Fuxianhuiid ventral nerve cord and early nervous system evolution in Panarthropoda

Panarthropods are typified by disparate grades of neurological organization reflecting a complex evolutionary history. The fossil record offers a unique opportunity to reconstruct early character evolution of the nervous system via exceptional preservation in extinct representatives. Here we describe the neurological architecture of the ventral nerve cord (VNC) in the upper-stem group euarthropod Chengjiangocaris kunmingensis from the early Cambrian Xiaoshiba Lagerstätte (South China). The VNC of C. kunmingensis comprises a homonymous series of condensed ganglia that extend throughout the body, each associated with a pair of biramous limbs. Submillimetric preservation reveals numerous segmental and intersegmental nerve roots emerging from both sides of the VNC, which correspond topologically to the peripheral nerves of extant Priapulida and Onychophora. The fuxianhuiid VNC indicates that ancestral neurological features of Ecdysozoa persisted into derived members of stem-group Euarthropoda but were later lost in crown-group representatives. These findings illuminate the VNC ground pattern in Panarthropoda and suggest the independent secondary loss of cycloneuralian-like neurological characters in Tardigrada and Euarthropoda.The nervous system represents a critical source of phylogenetic information and has been used extensively for exploring the evolutionary relationships of extant Panarthropoda (i.e., Onychophora, Tardigrada, Euarthropoda) (1–7). Identification of fossilized nervous tissues has provided a unique perspective on early euarthropod brain neuroanatomy and suggests that broad patterns of extant neurological diversity were already in place by the Cambrian (8–11). The ventral nerve cord (VNC) reflects fundamental aspects of panarthropod body organization that complement the organization of the brain and together illuminate the evolution of the CNS (1–3, 5, 7, 12–16). The early evolutionary history of the panarthropod postcephalic CNS, however, remains obscure due to the exclusive preservation of brains in most available fossils (8, 10, 11). Moreover, the unresolved phylogenetic relationships within Panarthropoda complicate accurate reconstruction of the CNS ground pattern (16–22). In this study, we demonstrate the exceptional preservation of postcephalic neurological features in the early Cambrian fuxianhuiid Chengjiangocaris kunmingensis, an upper stem-group euarthropod (17) from the Xiaoshiba Lagerstätte, South China (23). These fossils clarify the neurological organization of the VNC in early euarthropod ancestors, thereby polarizing the evolution of the panarthropod CNS.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.The β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3–10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11, 12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13–15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16–20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21, 22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23–25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26–30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic at greater depths within the membrane (31), and this environmental anisotropy is reflected in the amino acid composition of the barrel surface. Aliphatic side chains are prevalent toward the center of the membrane, and aromatic side chains are common in the lipid head group regions, where they encircle the barrel in external- and periplasmic-side girdles (32).Recently we developed E_zβ, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33, 34). E_zβ can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34). E_zβ and related statistical functions (35, 36) can recapitulate properties of natural outer membrane proteins (37, 38) and predict the effects of mutations on protein stability and oligomerization (39). Similar potentials have driven computational approaches that have fully redesigned α-helical membrane protein surfaces to convert membrane proteins into water-soluble ones (40–42).Here, we considered whether the complete redesign of the lipid-facing surface of an outer membrane protein using a statistical potential such as E_zβ preserves its structure and function. This approach allowed us to investigate whether membrane insertion requires only a lipid-facing surface composed of depth-appropriate hydrophobic residues, or whether folding requires sequence-specific interstrand interactions, chaperone-recruiting sequences, evolutionarily optimized aromatic girdles, folding nucleation sites, or other design features lost during the population averaging inherent in parameter fitting of statistical potentials.Previous studies have explored the sensitivity of the β-barrel fold and its chaperone recognition mechanisms to mutations. The canonical eight-stranded β-barrel membrane protein OmpA tolerates a limited number of mutations to the lipid-facing surface, provided hydrophobicity is maintained (43, 44). More radically, the eight-stranded barrel OmpX has been duplicated to form a 16-stranded barrel capable of membrane insertion (45). However, the lipid-facing residues of transmembrane β-strands are conserved across homologous β-barrel membrane proteins beyond the extent expected from hydrophobicity alone (46, 47), implying a functional role that has yet to be elucidated.To explore the sequence constraints on β-barrel membrane proteins, we extensively redesigned the lipid-facing surface of E. coli OmpA. We created a series of OmpA variants with entirely or partially redesigned lipid-facing surfaces and tested their ability to insert into the outer membrane of E. coli. Our results indicate that the surfaces of β-barrel membrane proteins are amenable to large-scale redesign, provided that energetically destabilizing substitutions are avoided.     

From the Cover: The point of no return in vetoing self-initiated movements

In humans, spontaneous movements are often preceded by early brain signals. One such signal is the readiness potential (RP) that gradually arises within the last second preceding a movement. An important question is whether people are able to cancel movements after the elicitation of such RPs, and if so until which point in time. Here, subjects played a game where they tried to press a button to earn points in a challenge with a brain–computer interface (BCI) that had been trained to detect their RPs in real time and to emit stop signals. Our data suggest that subjects can still veto a movement even after the onset of the RP. Cancellation of movements was possible if stop signals occurred earlier than 200 ms before movement onset, thus constituting a point of no return.It has been repeatedly shown that spontaneous movements are preceded by early brain signals (1–8). As early as a second before a simple voluntary movement, a so-called readiness potential (RP) is observed over motor-related brain regions (1–3, 5). The RP was found to precede the self-reported time of the “‘decision’ to act” (ref. 3, p. 623). Similar preparatory signals have been observed using invasive electrophysiology (8, 9) and functional MRI (7, 10), and have been demonstrated also for choices between multiple-response options (6, 7, 10), for abstract decisions (10), for perceptual choices (11), and for value-based decisions (12). To date, the exact nature and causal role of such early signals in decision making is debated (12–20).One important question is whether a person can still exert a veto by inhibiting the movement after onset of the RP (13, 18, 21, 22). One possibility is that the onset of the RP triggers a causal chain of events that unfolds in time and cannot be cancelled. The onset of the RP in this case would be akin to tipping the first stone in a row of dominoes. If there is no chance of intervening, the dominoes will gradually fall one-by-one until the last one is reached. This has been coined a ballistic stage of processing (23, 24). A different possibility is that participants can still terminate the process, akin to taking out a domino at some later stage in the chain and thus preventing the process from completing. Here, we directly tested this in a real-time experiment that required subjects to terminate their decision to move once a RP had been detected by a brain–computer interface (BCI) (25–31).     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl,oxygen-rebound mechanism

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η¹-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η¹) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.Carbohydrates are the most diverse set of biomolecules, and thus, many enzyme classes have evolved to assemble, modify, and depolymerize carbohydrates, including glycosyltransferases, glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases (1). Recently, a new enzymatic paradigm was discovered that employs copper-dependent oxidation to cleave glycosidic bonds in polysaccharides (2–13). These newly classified enzymes, termed lytic polysaccharide monooxygenases (LPMOs), broadly resemble other copper monooxygenases and some hydroxylation catalysts (14–21).The discovery that LPMOs use an oxidative mechanism has attracted interest both because it is a unique paradigm for carbohydrate modification that employs a powerful C–H activation mechanism, and also because LPMOs synergize with hydrolytic enzymes in biomass conversion to sugars because they act directly on the crystalline polysaccharide surface without the requirement for depolymerization (4, 22, 23), making them of interest in biofuels production. LPMOs were originally characterized as Family 61 glycoside hydrolases (GH61s, reclassified as auxiliary activity 9, AA9) or Family 33 carbohydrate-binding modules (CBM33s, reclassified as AA10), which are structurally similar enzymes found in fungi and nonfungal organisms (22), respectively. In 2005, Vaaje-Kolstad et al. described the synergism (24) of a chitin-active CBM33 (chitin-binding protein, CBP21) with hydrolases, but the mechanism was not apparent. Harris et al. demonstrated that a GH61 boosts hydrolytic enzyme activity on lignocellulosic biomass (2). Vaaje-Kolstad et al. subsequently showed that CBP21 employs an oxidative mechanism to cleave glycosidic linkages in chitin (4).Following these initial discoveries, multiple features of LPMOs have been elucidated. LPMOs use copper (5–7) and produce either aldonic acids or 4-keto sugars at oxidized chain ends, believed to result from hydroxylation at the C1 or C4 carbon, respectively. Hydroxylation at the C1 carbon is proposed to spontaneously undergo elimination to a lactone followed by hydrolytic ring opening to an aldonic acid, whereas hydroxylation and elimination at C4 yields a 4-keto sugar at the nonreducing end (5–12). The active site is a mononuclear type(II) copper center ligated by a “histidine brace” (5, 12), comprising a bidentate N-terminal histidine ligand via the amino terminus and an imidazole ring nitrogen atom and another histidine residue also via a ring nitrogen atom. Hemsworth et al. reported a bacterial LPMO structure wherein the active site copper ion was photoreduced to Cu(I) (12), and Aachmann et al. demonstrated that Cu(I) binds with higher affinity than Cu(II) in CBP21 (13). A structural study of a fungal LPMO revealed an N-terminal methylation on a nitrogen atom in the imidazole ring of unknown function (5), but some LPMOs are active without this modification (6, 11). LPMOs require reducing agents for activity such as ascorbate (2–8, 10–12), and cellobiose dehydrogenase (CDH), a common fungal secretome component, can potentiate LPMO activity in lieu of a small-molecule reducing agent (7, 8).Overall, many structural and mechanistic insights have been reported since the discoveries that LPMOs are oxidative enzymes (4–10). However, many questions remain regarding LPMO function (22, 25). Here, we examine the LPMO catalytic mechanism with density functional theory (DFT) calculations on an active site model (ASM) of a fungal LPMO. We seek to (i) understand the identity of the reactive oxygen species (ROS), (ii) compare two hypothesized catalytic mechanisms, and (iii) examine the role of N-terminal methylation in catalysis.     

Therapeutic mechanisms of high-frequency stimulation in Parkinson’s disease and neural restoration via loop-based reinforcement

High-frequency deep brain stimulation (HFS) is clinically recognized to treat parkinsonian movement disorders, but its mechanisms remain elusive. Current hypotheses suggest that the therapeutic merit of HFS stems from increasing the regularity of the firing patterns in the basal ganglia (BG). Although this is consistent with experiments in humans and animal models of Parkinsonism, it is unclear how the pattern regularization would originate from HFS. To address this question, we built a computational model of the cortico-BG-thalamo-cortical loop in normal and parkinsonian conditions. We simulated the effects of subthalamic deep brain stimulation both proximally to the stimulation site and distally through orthodromic and antidromic mechanisms for several stimulation frequencies (20–180 Hz) and, correspondingly, we studied the evolution of the firing patterns in the loop. The model closely reproduced experimental evidence for each structure in the loop and showed that neither the proximal effects nor the distal effects individually account for the observed pattern changes, whereas the combined impact of these effects increases with the stimulation frequency and becomes significant for HFS. Perturbations evoked proximally and distally propagate along the loop, rendezvous in the striatum, and, for HFS, positively overlap (reinforcement), thus causing larger poststimulus activation and more regular patterns in striatum. Reinforcement is maximal for the clinically relevant 130-Hz stimulation and restores a more normal activity in the nuclei downstream. These results suggest that reinforcement may be pivotal to achieve pattern regularization and restore the neural activity in the nuclei downstream and may stem from frequency-selective resonant properties of the loop.High-frequency (i.e., above 100 Hz) deep brain stimulation (HFS) of the basal ganglia (BG) and thalamus is clinically recognized to treat movement disorders in Parkinson’s disease (PD) (1–4), but its therapeutic mechanisms remain unclear (5, 6).Early hypotheses about HFS were derived from the rate-based model of the BG function (7, 8) and postulated the disruption of the output of the BG-thalamic system via either the inactivation of neurons in the stimulated site (target) (9–15), which would provide an effect similar to a surgical lesion, or the abnormal excitation of axons projecting out of the target (16–19), which would disrupt the neuronal activity in the structures downstream, including any pathophysiological activity (20).More recently, an ever-growing number of experiments in PD humans and animal models of Parkinsonism has indicated that HFS affects the firing patterns of the neurons rather than the mean firing rate both in the target and the structures downstream (18, 19, 21–31) and it replaces repetitive low-frequency (i.e., ≤50 Hz) bursting patterns with regularized (i.e., more tonic) patterns at higher frequencies (25, 26). It has been proposed that increased pattern regularity of neurons in the target may be therapeutic (5, 32–37), but it is still unknown how this regularity comes about with HFS.It has been suggested that an increased pattern regularity can deplete the information content of the target output and this lack of information would act as an “information lesion” (33) and prevent the pathological activity from being transmitted within the BG-thalamic system (22, 33, 36). As a result, an information lesion in the target [typically, one among the subthalamic nucleus (STN), internal globus pallidus (GPi), or thalamus] would have effects similar to those of a destructive lesion in the same site, which has been reported to alleviate the movement disorders (38).Instead, studies (32, 34, 35, 37) have suggested that an increased pattern regularity of the BG output partly compensates the PD-evoked impairment of the information-processing capabilities of the thalamo-cortical system, and this restores a more faithful thalamic relay of the sensorimotor information (35, 39).Although intriguing, these hypotheses remain elusive on (i) the neuronal mechanisms that would elicit pattern regularization (e.g., why regularization would be relevant only for HFS) and (ii) the effects that increased regularity would have on the cortico-BG-thalamo-cortical loop.It has been hypothesized that pattern regularization occurs because axons projecting out of the target follow the pattern of the stimulus pulses (40, 41) and, given the segregated organization of the BG-thalamic connections (42), it has been assumed that pattern regularization percolates straightforward from the target to the structures immediately downstream (34, 36). However, this representation of the pattern regularization as a “local” effect can hardly be reconciled with the fact that HFS of any structure of the cortico-BG-thalamo-cortical loop is therapeutic for at least some movement disorders (1–4, 43–47), nor does it explain why stimulation at frequencies above 160–180 Hz is not necessarily therapeutic despite the fact that the regularity of the axonal patterns may increase (48, 49). Moreover, coherence in the 8–30-Hz band among neurons across different structures may decrease under HFS but not for lower frequencies (26, 50–52), which suggests the emergence of diffused changes in neuronal activity that would be hardly accounted for with purely local effects.There is emerging evidence, instead, that HFS affects multiple structures simultaneously. First, it has been shown that deep brain stimulation (DBS) may antidromically activate afferent axons and fibers of passage (53–59), thus reaching structures not immediately downstream. Second, studies (57, 58) observed in 6-hydroxydopamine (6-OHDA)-intoxicated rats that the antidromic effects increase with the stimulation frequency and peak around 110–130 Hz. Third, it has been shown in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated nonhuman primates (NHPs) that STN DBS may evoke similar poststimulus responses in different BG structures, both downstream from and upstream to the STN (5, 27, 28, 30, 60). Finally, it has been reported that the cortico-BG-thalamo-cortical system consists of multiple sets of reentrant, interconnected, and partially overlapping neuronal loops (5, 42, 61, 62), which means that the structures upstream to the target (e.g., the striatum) may play an important role in the therapeutic mechanisms of HFS.Altogether, these results suggest that (A) pattern regularization is a global effect that exploits the closed-loop nature of the cortico-BG-thalamo-cortical system and selectively emerges only for specific HFS values, and that (B) the therapeutic merit of pattern regularization has to deal with the restoration of a more normal functionality of the entire cortico-BG-thalamo-cortical loop rather than with variations in the information content of one specific structure.We explored hypotheses (A) and (B) and assessed the system-wide effects of DBS by constructing a computational model of the cortico-BG-thalamo-cortical loop in both normal and parkinsonian conditions and by simulating the effects of STN DBS both at low (20–80 Hz) and high (100–180 Hz) frequencies. The model includes populations of single-compartment neurons and interneurons from motor cortex, striatum, GPi, and thalamus according to a network topology derived from the NHP anatomy, and it simulates both the orthodromic and antidromic effects of DBS. As a result, this model reproduced both average activity and discharge patterns of single units in NHP and rats under normal and parkinsonian conditions, with and without DBS, for all modeled structures.We show through numerical simulation that hypothesis (A) is significantly contributed by reinforcement mechanisms in the striatum. These mechanisms are selectively elicited by HFS, facilitate the percolation of regularized discharge patterns from the striatum to the GPi, and have a primary role in (B), because the percolated striato-pallidal input combines with the local effects of STN DBS to restore the thalamic relay function (63).     

Kinetics of protein–ligand unbinding: Predicting pathways,rates, and rate-limiting steps

The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate k_off is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate k_on. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design.Understanding the thermodynamics and kinetics of protein–ligand interactions is of paramount relevance in the early stages of drug discovery (1–3). So far the major emphasis has been placed on predicting the most likely binding pose as determined by the highest binding affinity (4, 5). In contrast, it has not been possible to predict the pathways for unbinding and the associated rates. However, it is by now well-recognized that one of the most pertinent factors for sustained drug efficacy and safety is not just its affinity, but possibly even more so, the mean lifetime of the protein–ligand complex (1–3). The latter property is strictly related to the time during which the ligand remains in the binding site (1, 2), and is typically expressed by its inverse, the dissociation rate k_off (2). In principle k_off should be amenable to calculations through all-atom molecular dynamics (MD) simulations. These simulations could give detailed and useful insights into the atomic interactions at work during unbinding, especially in the ephemeral but kinetically most relevant transition state ensemble (TSE) (6, 7). Such information is of great value in designing modifications of the ligand that might improve its pharmaceutical properties.However, despite the potential of MD simulations no such calculation has yet been reported. This is a consequence of the limited timescales of MD simulations. Even with the most modern purpose-built supercomputers or massive distributed computing, one can barely reach the timescale of milliseconds (3). Unfortunately most of the reported ligand–protein dissociation times far exceed this timescale (2). These timescales can be reached either by transition path sampling methods (8, 9), quasi-classical approximations (10), by the construction of Markov state models (11, 12), or through carefully designed enhanced sampling methods (8, 13–30) that make accessible the timescale of seconds and beyond in a controlled and accurate way. The enhanced sampling method we use in this work is based on metadynamics (13–15), which has been widely and successfully applied to a variety of systems including complex protein–ligand systems (25–30), and has been rigorously proven to converge to the correct free-energy surface (31, 32).Recently, we have extended the scope of metadynamics by showing that it can also be used to recover kinetic information (15). Furthermore, we showed that by using an a posteriori statistical analysis (33) one can also establish the reliability of the kinetics thus generated. The use of metadynamics for obtaining kinetic information is still in its infancy, however its usefulness has been tested by us and other groups in a range of systems (15, 33–36).In this work, we demonstrate that the scope of the method reported in ref. 15 can be extended to study protein–ligand dissociation pathways and to determine in an accurate way the ligand unbinding rates. We reach well into the hundreds of milliseconds regime and longer, maintaining at the same time full atomic resolution for protein, ligand, and solvent. Specifically, we study the unbinding of the inhibitor benzamidine from trypsin, a serine protease protein (27, 37, 38) using classical force fields (39, 40). Using our acceleration method (15, 33) we are able to harness 21 independent successful unbinding trajectories in which the ligand goes from the bound to the fully unbound state. We find that one of the most distinctive features of the unbinding process is the role played by the water molecules (41, 42). In particular, the solvent promotes unbinding by assisting in the breakage of shielded hydrogen bonds through the formation of water bridge interactions (41).From the analysis of the unbinding trajectories we find that along the unbinding pathways the ligand rests for times ranging from nanoseconds to milliseconds in a number of intermediate structures. We calculate the rates for all possible transitions between these intermediates and construct a Markov model for the unbinding process (11, 43, 44). The overall escape rate computed from this Markov model is in good agreement with the direct estimation of the mean unbinding time that comes from the metadynamics runs. Reassured by this agreement we use the Markov model to determine the dominant unbinding pathways and rate-limiting steps. To this end, starting from the metadynamics reactive trajectories, we perform a committor analysis and determine the TSE (6). Using the recently computed value of the binding affinity (27) we also estimate the binding rate constant k_on. Our calculated unbinding and binding rates compare reasonably well with the known experimental measurement (37), especially taking into account the margin of error in the experiment and the inaccuracy of the force field used in the simulations (42). Unprecedented structural features of the target are also disclosed. In particular, we find that in its apo state trypsin can exist in two forms. In the first form, loop Val207–Tyr224 (hereafter labeled loop L) oscillates around the crystallographic state. In the other form, a small distortion of this loop is stabilized. The mean lifetime of this distorted state is nearly 0.7 ms and during this time the ligand cannot reach the binding site.We believe that this metadynamics-based strategy is, to our knowledge, the first direct approach for calculating k_off from MD simulations of unbinding. Previous studies have focused on the calculation of k_on and the magnitude of k_off was only indirectly obtained (12, 38). Our strategy should be easily applicable for calculating unbinding pathways and rates for generic protein–ligand systems, thus complementing and extending the role of enhanced sampling-based simulations in drug discovery.     

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.     

Cytonuclear interactions affect adaptive traits of the annual plant Arabidopsis thaliana in the field

Although the contribution of cytonuclear interactions to plant fitness variation is relatively well documented at the interspecific level, the prevalence of cytonuclear interactions at the intraspecific level remains poorly investigated. In this study, we set up a field experiment to explore the range of effects that cytonuclear interactions have on fitness-related traits in Arabidopsis thaliana. To do so, we created a unique series of 56 cytolines resulting from cytoplasmic substitutions among eight natural accessions reflecting within-species genetic diversity. An assessment of these cytolines and their parental lines scored for 28 adaptive whole-organism phenotypes showed that a large proportion of phenotypic traits (23 of 28) were affected by cytonuclear interactions. The effects of these interactions varied from slight but frequent across cytolines to strong in some specific parental pairs. Two parental pairs accounted for half of the significant pairwise interactions. In one parental pair, Ct-1/Sha, we observed symmetrical phenotypic responses between the two nuclear backgrounds when combined with specific cytoplasms, suggesting nuclear differentiation at loci involved in cytonuclear epistasis. In contrast, asymmetrical phenotypic responses were observed in another parental pair, Cvi-0/Sha. In the Cvi-0 nuclear background, fecundity and phenology-related traits were strongly affected by the Sha cytoplasm, leading to a modified reproductive strategy without penalizing total seed production. These results indicate that natural variation in cytoplasmic and nuclear genomes interact to shape integrative traits that contribute to adaptation, thereby suggesting that cytonuclear interactions can play a major role in the evolutionary dynamics of A. thaliana.The genomes of eukaryotes originate from ancient endosymbiotic associations that eventually led to energy-harnessing organelles: mitochondria, common to all eukaryotes, and chloroplasts in the “green” lineage. The evolution of endosymbionts into cellular organelles was accompanied by massive gene loss, with a large proportion being transferred to the nucleus (1, 2). Nevertheless, mitochondria and chloroplasts retained a few (30–80) protein-encoding genes that play crucial roles in energy metabolism (respiration and photosynthesis). Mitochondrion and chloroplast metabolisms rely on the proper interaction of nuclear-encoded proteins and their counterparts encoded in the organelle genome. Consequently, the genes in nuclear and organellar compartments are expected to be coadapted (3).Cytonuclear coadaptation has been demonstrated by altered phenotypes observed on interspecific exchanges of cytoplasm between related species in mammals (4), yeast (5), arthropods (6), and plants, whose interspecific crosses are frequently successful (7). These alterations affect organelle function and even the organism phenotype, indicating epistasis between nuclear and cytoplasmic genes. Although cytonuclear coadaptation is generally studied at the interspecific level, the existence of intraspecific genetic diversity in organelle genomes suggests a potential for genomic coadaptation within species. A few studies have reported phenotypic effects of intraspecific cytonuclear epistasis in nonplant species (8–11). In plants, many studies have focused on cytoplasmic male sterility (CMS), an impairment of pollen production governed by nucleo-mitochondrial interactions in some hermaphroditic species (12), in particular in crops and their relatives (13). The phenotypic effects of intraspecific cytonuclear epistasis other than CMS have been reported in only a limited number of plant systems (14–17), with evidence that cytoplasmic variation contributes to local adaptation (18, 19).In recent years, several studies using reciprocal segregating populations of the model plant Arabidopsis thaliana have investigated the effect of cytonuclear epistasis on a number of laboratory-measured phenotypes such as the metabolome, defense chemistry and growth (17, 20, 21), water-use efficiency (22, 23), and seed germination (24, 25). Although some studies have reported significant effects of cytonuclear epistasis (17, 20, 21, 23, 25), others have found additive cytoplasmic effects but with weak or no cytonuclear epistasis (22). Each of these studies (with the exception of ref. 25) was, however, based on a single reciprocal cross between two natural accessions, thereby preventing the estimation of the prevalence of cytonuclear epistasis in this species. In addition, although these reports involve adaptive traits (26–30), the investigation of the effect of cytonuclear epistasis on adaptive phenotypes in field conditions is, at best, scarce in A. thaliana.Here, following the modern standards of ecological genomics (31), we explored the prevalence of cytonuclear interactions on adaptive whole-organism traits in the model plant A. thaliana in a field experiment. To do so, based on eight natural accessions of a core collection that covers a significant part of the species’ cytoplasmic and nuclear genetic diversity in A. thaliana (25, 32), we created eight series of seven cytolines. Cytolines are genotypes that combine the nuclear genome from one parent with the organelle genomes of another (33). We examined the cytolines and their parental accessions for effects of cytonuclear interactions on 28 field-measured traits related to germination, phenology, resource acquisition, plant architecture and seed dispersal, fecundity, and survival.     

Retinal waves regulate afferent terminal targeting in the early visual pathway

Current models of retinogeniculate development have proposed that connectivity between the retina and the dorsal lateral geniculate nucleus (dLGN) is established by gradients of axon guidance molecules, to allow initial coarse connections, and by competitive Hebbian-like processes, to drive eye-specific segregation and refine retinotopy. Here we show that when intereye competition is eliminated by monocular enucleation, blocking cholinergic stage II retinal waves disrupts the intraeye competition-mediated expansion of the retinogeniculate projection and results in the permanent disorganization of its laminae. This disruption of stage II retinal waves also causes long-term impacts on receptive field size and fine-scale retinotopy in the dLGN. Our results reveal a novel role for stage II retinal waves in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, allowing for correct laminar patterning and the even allocation of synaptic territory. These findings should contribute to answering questions regarding the role of neural activity in guiding the establishment of neural circuits.The brain employs several strategies to guide the establishment of correct neural connectivity (1, 2). It has been well recognized that the high specificity of connections between the retina and the dorsal lateral geniculate nucleus (dLGN) is established through several factors. These include gradients of axon guidance molecules that guide the initial coarse targeting of afferent terminals (3–6), and spontaneous retinal activity (retinal waves) that drives competitive processes important for the refinement and segregation of afferent terminal branches (2, 7–15).Retinal waves are spontaneous propagating bursts of correlated retinal ganglion cell (RGC) activity and have been classified into three developmental stages (1, 15). Stage II retinal waves (from here on also referred to as retinal waves) are extensively studied and have been found to be critical for the development of retinofugal pathways (1, 2, 15). They are mediated by cholinergic signaling from starburst amacrine cells onto RGCs (8, 13, 16–18) and have been hypothesized to drive the Hebbian-like remodeling of RGC afferent terminals (19, 20). Retinal waves play crucial roles in both the establishment of eye-specific segregation (8, 12, 14, 20, 21), through the removal of afferent branches from opposing putative eye-specific domains, and the refinement of afferent terminals within eye-specific laminae, which is believed to be necessary for the establishment of fine-scale retinotopy (12, 22). However, studies have suggested that retinal waves might play additional roles in the development of the retinogeniculate pathway. When retinal waves are blocked during early development, mature lamination in the adult is abnormal (23–25), while eye-specific segregation recovers (26, 27). These results uncovered a retinal wave-dependent window for the development of retinogeniculate lamination. However, the question remains open as to whether these lamination defects are due to abnormal late eye-specific segregation or the disruption of some form of retinal wave-dependent afferent terminal targeting.A potential retinal wave-dependent mechanism that could regulate retinogeniculate afferent terminal targeting is axon–axon competition originating from the same eye (i.e., intraeye competition). Classic studies in goldfish first demonstrated the principle of axon–axon competition at the optic tectum (28). These studies showed that RGC afferent terminals can undergo expansive or compressive rearrangements in their targeting in response to changes in afferent number, or retinorecipient target size, while maintaining correct retinotopy (28–32). Similarly, neonatal monocular enucleation in ferrets results in an expanded ipsilateral and contralateral projection by adulthood, while correct laminar organization is maintained (7, 10). This demonstrates that retinogeniculate afferent terminals can undergo an expansive and orderly rearrangement due to intraeye competition, and that intereye competition is not required for the establishment of proper retinogeniculate lamination.To investigate whether retinal waves play a role in regulating retinogeniculate afferent terminal targeting by way of intraeye competition, we monocularly enucleated ferrets one day after birth (P1), to eliminate intereye competition, while also pharmacologically blocking retinal waves (P1– P10) in the surviving eye with the cholinergic agonist epibatidine (EPI) (8, 13, 18). Effects on the targeting of retinogeniculate afferents terminals were assessed anatomically, to characterize impacts on retinogeniculate lamination, and functionally, to assess changes in receptive field (RF) structure and retinotopy in the dLGN. Our results demonstrate that retinal waves regulate afferent terminal targeting by way of intraeye competition during the development of the retinogeniculate pathway.     

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.The Gram-negative encapsulated bacterium Neisseria meningitidis causes severe sepsis and meningococcal meningitis. Invasive meningococcal disease (IMD) is associated with 5–15% mortality; furthermore, devastating long-term sequelae such as amputations, hearing loss, and neurodevelopmental disabilities are observed in 11–19% of IMD survivors (1). Meningococcal serogroups are distinguished by the composition of their capsular polysaccharides. The five serogroups most commonly associated with invasive disease are A, B, C, W, and Y. (2). Effective mono- or polyvalent-conjugated polysaccharide vaccines against N. meningitidis serogroups A, C, W, and Y have been available since the early 1990s (3). However, serogroup B meningococcus (MenB) is responsible for the majority of endemic and epidemic meningococcal disease in developed countries (4–6). The development of an efficient capsular polysaccharide-based vaccine against MenB has been hampered by potential autoimmunity issues, namely, the structural similarity between the MenB capsular polysaccharide and the neuraminic acid present on the surface of human fetal neural tissues (7).In early 2013 the European Medicines Agency approved 4CMenB, to our knowledge the first broadly protective vaccine against MenB, for the prevention of IMD in all age groups. 4CMenB is a multicomponent vaccine formulation composed of three surface-exposed meningococcal proteins originally identified by the reverse vaccinology approach (8) plus outer membrane vesicles from the New Zealand epidemic clone. The three antigenic proteins are factor H-binding protein (fHbp), neisserial heparin-binding antigen (NHBA), and neisserial adhesin A (NadA) (9, 10).The gene encoding NadA is present in ∼30% of pathogenic meningococcal isolates and is associated mostly with strains that belong to three of the four hypervirulent serogroup B lineages (11–14). NadA expression levels can vary among isolates by more than 100-fold, and its expression is up-regulated in vivo by niche-specific signals (15). NadA induces high levels of bactericidal antibodies in humans (16–18) and is recognized by serum antibodies of children convalescent after IMD (19), suggesting that it is expressed and is immunogenic during IMD. Two main genetically distinct groups of NadA have been identified that share overall amino acid sequence identities of 45–50%. Group I includes the three most common variants (NadA1, NadA2, and NadA3, the latter being the vaccine variant), which share ∼95% sequence identity and are immunologically cross-reactive (11). Group II includes three rarer variants: NadA4, primarily associated with carriage strains (11); NadA5, found mainly in strains of clonal complex 213 (20, 21); and NadA6 (Fig. S1A); these three share ∼90% sequence identity (Fig. S1B) (22).Functionally, NadA3 expressed on the surface of Escherichia coli promotes adhesion to and invasion of Chang epithelial cells (23). This adhesive activity has been mapped, at least partially, to an N-terminal region extending to residue T132 (23, 24). Recently, interactions of NadA3 with β-1 integrin (25) and with the heat shock protein Hsp90 (26) have been reported.Structurally, NadA belongs to the class of trimeric autotransporter adhesins (TAAs) (27, 28), which are known to mediate adhesion through interaction with extracellular matrix proteins and are involved in invasion of target cells (29). TAAs are obligate homotrimers, and accordingly the recombinant NadA3 vaccine antigen, lacking the C-terminal membrane anchor region, forms soluble, stable trimers (23, 30). TAAs generally are made of a conserved C-terminal integral membrane β-barrel, which anchors the proteins to the outer membrane, and an N-terminal “passenger” domain responsible for adhesion (31). The TAA passenger domain typically is made of a central α-helical domain (stalk) that forms coiled-coil structures and a distinct N-terminal domain (head) that is mainly responsible for binding to host cellular receptors.Here we present the X-ray structure of a large ectodomain fragment of NadA5 and a structural analysis by transmission electron microscopy (TEM) of the vaccine variant NadA3. In addition, epitope mapping shows that the head of NadA3 contains immunogenic regions responsible for the generation of a protective bactericidal response.     

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.Bacteria in the genus Wolbachia are maternally transmitted Rickettsia-like endosymbionts that infect an estimated 40–69% of arthropod species (1, 2). In many cases, Wolbachia manipulate host reproduction to spread throughout arthropod populations (3). Incongruence between Wolbachia and host phylogenies indicate that horizontal transfer of the symbiont has been commonplace over evolutionary time (4, 5), enabling Wolbachia to invade new species. However, there is a poor understanding of barriers to horizontal transmission and why some species remain uninfected. An understanding of these factors is important from an evolutionary perspective given that Wolbachia influences speciation (6, 7), and from an applied perspective as Wolbachia is being transinfected into vector species for the control of arthropod-borne disease (8–10).The ability to invade the host germ line is an important feature of Wolbachia biology that facilitates horizontal transmission, leading to the pervasive nature of this bacterium across invertebrate taxa. In order for Wolbachia to become established in a naïve host species, it must be acquired horizontally and successfully transmitted vertically (i.e., to offspring) to maintain the infection in the population. Multiple mechanisms of Wolbachia horizontal transmission have been proposed, including cohabitation, hemolymph transfer, predation, and parasitoid infection (11–15). After microinjection into Drosophila, Wolbachia infects the stem cell niches in the germ line (16, 17), and both Wolbachia-derived and host factors appear to influence tropism and bacterial density during oogenesis (17–20). Alternatively, somatic tissue may act as a reservoir for Wolbachia infection of the developing oocyte (20–23). Although pathways of horizontal transmission have been characterized in some species, identification of barriers to vertical transmission of the acquired Wolbachia infection remains elusive.Microbial conflict or incompatibility within arthropods is a potential barrier to transmission of heritable symbionts. Studies in the tick Dermacentor variabilis demonstrate competitive exclusion between maternally inherited bacteria. Transovarial transmission of Rickettsia montanensis (formerly Rickettsia montana) and Rickettsia rhipicephali is inhibited by infection with the reciprocal species (24). Similarly, infection exclusion has been observed in D. variabilis between conspecific strains of Anaplasma marginale where one strain inhibits the infection of the other (25). Competitive inter- and intraspecific microbial interactions have also been observed with Wolbachia (26, 27).Anopheles mosquitoes provide a unique system to examine microbial barriers to Wolbachia transmission. With few exceptions, Anophelines (which transmit the Plasmodium parasites that cause human malaria) are naturally uninfected with Wolbachia (28–31), suggesting the potential presence of innate barriers to infection in this genus. However, in vitro and in vivo studies indicate that Wolbachia are capable of infecting cultured Anopheles cells (32, 33), ex vivo cultured tissues (34), in vivo somatic tissue (35–37), and can stably infect the mosquito germ line (38). We investigated the ability of the native microbial community to influence vertical transmission of Wolbachia in Anopheles mosquitoes. We found that bacteria in the genus Asaia were responsible for inhibiting Wolbachia maternal transmission in this important mosquito genus.     

BMP signaling is required for the generation of primordial germ cells in an insect

Two modes of germ cell formation are known in animals. Specification through maternally inherited germ plasm occurs in many well-characterized model organisms, but most animals lack germ plasm by morphological and functional criteria. The only known alternative mechanism is induction, experimentally described only in mice, which specify germ cells through bone morphogenetic protein (BMP) signal-mediated induction of a subpopulation of mesodermal cells. Until this report, no experimental evidence of an inductive germ cell signal for specification has been available outside of vertebrates. Here we provide functional genetic experimental evidence consistent with a role for BMP signaling in germ cell formation in a basally branching insect. We show that primordial germ cells of the cricket Gryllus bimaculatus transduce BMP signals and require BMP pathway activity for their formation. Moreover, increased BMP activity leads to ectopic and supernumerary germ cells. Given the commonality of BMP signaling in mouse and cricket germ cell induction, we suggest that BMP-based germ cell formation may be a shared ancestral mechanism in animals.There are two well-characterized modes of animal germ cell specification. In the inheritance mode, observed in Drosophila melanogaster, Caenorhabditis elegans, and Xenopus laevis, maternally provided cytoplasmic determinants (germ plasm) specify a subset of early embryonic cells as germ cells. In contrast, mice specify their germ line through the induction mode, in which a zygotic cell–cell signaling mechanism specifies germ cells later in development. We previously hypothesized that the inductive mode was ancestral among metazoans and that the inheritance mode had evolved independently in multiple derived lineages (1, 2). Consistent with this hypothesis, multiple basally branching insects do not segregate maternally provided germ plasm, unlike the relatively derived Drosophila model (3, 4). However, experimental evidence for the inductive mode was available only for salamanders (5, 6) and mice (7–10), and to date, inferences of induction outside of vertebrates have been based on gene expression and cytological data (1, 11–16).Because Drosophila is highly derived with respect to many aspects of development (17), we examined germ cell development in the cricket Gryllus bimaculatus, a basally branching insect that may shed light on putative ancestral mechanisms of specifying germ cells. We previously showed that unlike Drosophila, Gryllus primordial germ cell (PGC) specification requires zygotic mechanisms rather than germ plasm or the oskar germ-line determinant (4, 18). However, the signals that might induce PGC formation in Gryllus remained unknown. Because mammals require the highly conserved bone morphogenetic protein (BMP) pathway to specify PGCs (8–10, 19, 20), we investigated BMP signaling as a candidate for regulating inductive germ cell specification in Gryllus.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.