humans and other animals make every day are deceptively complex and only appear easy because of extensive practice. For example, picking up an object involves several steps that must be precisely controlled, including reaching towards the item and holding it using the right amount of pressure to not crush it or drop it. Part of the brain called the motor cortex is thought to be important for learning and controlling these skilled movements, but its exact role in these processes is not clear. A technique called optogenetics allows the roles of individual parts of the brain to be studied by rapidly altering their activity, whilst minimizing the likelihood that the brain will compensate for these changes. By genetically modifying animals to produce light-sensitive channel proteins in certain brain cells, the activity of particular regions of the brain can be controlled by shining light onto them. Guo et al. have now used optogenetics to control the motor cortex as the mice performed a task they had been trained to do – reaching for and picking up a food pellet. Suddenly shutting down the motor cortex at the start of a trial prevented the mice from starting the task, and shut down part way through the task caused the front limbs of the mice to freeze in midair. However, only the learned, skilled task was frozen by motor cortex shutdown; mice could still move their limbs normally if the motor cortex was instead shut down during routine movements. When the cortex was reactivated, the mice instantly resumed trying to pick up the food pellet. Unexpectedly, even during rest periods when there was no food pellet and the mice were just waiting for the experiment to begin, turning the motor cortex off and then back on again suddenly caused the mice to perform the complete grabbing motion. This implies that the cortical activity evoked at the end of inactivation acts to trigger the full movement sequence. This was particularly likely to occur if the animal had been deprived of food before the test or was particularly well trained, but did not depend on the position of the limb. Overall, Guo et al.’s work opens the question of how the instructions that describe the learned movement are encoded within the motor cortex and its downstream networks. Future studies could also investigate how learning a set of movements affects the structure of cortical neurons and their connections, thus suggesting how these memories are stored. DOI: http://dx.doi.org/10.7554/eLife.10774.002

molA(HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB2C2 (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7-Å resolution, respectively. The MolA binding protein binds molybdate and tungstate but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate binding protein structurally solved. The ~100 μM binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate binding proteins that have nanomolar to low micromolar affinity for molybdate. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus. (Word count 123/150)

The high-affinity uptake of methionine by Escherichia coli is mediated by MetNI, a member of the methionine uptake transporter family of ATP-binding cassette (ABC) transporters. We previously reported the crystal structure of MetNI at 3.7 A resolution and a brief analysis of the methionine mediated trans-inhibition of the transporters' ATPase activity1. Here, we report two new crystal structures of the MetNI transporter, solved at 2.8 and 4.0 A resolution. While both structures of MetNI reveal the transporter adopting an inward-facing conformation, significant changes are observed in the conformation of the MetI transmembrane, MetN nucleotide binding, and MetN-C2 carboxyl-terminal regulatory domains. The conformational changes can be described primarily as rigid-body movements which result in a partial closing of the MetN nucleotide binding domains, and a simultaneous rotational rearrangement of the MetN-C2 regulatory domain. The kinetic properties of trans-inhibition have also been characterized by an analysis of ligand binding on the ATPase activity of wild type and specific MetN-C2 regulatory domain mutants.1. Kadaba N, Kaiser J, Johnson E, Lee A, and Rees D C. The high affinity E. coli methionine ABC transporter: structure and allosteric regulation. Science. 2008 Jul 11;321(5886):250-3.

The crystal structure of a putative metal-chelate–type adenosine triphosphate (ATP)–binding cassette (ABC) transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B 12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.

The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of theEscherichia coliBtuCD protein, an ABC transporter mediating vitamin B12uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for theE. colilipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters.

Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane α helices and a third cytoplasmic α helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.

The propellant spray resulting from the impingement of liquid jets of hydrazine and nitrogen tetroxide has been studied at atmospheric pressure by means of streak photography. The streak photographs show periodic small explosions that originate near the impingement point and propagate through the propellant spray at velocities of 3000 to 5000 fps, consuming all propellant droplets over a distance of up to 6 in. Typical streak photographs are presented. The frequency of the explosions or pops ranged up to several hundred cps, and could be controlled by varying the contact time in the liquid phase. Below a minimum threshold contact time, popping did not take place. At high values of contact time, the popping rate was controlled by the transit time of the free jet before impingement. Flashing of the oxidizer prior to impingement prevented popping under certain conditions. It is postulated that popping is initiated by liquid phase reactions. A mechanism for the occurrence of popping and its relation to reactive stream separation is suggested.