Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range.Filopodia are actin-rich cell membrane protrusions, involved in processes as diverse as cell migration, wound closure, and cell invasion by pathogens (1–3). During cell migration, filopodia can exert forces on the substrate (4, 5) and act as precursors of focal adhesions (6–8). Filopodia initiate contacts during wound closure and contribute to dorsal closure of the fruit fly embryo in a zipper-like fashion (9–12). Viruses can hijack filopodia and filopodia-like cell–cell bridges to surf toward the cell body (13, 14). Filopodia from macrophages and epithelial cells actively pull pathogens bound to their tips (15–18). In all these examples filopodial retraction and retrograde force production are crucial. However, although filopodia formation and growth have been well studied (1–3), the mechanisms underlying their retraction are poorly understood.Filopodia show continuous rearward movement of their actin filaments in a process called “retrograde flow” (3, 19). In the lamellipodium, from which filopodia often emanate, the retrograde flow originates from actin treadmilling due to actin depolymerization at the rear and polymerization at the front of the lamellipodium. This retrograde flow is further amplified by the motor activity of myosins (20–23). In neurons, the filopodial shaft is deeply anchored in the growth cone and filopodial dynamics depends on the balance between actin polymerization at the filopodial tip and its retrograde flow (19). In other cell types actin depolymerization at the tip has been associated with retracting filopodia (24).Different contributions to filopodial force production during retraction can be considered. A connection between the filopodial tip and retracting actin filaments through transmembrane receptors such as integrins could transduce cortical forces applied on the actin shaft. In macrophages, force measurements on retracting filopodia suggested a major role for cortical myosins pulling on filopodial actin bundles (16). These measurements showed that retraction could be slowed down for forces below 20 pN. Applied forces higher than 20 pN inverted filopodial retraction of macrophages (25).Filopodial force production can also be due to membrane mechanics (26). Forces exerted by actin-free tubes extruded from the cell plasma membrane typically range between 5 pN and 30 pN (27). Membrane tension could drive filopodial retraction by exerting inward forces against the actin filaments. Moreover, filopodial actin filaments have been found disconnected from the membrane at the tip (28, 29), underlining the importance of membrane properties in filopodial mechanics. The contributions of membrane- and actin-based forces, as well as the mechanical links controlling force production during filopodial retraction, are still unclear.Here, we studied the retraction dynamics and the forces exerted by a single filopodium that is contacting an optically trapped bead at its tip. We found that filopodia retracted in association with a reduced actin polymerization at their tip at rates below those needed to compensate for the retrograde flow. The speed of filopodial retraction was only marginally affected by counteracting forces up to 50 pN, suggesting that the driving forces for retraction were not limiting within this range. We argue that actin treadmilling in the cell cortex, that functions far from its stall regime, transduces inward forces to the filopodial actin shaft at the base via high friction. In addition we found that filopodia can exert passive inward forces of 15 pN by using cell membrane-based forces. External counterforces that are only 5 pN higher than the membrane force can lead to rupture of connections between the actin shaft and the membrane at the filopodial tip. These weak contacts at the tip define the maximal pulling force of filopodia and allow cytoskeletal inward forces to operate only for short time intervals (<25 s). We found that the mechanical disconnection between membrane and actin filaments is only transient as actin dynamics at the tip are altered after disconnection. A continuous load-and-fail behavior allows thus tip-bound filopodia to probe the mechanics of their environment.     

Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin

Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena’s actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.The actin cytoskeleton facilitates fundamental cellular processes including division, polarization, and motility. The organization and dynamics of particular F-actin networks are determined by the coordinated action of specific subsets of actin-binding proteins with complementary biochemical properties such as sequestering, nucleating, elongating, bundling/crosslinking, and severing (1–3).Cell motility is driven primarily by lamellipodia, protrusive structures at the cell’s leading edge composed of a dendritic network of short-branched filaments produced by the rapid capping of filaments nucleated by the actin-related proteins 2 and 3 (Arp2/3) complex (4). Filopodia are exploratory finger-like projections composed of uniformly long, straight, parallel-bundled filaments that extend from lamellipodia. One current model for filopodia assembly is convergent elongation (5). Formin and/or Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins gather and rapidly elongate subpopulations of lamellipodial actin-barbed ends, antagonizing inhibition by capping protein (CP), and the parallel actin-crosslinking protein Fascin aligns and bundles elongating filopodial filaments (6).Ena/VASP proteins are large, multidomain actin-assembly factors (7, 8). The N-terminal Ena/VASP homology 1 (EVH1) domain binds FP4 motifs for proper localization (9). The central proline-rich region binds the G-actin–binding (GAB) protein profilin and SH3 domains (10–12). The EVH2 domain comprises a WH2 GAB domain (11, 13) and an F-actin–binding (FAB) domain (14). The C-terminal coiled-coil facilitates tetramerization (13, 15) and is necessary for processive actin filament elongation and proper function in vivo (11, 12, 15, 16).The importance of Ena/VASP proteins in filopodia formation and maintenance is well established (17), and Ena/VASP’s general actin-assembly properties have been defined. Most in vitro studies agree that Ena/VASP allows actin filament elongation in the presence of CP (12, 18–21), although the specific underlying mechanism(s) differ (9). Breitsprecher et al. (20) proposed that multiple mammalian or Dictyostelium Ena/VASP tetramers must be clustered on a surface to inhibit CP and allow long runs of processive filament elongation. Hansen and Mullins (12) proposed that individual human VASP (hVASP) tetramers protect barbed ends from CP, but only for very short processive runs (∼1.4 s = 40 subunits) (12). Furthermore, how Ena/VASP proteins and Fascin assemble robust filopodia composed of bundled filaments of uniform length is not known.The namesake Enabled (Ena), the sole Drosophila family member, was identified as a mutant ameliorating defects caused by loss of the tyrosine kinase Abl (22). Ena regulates many morphogenetic processes; for example, ena mutants display defects in axon guidance (23–25) and reduced filopodia length and number during dorsal closure; these defects disrupt the correct pairing of contralateral cells during epithelial zippering (26). Conversely, Ena overexpression induces filopodia assembly (26).We used single- and two-color total internal reflection fluorescence microscopy (TIRFM) to observe the assembly of both single and Fascin-bundled filaments in the presence of Drosophila Ena. We discovered that Ena possesses a unique combination of actin assembly properties that in combination with Fascin drive the self-organization and maintenance of robust filopodia-like bundles composed of coherently elongating actin filaments of uniform length.     

From the Cover: PNAS Plus: Cell-cycle regulation of formin-mediated actin cable assembly

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.Eukaryotic cells contain populations of actin structures with distinct architectures and protein compositions, which mediate varied cellular processes (1). Understanding how F-actin polymerization is regulated in time and space is critical to understanding how actin structures provide mechanical forces for corresponding biological processes. Branched actin filament arrays, which concentrate at sites of clathrin-mediated endocytosis (2, 3) and at the leading edge of motile cells (4), are nucleated by the actin-related protein-2/3 (Arp2/3) complex. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking or form myosin-containing contractile bundles, often are nucleated by formin proteins (5–14).Much has been learned about how branched actin filaments are polymerized by the Arp2/3 complex and how these filaments function in processes such as endocytosis (2, 15). In contrast, relatively little is known about how actin cables are assembled under physiological conditions. In previous studies, branched actin filaments derived from the Arp2/3 complex have been reconstituted using purified proteins (16–19) or cellular extracts (20–25). When microbeads were coated with nucleation-promoting factors for the Arp2/3 complex and then were incubated in cell extracts, actin comet tails were formed by sequential actin nucleation, symmetry breaking, and tail elongation. Importantly, the motility behavior of F-actin assembled by the Arp2/3 complex using defined, purified proteins differs from that of F-actin assembled by the Arp2/3 complex in the full complexity of cytoplasmic extracts (19, 26–28).Formin-based actin filament assembly using purified proteins also has been reported (29, 30). However, reconstitution of formin-derived actin cables under the more physiological conditions represented by cell extracts has not yet been reported.The actin nucleation activity of formin proteins is regulated by an inhibitory interaction between the N- and C-terminal domains, which can be released when GTP-bound Rho protein binds to the formin N-terminal domain, allowing access of the C terminus (FH1-COOH) to actin filament barbed ends (31–40). In yeast, the formin Bni1 N terminus also has an inhibitory effect on actin nucleation through binding to the C terminus (41).Interestingly, several recent reports provided evidence for cell-cycle regulation of F-actin dynamics in oocytes and early embryos (42–45). However, which specific types of actin structures are regulated by the cell cycle and what kind of nucleation factors and actin interacting-proteins are involved remain to be determined.Here, we report a reconstitution of actin cables in yeast extracts from microbeads derivatized with Bni1 FH1-COOH, identifying the proteins involved, increasing the inventory of the proteins that regulate actin cable dynamics and establishing that the actin cable reconstitution in cytoplasmic extracts is cell-cycle regulated.     

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.The cell surface mediates interactions between the cell and the outside world by serving as the site for signal transduction. It also facilitates the uptake and release of cargo and supports adhesion to substrates. These diverse roles require that the cell surface components involved in each function are spatially and temporally organized into domains spanning a few nanometers (nanoclusters) to several micrometers (microdomains). The cell surface itself may be considered as a fluid–lipid bilayer wherein proteins are embedded (1). In the living cell, this multicomponent system is supported by an actin cortex, composed of a branched network of actin and a collection of filaments (2–4).Current models of membrane organization fall into three categories: those invoking lipid–lipid and lipid–protein interactions in the plasma membrane [e.g., the fluid mosaic model (1, 5) and the lipid raft hypothesis (6)], or those that appeal to the membrane-associated actin cortex (e.g., the picket fence model) (7), or a combination of these (8, 9). Although these models based on thermodynamic equilibrium principles have successfully explained the organization and dynamics of a range of membrane components and molecules, there is a growing class of phenomena that appears inconsistent with chemical and thermal equilibrium, which might warrant a different explanation. These include aspects of the organization and dynamics of outer leaflet glycosyl-phosphatidylinositol-anchored proteins (GPI-anchored proteins) (10–13), inner leaflet Ras proteins (14), and actin-binding transmembrane proteins (13, 15, 16).Recent experimental and theoretical work has shown that these features can be explained by taking into account that many cortical and membrane proteins are driven by ATP-consuming processes that drive the system out of equilibrium (13, 15, 17). The membrane models mentioned above have by-and-large neglected this active nature of the actin cortex where actin filaments are being continuously polymerized and depolymerized (18–21), in addition to being persistently acted upon by a variety of myosin motors (22–24) that consume ATP and exert contractile stresses on cortical actin filaments, continually remodeling the architecture of the cortex (4, 21, 25). These active processes in turn can generate tangential stresses and currents on the cell surface, which could drive the dynamics and local composition of membrane components at different scales (22, 26–29).Actin polymerization is proposed to be driven at the membrane by two nucleators, the Arp2/3 complex, which creates a densely branched network, as well as formins that nucleate filaments (18, 21, 30). A number of myosin motors are also associated with the juxtamembranous actin cortex, of which nonmuscle myosin II is the major component in remodeling the cortex and creating actin flows (4, 23, 25, 26, 31, 32). Based on our observations that the clustering of cell surface components that couple directly or indirectly to cortical actin [e.g., GPI-anchored proteins, proteins of the Ezrin, Radaxin, or Moesin (ERM) family (13, 15)] depends on myosin activity, we proposed that this clustering arises from the coupling to contractile actomyosin platforms (called “actin asters”) produced at the cortex (15, 33).A coarse-grained theory describing this idea has been put forward and corroborated by the verification of its key predictions in live cells (15, 33), but a systematic identification of the underlying microscopic processes is lacking. Given the complexity of numerous processes acting at the membrane of a living cell, we use an in vitro approach to study the effect of an energy-consuming actomyosin network on the dynamics of membrane molecules that directly interact with filamentous actin.A series of in vitro studies have explored the organization of confined, dynamic filaments (both actin and microtubules) (34–39) or the role of actin architecture on membrane organization (40–46). Indeed, these studies have yielded insights into the nontrivial emergent configurations that mixtures of polar filaments and motors can adopt when fueled by ATP (34–37), in particular constitutively remodeling steady states that display characteristics of active mechanics (38, 39, 47). However, the effect of linking these mechanics to the confining lipid bilayer and its organization has not been studied.The consequences of actin polymerization on membrane organization, in particular on giant unilamellar vesicles (GUVs), have been addressed in a number of studies on the propulsion of GUVs by an actin comet tail (40, 45, 46). In those experiments, the apparent advection of membrane bound ActA or WASP toward the site of actin polymerization is mainly due to the change in binding affinity of WASP to actin through Arp2/3 (44) and the spherical geometry resulting in the drag of actin to one pole of the vesicle after symmetry break of the actin shell. That this dynamic process changes the bulk properties of the bilayer, namely the critical temperature of a phase-separating lipid bilayer, was shown by Liu and Fletcher (40) when the actin nucleator N-WASP was connected to a lipid species (PIP₂) that was capable of partitioning into one of the two phases.Besides these pioneering studies on the effects of active processes on membrane organization, little was done to directly test the effect of active lateral stresses as well as actomyosin remodeling at the membrane, particularly on the dynamics and organization of membrane-associated components.To this end, we build an active composite in vitro by stepwise addition of components: a supported lipid bilayer with an actin-binding component, actin filaments, and myosin motors. By systematically varying the concentrations of actin and myosin as well as the average actin filament length, we find distinct states of actomyosin organization at the membrane surface upon complete ATP consumption. More importantly, we find that the ATP-fueled contractile actomyosin currents induce the transient accumulation of actin-binding membrane components. As predicted, the active mechanics of actin and myosin at physiologically relevant ATP concentrations drives the system into a nonequilibrium steady state with anomalous density fluctuations and the transient clustering of actin-binding components of the lipid bilayer (15, 33). Finally, connection of this active layer of actomyosin to a phase-segregating bilayer, influences its phase behavior and coarsening dynamics.     

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.     

From the Cover: Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.Self-organization is one of the principal mechanisms of biological pattern formation at the molecular, cellular, and tissue scale. Although the pioneering work of Turing (1) has suggested reaction–diffusion as a generic route toward pattern generation (2), a concrete biomolecular or mechanical understanding of how this might occur in vivo remains elusive, except in a few specific cases (3–5). For instance, Kondo and coworkers (6) demonstrated that pigment patterning on the skin of the Pomocanthus imperator can be understood quantitatively from the simple attraction–repulsion kinetics of two cell types.At the cellular level, active structures, such as the cytoskeleton, are generically expected to display a large variety of structures from a theoretical perspective (7–12), many of which have been reproduced in elegant in vitro studies (13–15). In the case of actomyosin gels, the contractile stresses arising from molecular motors have been shown to create large actin flows that can reorganize the cortex (16, 17). Because actin filaments and motors are “self-advected,” or transported, by their own flow (18), there is a self-reinforcing loop in gel density, capable of creating patterns. Nevertheless, most theoretical studies do not consider the cross-effects of polymerization and diffusion, which resist pattern formation. Interestingly, in the past years, several groups have reported in vivo examples of actin patterns: mammalian axons (19), Caenorhabditis elegans embryo (20), and Drosophila trachea (21) are all cellular cylinders that display a regular array of concentric actin rings on their cortex.In this article, we study the example of ring formation in the Drosophila trachea and propose a generic mechanism for stable actin pattern formation, arising from the interplay of actin turnover and myosin activity. The model makes clear predictions, which we test through fly genetics and drug experiments.     

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.     

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase–, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion.Cofilin is one crucial mediator of actin cytoskeletal dynamics during cell motility (1–5). At the cell edge, cofilin severs F-actin filaments, generating substrates for Arp2/3-mediated branching activity and contributing to F-actin depolymerization by creating a new pointed end and F-actin assembly by increasing the pool of polymerization-competent actin monomers (G-actin) (6, 7). Because of its ability to sever actin filaments and thus, modulate actin dynamics, the precise spatial and temporal regulation of cofilin activity at the cell leading edge is crucial to cell protrusion, chemotaxis, and motility both in vitro and in vivo (2, 8–13). Misregulation of cofilin activity and/or expression is directly related to diseases, including tumor metastasis (14–18) and Alzheimer’s disease (19).Several mechanisms regulate tightly the activation of cofilin in response to upstream stimuli, including interaction with phosphatidylinositol (4,5)-bisphosphate (20–22), local pH changes (23, 24), and phosphorylation at a single regulatory serine (Ser3) (8, 25). The phosphorylation of cofilin, leading to its inactivation, is catalyzed by two kinase families: the LIM-kinases [LIMKs(Lin11, Isl-1, and Mec-3 domain)] and the testicular kinases (25–27). Two primary families of ser/thr phosphatases dephosphorylate and reactivate the actin-depolymerizing and -severing functions of cofilin: slingshot (SSH) (28) and chronophin (CIN) (29).SSH was identified as a cofilin phosphatase through genetic studies in Drosophila (28). The most active and abundant SSH isoform, SSH-1L, has been implicated in such biological processes as cell division, growth cone motility/morphology, neurite extension, and actin dynamics during membrane protrusion (30). SSH dephosphorylates a number of actin regulatory proteins in addition to cofilin, including LIMK1 (31) and Coronin 1B (32). CIN is a haloacid dehydrogenase-type phosphatase, a family of enzymes with activity in mammalian cells that has been poorly characterized. CIN dephosphorylates a very limited number of substrates (33) and as opposed to SSH, has little phosphatase activity toward LIMK both in vitro and in vivo; thus, it seems to be the more specific activator of cofilin (29, 30). CIN exhibits several predicted interaction motifs potentially linking it to regulation by PI3-kinase and phospholipase Cγ (PLCγ), both of which have been implicated in signaling to cofilin activation in vivo in MTLn3 adenocarcinoma cells (10, 34). CIN has been involved in cell division (29), cofilin–actin rod formation in neurons (35), and chemotaxing leukocytes (36, 37). The molecular mechanisms that control the activity and localization of CIN in cells are still not well-understood. In neutrophils, CIN mediates cofilin dephosphorylation downstream of Rac2 (36), and stimulation of protease-activated receptor2 results in recruitment of CIN and cofilin at the cell edge by β-arrestins to promote localized generation of free actin barbed ends, membrane protrusion, and chemotaxis (37). Chemotaxis to EGF by breast tumor cells is directly correlated with cancer cell invasion and metastasis (38, 39). Although cofilin activity is required for tumor cell migration, the contribution(s) of CIN to the regulation of actin dynamics at the leading edge has not yet been investigated.The importance of cofilin in regulating tumor cell motility has been extensively studied using MTLn3 mammary carcinoma cells as a model system. The initial step of MTLn3 cell chemotaxis to EGF consists of a biphasic actin polymerization response resulting from two peaks of free actin barbed end formation (34, 40, 41). The first or early peak of actin polymerization occurs at 1 min after EGF stimulation and requires both cofilin and PLCγ activities (34), but it is not dependent on cofilin dephosphorylation (42). This first transient allows the cells to sense EGF gradients and initiate small-membrane protrusions (11). The second or late peak of actin polymerization occurs at 3 min and is dependent on both cofilin and PI3-kinase activities (43, 44). Cofilin activity in this late transient has been associated with full protrusion of lamellipodia (34). The mechanism by which cofilin becomes activated at the 3-min peak has not been identified, although it is likely to involve the phosphoregulation of Ser3 (42, 45).In this work, we determine the molecular mechanisms involved in the full protrusion of the leading edge upon EGF stimulation. We have identified CIN as a critical regulator of cofilin activation to coordinate leading edge dynamics. Our results yield insights into how CIN controls cell protrusion, a key step in the process of cell migration and metastasis.     

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).     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM.The dynamic connection between the forces generated in the actomyosin cytoskeleton and integrin-mediated focal adhesions (FAs) to the extracellular matrix (ECM) is essential for many physiological processes including cell migration, vascular formation and function, the immune response, and tissue morphogenesis. These diverse functions are mediated by distinct cellular structures including protruding lamellipodia containing nascent FAs that mediate haptotaxis (1), ventral adhesive actin waves that mediate leukocyte transmigration through endothelia (2, 3), and stress fibers (SFs) and FAs that drive fibrillarization of ECM in developing embryos (4, 5). The coordination and interdependence of actin and integrin-based adhesion in these specialized cellular structures are rooted in their biochemical interdependence. Activation of integrins to their high-affinity ECM binding state requires the actin cytoskeleton (6). In turn, integrin engagement with ECM induces signaling that mediates actin polymerization and contractility downstream of Rho GTPases (6, 7). ECM-engaged integrins also affect cytoskeletal organization by physically linking the contractile actomyosin system to extracellular anchorage points (7). Thus, adhesion-associated actin structures are integrated systems that mediate cellular functions requiring coordination of intracellular cytoskeletal forces with ECM binding.Mesenchymal cells generally possess two main types of adhesion-associated actin structures: protruding lamellipodia containing nascent FAs at the cell edge and linear actin bundles in the cell body connected to FAs. Compared with architecturally invariant lamellipodia, adhesion-associated actin bundle structures, including filopodia, the perinuclear actin cap/transmembrane actin-associated nuclear lines, trailing edge bundles, and dorsal SFs, are more diverse in their morphology and less well understood in their architecture and function (8–10). The most-studied actin bundle structure is perhaps dorsal SFs, noncontractile bundles associated at one end with a ventral FA near the cell edge and that extend radially toward the cell center and join with dorsal actin arcs on their other end. How the functional specificity of dorsal SFs is generated apart from the many other distinct adhesion-associated actin bundle structures is not well understood.The functional specificity of adhesion-associated actin structures could be generated either on the adhesion side by compositional differences in FA proteins or on the actin side by differences in the nucleation mechanism and actin binding proteins. On the adhesion side, it is well known that different integrin family members bind distinct types of ECM (11, 12). However, cells adhered to different ECMs all form common structures including lamellipodia, filopodia, and multiple types of SFs. In addition to different integrins, FA function could be regulated by the process of “maturation” in which FAs undergo stereotypical dynamic changes in composition and morphology driven by actomyosin-mediated cellular tension (13, 14). Nascent FAs contain integrins, focal adhesion kinase (FAK), a-actinin, and paxillin (13, 15). When tension is applied, nascent FAs grow and recruit hundreds of proteins, including talin, vinculin, and zyxin (16). These mature FAs then either disassemble or further mature into tensin-containing fibrillar FAs that are responsible for fibronectin fibrillogenesis (17). Thus, the changes in FA size and protein content that accompany FA maturation could give rise to functional specialization of adhesion/actin systems.On the other hand, actin filaments in migrating cells are generated by two main classes of nucleators: the Arp2/3 complex and formins (18). Different nucleating proteins generate different actin organization and geometries, which could in turn dictate functional specificity of adhesions. Arp2/3 forms the branched network in lamellipodia and is thought to be linked to nascent FAs through interaction with FAK (19–21) or vinculin (22). The formin family of actin nucleators, which generates linear actin bundles (23), is more diverse, although formins share a common actin assembly core domain (24), (25). Recent work has begun to ascribe the generation of particular actin structures to some of the 15 formins in mammalian cells, particularly members of the diaphanous family and FHOD1 (26–29). Specifically regarding dorsal SFs, evidence points strongly to polymerization by a formin family member (23, 30–32) but no formin has ever been localized to these SFs or their associated FAs in motile cells. Thus, although formins are clearly critical for forming distinct actin structures, whether they cooperate with FA proteins to specify the function of adhesion-associated actin structures in the cell is unclear.We hypothesized that inverted formin 2 (INF2), found in our recent FA proteome (33), may play a critical role in the formation and functional specificity of adhesion-associated actin structures. INF2 is expressed in cells in two isoforms, one containing a membrane-targeting CAAX-motif that plays a role in mitochondrial fission (34) and a non-CAAX isoform whose function is not well characterized. INF2 is an unusual formin insofar as it contains, in addition to the FH1–FH2 domains that polymerize actin, a WH2-like domain at the C terminus (35) that binds actin monomers to regulate autoinhibition, and also mediates filament severing (35, 36). INF2 also interacts with and inhibits members of the diaphanous family of formin proteins (37). INF2 therefore could have multiple possible roles at FAs in local modulation of actin.Here we explore the role of INF2 in mouse embryonic fibroblasts (MEFs). We find for the first time to our knowledge strong localization of an endogenous formin to FAs at the distal tips of dorsal SFs where it is required for actin polymerization at FAs to form dorsal SFs. We show that INF2 plays a role in controlling morphological, but not compositional maturation of FAs. Strikingly, INF2 is responsible for the formation of one specific class of FAs, the fibrillar FAs that organize the ECM; disruption of INF2 leads to defects in ECM fibrillogenesis. Thus, our study demonstrates that INF2 mediates the formation of dorsal SFs and fibrillar FAs, which together comprise a specific integrated adhesion-associated actin structure responsible for the fibrillogenesis of ECM by fibroblasts.     

Microscopic identification of the order parameter governing liquid–liquid transition in a molecular liquid

A liquid–liquid transition (LLT) in a single-component substance is an unconventional phase transition from one liquid to another. LLT has recently attracted considerable attention because of its fundamental importance in our understanding of the liquid state. To access the order parameter governing LLT from a microscopic viewpoint, here we follow the structural evolution during the LLT of an organic molecular liquid, triphenyl phosphite (TPP), by time-resolved small- and wide-angle X-ray scattering measurements. We find that locally favored clusters, whose characteristic size is a few nanometers, are spontaneously formed and their number density monotonically increases during LLT. This strongly suggests that the order parameter of LLT is the number density of locally favored structures and of nonconserved nature. We also show that the locally favored structures are distinct from the crystal structure and these two types of orderings compete with each other. Thus, our study not only experimentally identifies the structural order parameter governing LLT, but also may settle a long-standing debate on the nature of the transition in TPP, i.e., whether the transition is LLT or merely microcrystal formation.Liquid-liquid transition (LLT) is an intriguing phenomenon in which a liquid transforms into another one via a first-order transition. This means that there can be more than two liquid states for a single-component substance. Despite its counterintuitive nature, there have recently been many pieces of experimental and numerical evidence for the existence of LLT, for various liquids such as water (1–5), aqueous solutions (6–8), triphenyl phosphite (9–12), l-butanol (13), phosphorus (14), silicon (15, 16), germanium (17), and Y₂O₃–Al₂O₃ (18, 19). This suggests that the LLT may be rather universally observed for various types of liquids. However, none of the LLTs reported so far is free from criticisms (20, 21), mainly because these LLTs take place under experimentally difficult conditions [e.g., at high temperature and pressure (14, 15, 17–19)] or in a supercooled state below the melting point (1–3, 5–7, 9, 10), where the transition is inevitably contaminated by microcrystal formation. The latter is not limited to experiments but arises in numerical simulations, often causing many controversies [LLT (22–25) vs. crystallization (26–28)]. For ST2 water, however, this issue has recently been settled by an extensive simulation study by Palmer et al. (4).One of the hottest and long-standing debates is on the nature of the transition found in a molecular liquid, triphenyl phosphite (TPP), by Kivelson and his coworkers (29). The transition is very easy to access experimentally, because it takes place at ambient pressure and at a temperature range between 230 and 210 K and the transformation speed is slow enough to follow the kinetics. Since the finding of this transition (29, 30), many researchers thus have been interested in this intriguing phenomenon and there have been hot discussions on the nature of the transition (20, 21). Some people interpreted this as a liquid-associated phenomenon (9, 10, 31, 32), but others interpret it differently. All of the controversies come from the fact that this transition accompanies microcrystal formation and thus the final state, which is called “glacial phase,” often contains microcrystallites. This led many researchers to explain the transition by non-LLT scenarios, which include a defect-ordered phase scenario predicted by a frustration limited domain theory (29, 30, 33, 34), a microcrystallization scenario (35–38), and a liquid-crystal or plastic-crystal phase scenario (39). Each scenario captures a certain feature of the glacial phase, but fails in explaining all of the experimental results in a consistent manner. Similar situations are often seen in other candidates of LLTs, such as l-butanol [LLT (13) vs. microcrystallization (40–43)], confined water [LLT (5) vs. other phenomena (44–46)], and aqueous solutions [LLT (6, 7) vs. microcrystallization (8, 28, 47, 48)]. For TPP, however, some pieces of experimental evidence supportive of the LLT scenario rather than the microcrystallization scenario have recently been reported (11, 12).We propose a two-order-parameter (TOP) model of a liquid to explain LLT (20, 49). The main point of this model is that it is necessary to consider the spatiotemporal hierarchical nature of a liquid to understand LLT. More specifically, we argue that in addition to density order parameter ρ describing a gas–liquid transition, we need an additional scalar order parameter S, which is the number density of locally favored structures (LFS). In this model, LLT is a consequence of the cooperative ordering of the scalar nonconserved order parameter S, i.e., the cooperative formation of LFS. In other words, LLT is regarded as a gas–liquid-like transition of LFS: one liquid is a gas state of LFS (low-S state), and the other is its liquid state (high-S state). Recently, it was proposed by Anisimov and coworkers (50, 51) that the thermodynamic ordering field conjugate to the order parameter is the conversion equilibrium constant, which further characterizes the nature of LLT. We explained our experimental observation of LLT in TPP in terms of this model (9, 10). We also studied the phase transition dynamics and the physical and chemical properties of the second liquid state (liquid II), which were also explained by the model (20, 21).However, we have not had any direct experimental evidence for the formation of such LFS up to now; thus, an open question is, what is the relevant order parameter governing LLT, although the link of the order parameter to the enthalpy (9, 10), the refractive index (or, density) (9, 10, 29, 30), and the polarity associated with local molecular ordering (12) has been suggested for LLT in TPP. There have been structural studies on LLT by X-ray and neutron scattering measurements, focusing on local liquid structures at an inter- and intramolecular scale (36, 38, 52–54) and mesoscopic structures (34, 55). However, there has been no experimental evidence for the presence of locally favored structures, which characterize the liquid state uniquely, or the order parameter has still not been identified from a microscopic viewpoint.Here we study the structural change of TPP during LLT by time-resolved small- and wide-angle X-ray scattering measurements, which cover a length scale from a single molecule size ( ～  1 nm) to more than tens of nanometers. We show, to our knowledge, the first direct evidence for the presence of LFS and the temporal increase upon the liquid I-to-liquid II transformation. Furthermore, we also find an indication of the formation of microcrystallites during LLT. However, we reveal that LFS and microcrystallites have different sizes and growth kinetics, indicating that although they sometimes appear simultaneously during the process of LLT, LLT itself is driven by the formation of LFS and not by that of microcrystallites. We also discover that LFS are destroyed upon crystallization, clearly indicating not only that these two types of orderings are competing with each other but also that LFS is a structure unique to the liquid state. Our findings provide a comprehensive view on the long-standing controversy on the origin of the glacial phase, which was discovered by Kivelson and his coworkers (29, 30), and show that the fraction of LFS may be the relevant order parameter of LLT. This suggests that a liquid can have a spatiotemporal hierarchical structure at a low temperature, contrary to the common picture of a high-temperature liquid where the structure is random and homogeneous beyond the molecular size.     

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).     

Site-specific cation release drives actin filament severing by vertebrate cofilin

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.Actin polymerization powers the directed motility of eukaryotic cells and some pathogenic bacteria (1–3). Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. Sustained motility requires filament disassembly and subunit recycling. The essential regulatory protein cofilin severs actin filaments (4–6), which accelerates actin network reorganization by increasing the concentration of filament ends available for subunit exchange (7).Cofilin binding alters the structure and mechanical properties of filaments, which effectively introduces local “defects” that compromise filament integrity and promote severing (5). Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10–13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5, 12, 14–18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21). These cation binding sites are referred to as “polymerization” and “stiffness” sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar for divalent and tens of millimolar for monovalent cations) (19, 21) but are predominantly occupied by Mg²⁺ and K⁺ under physiological conditions. Here we demonstrate that cation release from the stiffness site plays a central role in filament severing by vertebrate cofilin, both in vitro and in cells.     

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.     

From the Cover: Mother’s voice and heartbeat sounds elicit auditory plasticity in the human brain before full gestation

Brain development is largely shaped by early sensory experience. However, it is currently unknown whether, how early, and to what extent the newborn’s brain is shaped by exposure to maternal sounds when the brain is most sensitive to early life programming. The present study examined this question in 40 infants born extremely prematurely (between 25- and 32-wk gestation) in the first month of life. Newborns were randomized to receive auditory enrichment in the form of audio recordings of maternal sounds (including their mother’s voice and heartbeat) or routine exposure to hospital environmental noise. The groups were otherwise medically and demographically comparable. Cranial ultrasonography measurements were obtained at 30 ± 3 d of life. Results show that newborns exposed to maternal sounds had a significantly larger auditory cortex (AC) bilaterally compared with control newborns receiving standard care. The magnitude of the right and left AC thickness was significantly correlated with gestational age but not with the duration of sound exposure. Measurements of head circumference and the widths of the frontal horn (FH) and the corpus callosum (CC) were not significantly different between the two groups. This study provides evidence for experience-dependent plasticity in the primary AC before the brain has reached full-term maturation. Our results demonstrate that despite the immaturity of the auditory pathways, the AC is more adaptive to maternal sounds than environmental noise. Further studies are needed to better understand the neural processes underlying this early brain plasticity and its functional implications for future hearing and language development.One of the first acoustic stimuli we are exposed to before birth is the voice of the mother and the sounds of her heartbeat. As fetuses, we have substantial capacity for auditory learning and memory already in utero (1–5), and we are particularly tuned to acoustic cues from our mother (6–9). Previous research suggests that the innate preference for mother’s voice shapes the developmental trajectory of the brain (10, 11). Prenatal exposure to mother’s voice may therefore provide the brain with the auditory fitness necessary to process and store speech information immediately after birth (12, 13).There is evidence to suggest that prenatal exposure to the maternal voice and heartbeat sounds can pave the neural pathways in the brain for subsequent development of hearing and language skills (14). For example, the periodic perception of the low-frequency maternal heartbeat in the womb provides the fetus with an important rhythmic experience (15, 16) that likely establishes the neural basis for auditory entrainment and synchrony skills necessary for vocal, gestural, and gaze communication during mother–infant interactions (17, 18).Studies examining the neural response to the maternal voice soon after birth have found activation in posterior temporal regions, preferentially on the left side, as well as brain areas involved in emotional processing including the amygdala and orbito-frontal cortex (19). Similarly, Beauchemin et al. have found activation in language-related cortical regions when newborns listened to their mother’s voice, whereas a stranger’s voice seemed to activate more generic regions of the brain (20). In addition, Partanen et al. have shown that the neural response to maternal sounds depends on experience as full-term newborns react differentially to familiar vs. unfamiliar sounds they were exposed to as fetuses, suggesting correlation between the amount of prenatal exposure and brain activity (21). Taken together, the above studies suggest that the mother’s voice plays a special role in the early shaping of auditory and language areas of the brain.Numerous animal studies have shown that brain development relies on developmentally appropriate acoustic stimulation early in life (22–32). Auditory deprivation during critical periods can adversely affect brain maturation and lead to long-lasting neural despecialization in the auditory cortex (AC), whereas auditory enrichment in the early postnatal period can enhance neural sensitivity in the primary AC, as well as improve auditory recognition and discrimination abilities.Preterm infants are born during a critical period for auditory brain development. However, the maternal auditory nursery provided by the womb vanishes after a premature birth as the preterm newborn enters the neonatal intensive care unit (NICU). The abrupt transition of the fetus from the protected environment of the womb to the exposed environment of the hospital imposes significant challenges on the developing brain (33). These challenges have been associated with neuropathologic consequences, including reduction in regional brain volumes, white matter microstructural abnormalities, and poor cognitive and language outcomes in preterm compared with full-term newborns (34–41).Considering the acoustic gap between the NICU environment and the womb, it is not surprising that auditory brain development is compromised in preterm compared with full-term infants (42, 43). Numerous studies have suggested that the auditory environment available for preterm infants in the NICU may not be conducive for their neurodevelopment (44–47). These concerns are derived from the frequent reality that hospitalized preterm newborns are overexposed to loud, toxic, and unpredictable environmental noise generated by ventilators, infusion pumps, fans, telephones, pagers, monitors, and alarms (48–51), whereas at the same time they are also deprived of the low-frequency, patterned, and biologically familiar sounds of their mother’s voice and heartbeat, which they would otherwise be hearing in utero (33, 45). In addition, the hospital environment contains a significant amount of high-frequency electronic sounds (52, 53) that are less likely to be heard in the womb because of the sound attenuation provided by maternal tissues and fluid within the intrauterine cavity (54–56). Efforts to improve the hospital environment for preterm neonates have primarily focused on reducing hospital noise and maintaining a quiet environment. However, exposing medically fragile preterm newborns to low-frequency audio recordings of their mothers on a daily basis has been less acknowledged to be of necessity, and the extent to which such maternal sound exposure can influence brain maturation after an extremely premature birth has been a matter of much debate.The present study aimed to determine whether enriching the auditory environment for preterm newborns with authentic recordings of their mother’s voice and heartbeat sounds in the first month of life would result in structural alterations in the AC. The rationale driving this question lies in the fact that such enriched maternal sound stimulation would otherwise be present had the baby not been born prematurely.     

Convergent evolution toward an improved growth rate and a reduced resistance range in Prochlorococcus strains resistant to phage

Prochlorococcus is an abundant marine cyanobacterium that grows rapidly in the environment and contributes significantly to global primary production. This cyanobacterium coexists with many cyanophages in the oceans, likely aided by resistance to numerous co-occurring phages. Spontaneous resistance occurs frequently in Prochlorococcus and is often accompanied by a pleiotropic fitness cost manifested as either a reduced growth rate or enhanced infection by other phages. Here, we assessed the fate of a number of phage-resistant Prochlorococcus strains, focusing on those with a high fitness cost. We found that phage-resistant strains continued evolving toward an improved growth rate and a narrower resistance range, resulting in lineages with phenotypes intermediate between those of ancestral susceptible wild-type and initial resistant substrains. Changes in growth rate and resistance range often occurred in independent events, leading to a decoupling of the selection pressures acting on these phenotypes. These changes were largely the result of additional, compensatory mutations in noncore genes located in genomic islands, although genetic reversions were also observed. Additionally, a mutator strain was identified. The similarity of the evolutionary pathway followed by multiple independent resistant cultures and clones suggests they undergo a predictable evolutionary pathway. This process serves to increase both genetic diversity and infection permutations in Prochlorococcus populations, further augmenting the complexity of the interaction network between Prochlorococcus and its phages in nature. Last, our findings provide an explanation for the apparent paradox of a multitude of resistant Prochlorococcus cells in nature that are growing close to their maximal intrinsic growth rates.Large bacterial populations are present in the oceans, playing important roles in primary production and the biogeochemical cycling of matter. These bacterial communities are highly diverse (1–4) yet form stable and reproducible bacterial assemblages under similar environmental conditions (5–7).These bacteria are present together with high abundances of viruses (phages) that have the potential to infect and kill them (8–11). Although studied only rarely in marine organisms (12–16), this coexistence is likely to be the result of millions of years of coevolution between these antagonistic interacting partners, as has been well documented for other systems (17–20). From the perspective of the bacteria, survival entails the selection of cells that are resistant to infection, preventing viral production and enabling the continuation of the cell lineage. Resistance mechanisms include passively acquired spontaneous mutations in cell surface molecules that prevent phage entry into the cell and other mechanisms that actively terminate phage infection intracellularly, such as restriction–modification systems and acquired resistance by CRISPR-Cas systems (21, 22). Mutations in the phage can also occur that circumvent these host defenses and enable the phage to infect the recently emerged resistant bacterium (23).Acquisition of resistance by bacteria is often associated with a fitness cost. This cost is frequently, but not always, manifested as a reduction in growth rate (24–27). Recently, an additional type of cost of resistance was identified, that of enhanced infection whereby resistance to one phage leads to greater susceptibility to other phages (14, 15, 28).Over the years, a number of models have been developed to explain coexistence in terms of the above coevolutionary processes and their costs (16, 29–32). In the arms race model, repeated cycles of host mutation and virus countermutation occur, leading to increasing breadths of host resistance and viral infectivity. However, experimental evidence generally indicates that such directional arms race dynamics do not continue indefinitely (25, 33, 34). Therefore, models of negative density-dependent fluctuations due to selective trade-offs, such as kill-the-winner, are often invoked (20, 33, 35, 36). In these models, fluctuations are generally considered to occur between rapidly growing competition specialists that are susceptible to infection and more slowly growing resistant strains that are considered defense specialists. Such negative density-dependent fluctuations are also likely to occur between strains that have differences in viral susceptibility ranges, such as those that would result from enhanced infection (30).The above coevolutionary processes are considered to be among the major mechanisms that have led to and maintain diversity within bacterial communities (32, 35, 37–39). These processes also influence genetic microdiversity within populations of closely related bacteria. This is especially the case for cell surface-related genes that are often localized to genomic islands (14, 40, 41), regions of high gene content, and gene sequence variability among members of a population. As such, populations in nature display an enormous degree of microdiversity in phage susceptibility regions, potentially leading to an assortment of subpopulations with different ranges of susceptibility to coexisting phages (4, 14, 30, 40).Prochlorococcus is a unicellular cyanobacterium that is the numerically dominant photosynthetic organism in vast oligotrophic expanses of the open oceans, where it contributes significantly to primary production (42, 43). Prochlorococcus consists of a number of distinct ecotypes (44–46) that form stable and reproducible population structures (7). These populations coexist in the oceans with tailed double-stranded DNA phage populations that infect them (47–49).Previously, we found that resistance to phage infection occurs frequently in two high-light–adapted Prochlorococcus ecotypes through spontaneous mutations in cell surface-related genes (14). These genes are primarily localized to genomic island 4 (ISL4) that displays a high degree of genetic diversity in environmental populations (14, 40). Although about a third of Prochlorococcus-resistant strains had no detectable associated cost, the others came with a cost manifested as either a slower growth rate or enhanced infection by other phages (14). In nature, Prochlorococcus seems to be growing close to its intrinsic maximal growth rate (50–52). This raises the question as to the fate of emergent resistant Prochlorococcus lineages in the environment, especially when resistance is accompanied with a high growth rate fitness cost.To begin addressing this question, we investigated the phenotype of Prochlorococcus strains with time after the acquisition of resistance. We found that resistant strains evolved toward an improved growth rate and a reduced resistance range. Whole-genome sequencing and PCR screening of many of these strains revealed that these phenotypic changes were largely due to additional, compensatory mutations, leading to increased genetic diversity. These findings suggest that the oceans are populated with rapidly growing Prochlorococcus cells with varying degrees of resistance and provide an explanation for how a multitude of presumably resistant Prochlorococcus cells are growing close to their maximal known growth rate in nature.     

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.