刚地弓形虫肌动蛋白解聚因子/丝切蛋白家族的研究进展

肌动蛋白解聚因子(actin depolymerizing factor,ADF)/丝切蛋白(cofilin)家族是一类肌动蛋白结合蛋白(actin-binding proteins,ABP),其家族成员均可与肌动蛋白结合,具有促进微丝解聚的作用,从而提高肌动蛋白单体水平,在重塑肌动蛋白骨架中起重要作用。ADF在顶端复合门寄生虫侵入宿主细胞过程中扮演重要角色。本文综述了刚地弓形虫ADF/cofilin家族的结构特点、调控肌动蛋白动力学机制、参与弓形虫侵入宿主及其在抗弓形虫感染等方面的作用。     

Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails

The intracellular bacterial pathogen Listeria monocytogenes is capable of remodelling the actin cytoskeleton of its host cells such that “comet tails” are assembled powering its movement within cells and enabling cell-to-cell spread. We used cryo-electron tomography to visualize the 3D structure of the comet tails in situ at the level of individual filaments. We have performed a quantitative analysis of their supramolecular architecture revealing the existence of bundles of nearly parallel hexagonally packed filaments with spacings of 12–13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility; presumably, they provide the necessary stiffness. We propose a mechanism for the initiation of comet tail assembly and two scenarios that occur either independently or in concert for the ensuing actin-based motility, both emphasizing the role of filament bundling.Several pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsiae, have developed means to hijack the actin cytoskeleton of their host cells to move inside the host’s cytosol and to spread from cell to cell (1, 2). The cytoplasmic comet tails assembled from actin and actin-interacting proteins propel the bacteria forward and form protrusions emanating from the cell surface, which then become engulfed by neighboring cells.Several studies using light microscopy and EM have attempted to visualize the supramolecular organization of Listeria cytoplasmic comet tails and protrusions (1–8). Despite advances in superresolution fluorescence microscopy, this technique has not yet resolved individual actin filaments in crowded environments. Furthermore, conventional EM applied to detergent-extracted and dehydrated samples suffers from artifacts or a complete collapse of the delicate cytoskeletal networks. Cytoplasmic comet tails were reported to consist of multiple short actin filaments forming a cross-linked and branched network (1, 2) with some degree of alignment at their periphery (7). Branching occurs at the bacterial surface through the interaction of the bacterial surface protein ActA with the Arp2/3 complex (9, 10), which nucleates daughter filaments at an angle of 70° from preexisting filaments (10–12). Isolated Listeria protrusions were described as containing bundles of long, axial filaments, interspersed by short, randomly oriented filaments (3). To date, the detailed molecular architecture of these networks, which is key to understanding actin-based motility, has remained elusive.We examined Listeria comet tails, stress fibers, and filopodia, in their native cellular environment using cryo-electron tomography (CET). CET combines the power of 3D imaging with a close-to-life preservation of cellular structures (13). We cultivated epithelial Potoroo kidney Ptk2 cells on EM grids and infected them with Listeria monocytogenes (SI Text). Uninfected, as well as infected, cells were subjected to plunge-freezing (13), and tomographic datasets of filopodia, stress fibers, and Listeria cytoplasmic comet tails near the periphery of cells or in protrusions were recorded (SI Text).For the interpretation of the tomograms, we applied an automated segmentation algorithm developed specifically for tracking actin filaments (14). Unlike manual segmentation, automated segmentation is fast and unbiased. Due to the low signal-to-noise ratio of CET data, we applied the algorithm conservatively, which tends to underestimate the frequency of branching and the length of filaments. Furthermore, short filaments (<100 nm in comet tails and <70 nm in stress fibers and filopodia) were excluded from the analysis to avoid false-positive results. Tomograms of five Listeria cytoplasmic comet tails (Fig. 1A and Figs. S1A and S2A), nine Listeria protrusions (Fig. 2A and Figs. S3B and S4 A and D), eight stress fibers (Fig. 3C and Fig. S5 A and D), and four filopodia (Fig. 3A and Fig. S6 A and F) were subjected to segmentation. The localization of individual filaments and the analysis of their local neighborhood were used to describe quantitatively the architecture of the networks.Open in a separate windowFig. 1.Hollow Listeria cytoplasmic comet tail contains closely packed parallel filaments. (A) Slice through the tomogram of a cytoplasmic comet tail (tail) in a PtK2 cell (cytoplasm is indicated by cyt) infected by Listeria (b). (Scale bar: 200 nm.) Distribution of XY-filaments (B) and Z-filaments (C) in the XZ plane, projected over the Y axis. The color scale ranges from high occurrence (red) to low occurrence (blue) (same color code in all relevant panels). (D) Two-dimensional histogram of interfilament distances, weighted by the distance, and relative orientations between the filaments. deg, degrees. (E) Two-dimensional histogram of the (ξ, ζ) coordinates of the neighboring filaments in an XY-bundle in the local plane perpendicular to the central filament (dark gray, drawn to scale). (F) XY-filaments projected into the XY plane. The color of the filaments corresponds to their angle with respect to the Y axis: 0–15° (blue), 15–30° (green), 30–45° (red). The cell wall of the bacterium is shown in gray. (G) XY-pairs of parallel filaments (black) among XY-filaments (orange).Open in a separate windowFig. 2.Listeria protrusion shows hexagonal bundles in the vicinity of the plasma membrane. (A) Slice through the tomogram of a protrusion formed by Listeria at the surface of a PtK2 cell. The electron micrograph is shown in Fig. S3A. (Scale bar: 200 nm.) Distribution of XY-filaments (B) and Z-filaments (C) in the XZ plane, projected over the Y axis. (D) Two-dimensional histogram of interfilament distances, weighted by the distance, and relative orientations between the filaments. (E) Two-dimensional histogram of the (ξ, ζ) coordinates of the neighboring filaments in an XY-bundle in the local plane perpendicular to the central filament (dark gray, drawn to scale). (F) XY-filaments projected into the XY plane (same color code as in Fig. 1F). The plasma membrane of the protrusion is shown in gray. (G) XY-pairs of parallel filaments (black) among XY-filaments (orange).Open in a separate windowFig. 3.Filopodia and stress fibers contain hexagonal bundles. Slices through the tomograms of a filopodium (A) and a stress fiber (C) of a PtK2 cell. (Scale bar: 200 nm.) (B and D) XY-pairs of parallel filaments (black) among XY-filaments (orange). The plasma membrane is shown in gray. (E and G) Two-dimensional histogram of interfilament distances, weighted by the distance, and relative orientations between the filaments. (F and H) Two-dimensional histogram of the (ξ, ζ) coordinates of the neighboring filaments in an XY-bundle in the local plane perpendicular to the central filament (dark gray, drawn to scale).We found that many filaments in the comet tails, stress fibers, and filopodia are organized into bundles with spacings between 12 and 13 nm. Moreover, we discovered an arrangement in which filaments are assembled into hexagonal close-packed arrays. Based on our results, we propose a mechanism for the initiation of comet tail assembly and two scenarios enabling Listeria motility, both illuminating the role of filament bundling.     

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1

Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actin's polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly, having a substantially reduced filament-binding loop and C-terminal α4 helix. Despite this divergence in structure, we demonstrate that PfADF1 is capable of efficient actin filament severing. Furthermore, this severing occurs despite PfADF1's low binding affinity for filaments. Comparative structural analysis along with biochemical and microscopy evidence establishes that severing is reliant on the availability of an exposed basic residue in the filament-binding loop, a conserved minimal requirement that defines AC-mediated filament disassembly across eukaryotic cells.     

Actin disassembly clock determines shape and speed of lamellipodial fragments

A central challenge in motility research is to quantitatively understand how numerous molecular building blocks self-organize to achieve coherent shape and movement on cellular scales. A classic example of such self-organization is lamellipodial motility in which forward translocation is driven by a treadmilling actin network. Actin polymerization has been shown to be mechanically restrained by membrane tension in the lamellipodium. However, it remains unclear how membrane tension is determined, what is responsible for retraction and shaping of the rear boundary, and overall how actin-driven protrusion at the front is coordinated with retraction at the rear. To answer these questions, we utilize lamellipodial fragments from fish epithelial keratocytes which lack a cell body but retain the ability to crawl. The absence of the voluminous cell body in fragments simplifies the relation between lamellipodial geometry and cytoskeletal dynamics. We find that shape and speed are highly correlated over time within individual fragments, whereby faster crawling is accompanied by larger front-to-rear lamellipodial length. Furthermore, we find that the actin network density decays exponentially from front-to-rear indicating a constant net disassembly rate. These findings lead us to a simple hypothesis of a disassembly clock mechanism in which rear position is determined by where the actin network has disassembled enough for membrane tension to crush it and haul it forward. This model allows us to directly relate membrane tension with actin assembly and disassembly dynamics and elucidate the role of the cell membrane as a global mechanical regulator which coordinates protrusion and retraction.     

Tyrosine phosphorylation of WASP promotes calpain-mediated podosome disassembly

Podosomes are actin-based adhesions involved in migration of cells that have to cross tissue boundaries such as myeloid cells. The Wiskott Aldrich Syndrome Protein regulates de novo actin polymerization during podosome formation and it is cleaved by the protease calpain during podosome disassembly. The mechanisms that may induce the Wiskott Aldrich Syndrome Protein cleavage by calpain remain undetermined. We now report that in myeloid cells, tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein-tyrosine291 (Human)/tyrosine293 (mouse) not only enhances Wiskott Aldrich Syndrome Protein-mediated actin polymerization but also promotes its calpain-dependent degradation during podosome disassembly. We also show that activation of the Wiskott Aldrich Syndrome Protein leading to podosome formation occurs independently of tyrosine phosphorylation in spleen-derived dendritic cells. We conclude that tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein integrates dynamics of actin and cell adhesion proteins during podosome disassembly required for mobilization of myeloid cells during the immune response.     

Power transduction of actin filaments ratcheting in vitro against a load

The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.The actin cytoskeleton forms a signal-responsive protein system made of filaments that undergo constant remodeling via directional assembly and disassembly processes. At the leading edge of a migrating cell, protrusive force results from insertional polymerization of actin filament barbed ends against the membrane, pushing it forward (1, 2). A wealth of regulatory proteins adapts the organization of the filaments and their mechanical properties to the movement the cell needs to make. At the single filament level, Hill was the first to propose that the free energy of polymerization could be transduced into mechanical work against the membrane (3). Oster and colleagues transcribed Hill’s conceptual model into a mechanistic one coined Brownian ratchet (4), later refined in the tethered ratchet (5). They showed that a single filament can push against a load because thermal fluctuations of either the load (4) or the filament (5) allow for stochastic insertion of monomers at the polymerizing tip. The amplitude of the fluctuations are force dependent, making the elongation velocity of the filament force dependent as well. The whole force-velocity profile of a single actin filament was never measured experimentally. However, the stalling force of a filament, at which the elongation velocity drops to zero, could be estimated to a few piconewtons (6–8). Comparatively, forces of a few nanonewtons are required to stall the migration of cells (9, 10) or Listeria comet tails (11). This difference of three orders of magnitude points to the need for a large number of cooperating filaments to generate high forces in protrusive structures. Exploring the cooperation within an assembly of filaments polymerizing together against a load remains a hard task for experimentalists. Actin gels (12–14) or brushes (15) have been reconstituted in vitro to measure the amount of force they can generate. In these complex structures, regulatory proteins can cause tethering of the filaments to the load (14), rearrangements under force (16), or variations of filaments number in reaction to force (17). These phenomena shed light on the strong influence of regulatory proteins on force production but impede to draw information on the physical mechanism of force generation.Here we present an experimental setup that was designed to closely resemble the conceptual view of an array of about 100 independent filaments polymerizing perpendicularly to a load, their tip remaining nontethered to that load. Because the number of parameters affecting the force generation is minimal, we are able to concentrate on the physical interaction of filaments with the load and show that it is compatible with the Brownian ratchet. However, if the Brownian ratchet model is relevant to describe force generation at the single filament level, it gives no information about the collective behavior of the filaments population. This collective aspect of force production is further developed using an analytical model, revealing that filaments cooperation is optimal in a specific regime which ensures maximum transduction of mechanical power to the load.     

ARF1-mediated actin polymerization produces movement of artificial vesicles

Vesicular trafficking and actin dynamics on Golgi membranes are both regulated by ADP-ribosylation factor 1 (ARF1) through the recruitment of various effectors, including vesicular coats. Actin assembly on Golgi membranes contributes to the architecture of the Golgi complex, vesicle formation, and trafficking and is mediated by ARF1 through a cascade that leads to Arp2/3 complex activation. Here we addressed the role of Golgi actin downstream of ARF1 by using a biomimetic assay consisting of liposomes of defined lipid composition, carrying an activated form of ARF1 incubated in cytosolic cell extracts. We observed actin polymerization around the liposomes resulting in thick actin shells and actin comet tails that pushed the ARF1 liposomes forward. The assay was used to characterize the ARF1-dependent pathway, leading to actin polymerization, and confirmed a dependency on CDC42 and its downstream effector N-WASP. Overall, this study demonstrates that actin polymerization driven by the complex multicomponent signaling cascade of the Golgi apparatus can be reproduced with a biomimetic system. Moreover, our results are consistent with the view that actin-based force generation at the site of vesicle formation contributes to the mechanism of fission. In addition to its well established function in coat recruitment, the ARF1 machinery also might produce movement- and fission-promoting forces through actin polymerization.     

Nitric oxide mediates mechano- and chemoreceptor-activated intestinal feedback control of gastric emptying

The effect of nitric oxide (NO) synthase inhibition on the gastric emptying of nutrient and nonnutrient meals was investigated in nine dogs. The inhibition of NO synthase delayed the gastric emptying time of both nutrient and nonnutrient meals, but the percentage delay of nutrient meals was significantly greater than that of nonnutrient meals. The inhibition of NO synthase during the emptying of nonnutrient meals enhanced mainly the amplitude of antral, pyloric, and distal duodenal contractions. However, NO synthase inhibition during the emptying of nutrient meals stimulated several spatial and temporal parameters of gastropyloroduodenal contractions. We conclude that NO is one of the neurotransmitters of intestinal feedback that regulates the gastric emptying of both nutrient and nonnutrient meals. The nature and intensity of intestinal feedback by the stimulation of both chemo- and mechanoreceptors by nutrient meals is different from that by the stimulation of mechanoreceptors only by the nonnutrient meals.Supported in part by grants from the Department of Veterans Affairs Research Service and National Institutes of Diabetes, Digestive and Kidney Diseases, DK32346.     

Myosin VI deafness mutation prevents the initiation of processive runs on actin

Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (P_i), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of P_i. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.Myosin VI is unique among the known myosins of animal cells in that it traffics toward the minus end of actin filaments (1). This unique directionality, coupled with its ability to act as both a processive transporter (2, 3) and load-dependent anchor (4), allow myosin VI to play a number of cellular roles that cannot be compensated for by any other myosin motor (5–15). To accomplish these cellular functions, myosin VI has a number of unique structural and functional adaptations, many of which have been debated in the literature, and some are still the subject of controversy (8). This is not surprising because its design features represent significant departures from other characterized myosin motors.Mutations in myosin VI can result in deafness in humans (16–20). There are three published human mutations (16–18) that cause deafness and result in amino acid changes in the myosin VI motor domain: C442Y, H246R, and E216V. In the mouse, there is one characterized missense mutation (20) in the motor (D179Y) as well as a null mutation (19), both of which result in deafness. All of these mutations likely lead to disruption of the normal organization and maintenance of the stereocilia, the mechanosensing organelles of hair cells, present in the cochlear apparatus, as has been documented in the case of the mouse mutations (19).Myosin VI achieves its ability to walk hand-over-hand along a single actin filament (21) by having a motor that has been kinetically tuned to spend the majority of its time strongly bound to actin (22). Thus, the probability that at least one head will be strongly bound to actin at all times is very high. (The ratio of the occupancy of the strongly bound actin states of the actin–myosin ATPase cycle to that of the weak + dissociated + strongly bound states is called the duty ratio.) Although a high duty ratio is sufficient for processive movement, processivity can be further enhanced by a mechanism known as “gating” whereby strain between the heads essentially stalls the lead head on actin until the rear head detaches (23). Our proposed mechanism of gating by myosin VI involves blocking of ATP binding to the lead head of a dimer (24, 25). With both a high duty ratio and gating a single myosin VI can move on the order of a micrometer or more along an actin filament. However, the myosin VI dimer can also function as a load-dependent anchor (4) on actin filaments (as load increases, the attachment time of the dimer greatly increases). This enables myosin VI to play a number of structural roles in cells, such as in the overall organization of the Golgi (26) and in the last step of secretion (27), as well as in formation and maintenance of the stereocilia of the hair cells (16, 19, 20, 28).Preliminary characterization of the mouse deafness mutation referred to as “tailchaser,” D179Y, revealed an apparent loss of coordination, or gating, between the two heads (20). In the cell, this mutation disrupts endocytosis and results in loss of stereocilia maintenance (20). The impact on endocytosis suggests that the mutation may disrupt processive movement of myosin VI. However, recent in vitro experiments suggest that loss of gating should not destroy processive movement (29), and therefore loss of gating would not necessarily lead to deafness. This prompted us to do a much more extensive functional and structural analysis of the impact of the D179Y mutation.Although myosin VI is unusual in its directionality, structural and kinetic characterizations of the myosin VI actin-activated ATPase cycle have shown that the motor domain of myosin VI adopts structural states and kinetic transitions similar to those of plus-end myosin motors (Fig. 1A). The D179Y mutation belongs to an important region of the motor, which we have called the transducer (30), that lies near the nucleotide-binding site (Fig. 1 B and C). This region has been proposed from structural studies to be responsible for the kinetic tuning of actin-catalyzed product release following ATP hydrolysis (30, 31). However, the structural changes that underlie these force-producing transitions on actin are poorly understood. Herein, the characterization of the D179Y myosin allows us to infer for the first time to our knowledge which of these transitions in the motor domain require rearrangements of the central transducer region of the motor. Ultimately we seek to understand how this region controls the timing of the sequential conformational changes required upon the working stroke that are critical to tune a myosin for a particular cellular function. The transducer plays a critical role for these transitions and has a major contribution for the control of the duration of the force-bearing states.Open in a separate windowFig. 1.Actin–myosin chemomechanical transduction cycle and the subdomain structure of the myosin motor. (A) Actin–myosin cycle. The three structural states crystallized for myosin VI are represented along the motor (ATPase) cycle. (B) Overall structure of the myosin VI D179Y motor domain in the postrigor state. The position of D179Y is highlighted. The four subdomains of the motor: N-terminal, U50, L50, and converter are colored gray, blue, white, and green, respectively. The lever arm that amplifies conformational changes of the motor includes the converter and the following elongated region, part of which is represented here as a green helix (insert-2 of myosin VI) to which calmodulin with four Ca²⁺ ions (pink) is bound. (C) Close-up view of the motor domain in a similar orientation as in B. The transducer region lies near the nucleotide-binding site at the junction between the N- and the U50 subdomains. It includes the last three strands of the seven-stranded beta sheet, helices HF and HG, as well as flexible elements such as loop 1, the β-bulge between β6 and β7, and the HO-linker. Important distortion occurs in the β-sheet as well as rearrangements in the rest of the transducer upon the transitions between states of the actin–myosin cycle.These results demonstrate that the D179Y mutation in myosin VI does not in fact destroy gating between the heads of a dimer, but rather prevents initiation of processive runs by allowing premature release of inorganic phosphate (P_i) from a head that is not attached to actin. We were able to rescue processivity by initiating processive runs with both heads bound to actin (in the absence of nucleotide) before addition of ATP or by lowering the ATP concentration. We also demonstrate that a drug that decreases the rate of P_i release in the absence of actin rescues processive movement on actin.     

Pattern formation and polarity sorting of driven actin filaments on lipid membranes

Collective motion of active matter is ubiquitously observed, ranging from propelled colloids to flocks of bird, and often features the formation of complex structures composed of agents moving coherently. However, it remains extremely challenging to predict emergent patterns from the binary interaction between agents, especially as only a limited number of interaction regimes have been experimentally observed so far. Here, we introduce an actin gliding assay coupled to a supported lipid bilayer, whose fluidity forces the interaction between self-propelled filaments to be dominated by steric repulsion. This results in filaments stopping upon binary collisions and eventually aligning nematically. Such a binary interaction rule results at high densities in the emergence of dynamic collectively moving structures including clusters, vortices, and streams of filaments. Despite the microscopic interaction having a nematic symmetry, the emergent structures are found to be polar, with filaments collectively moving in the same direction. This is due to polar biases introduced by the stopping upon collision, both on the individual filaments scale as well as on the scale of collective structures. In this context, positive half-charged topological defects turn out to be a most efficient trapping and polarity sorting conformation.

Collective motion of active matter is ubiquitous, with observations ranging from flocks of birds (1) and schools of fish (2) to propelled colloids (3). The interactions between agents in such systems lead to the formation of complex structures including clusters, swirls, or lanes of agents moving coherently (4). The structure of the emerging patterns strongly depends on both the agents’ shape and their velocity alignment mechanism. A particular case is that of elongated microscopic particles that translate along their major axis in a quasi-two-dimensional environment and only interact upon collision (5, 6). In the context of cytoskeletal systems, gliding actin filaments or microtubules propelled by molecular motors are found to be able to readily crawl over each other and only retain a weak level of alignment upon binary collisions, which eventually leads at high densities to a diverse set of patterns (7). Such resulting patterns are found to be strongly dependent on this weak microscopic alignment interaction, and therefore, even slightly tuning it causes the system to switch between polar and nematic phases, separated by a coexistence regime (8, 9). Observed structures in cytoskeletal systems with weak to moderate interactions include nematic lanes, polar waves, and vortices (10–12). Conversely, pattern formation in systems of elongated bacteria or granular matter is often based on hard interactions with a strong steric component (13–18). In this repulsion-dominated regime, particles are unable to crawl over each other and must stop upon collision. In the limiting case of spherical self-propelled particles, this kind of steric interaction can lead to a stable phase separation between stuck and moving particles, the so-called motility induced phase separation (MIPS) (19). On the other hand, in the case of elongated particles, steric effects can still act as velocity aligning mechanisms. As orientation mismatches are unstable, particles end up aligning and this leads to flocking behavior rather than to phase separation (5, 20–25). This latter case, in which strong steric constraints dominate binary interactions but alignment is still present, is poorly understood, and how modeling has to be extended to account for the emergent collective behavior of elongated, flexible agents with volume exclusion also remains still under debate (26–30). This is partly due to the lack of microscopic experimental systems allowing to explore this regime. Semiflexible cytoskeletal filaments would be the best candidate, but their volume exclusion is usually too weak. However, having them propelled by motors immobilized on a fluid membrane would be a promising route to bridge this experimental gap (31).Here, we enforce a steric repulsion-dominated interaction, leading to alignment between actin filaments by coupling myosin motors to a fluid-supported lipid bilayer. Because of the slippage of the motors on the membrane, the force propelling the filaments is too weak to enable filaments to crawl over each other and thus effectively implements a repulsion-dominated regime, with filaments stopping upon collisions. Eventually, however, because of the thermal fluctuations of their tips, filaments can align and resume motion. The experimental realization of such a microscopic binary interaction, based on volume exclusion, enables us to observe and quantify the resulting pattern formation process in a system of active semiflexible filaments. We then first characterize the interaction at the single filament scale, showing that it leads to nematic alignment. As the filaments’ density is increased, patterns of collective motion emerge, ranging from clusters to thick streams and vortices. Despite the nematic collision rule, we find the emerging structures to be locally polar. The repulsion-dominated interaction indeed introduces a polar bias not only due to the tendency of active filaments or clusters to keep moving together after a polar collision but also by forcing filaments with similar orientation to stop and accumulate when encountering an obstacle. In particular, at high densities, such an interaction leads to the formation of transient local +1/2 topological defects, which act as wedges and, therefore, effectively trap and polarity-sort motile filaments. We interpret this trapping mechanism as an analog of MIPS for elongated self-propelled particles.     

EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt

The cadherin-catenin complex is the major machinery for cell-cell adhesion in many animal species. This complex in general associates with actin fibers at its cytoplasmic side, organizing the adherens junction (AJ). In epithelial cells, the AJ encircles the cells near their apical surface and forms the "zonula adherens" or "adhesion belt." The mechanism as to how the cadherin-catenin complex and F-actin cooperate to generate these junctional structures, however, remains unknown. Here, we show that EPLIN (epithelial protein lost in neoplasm; also known as Lima-1), an actin-binding protein, couples with alpha-catenin and, in turn, links the cadherin-catenin complex to F-actin. Without EPLIN, this linkage was unable to form. When EPLIN had been depleted in epithelial cells, the adhesion belt was disorganized and converted into zipper-like junctions in which actin fibers were radially arranged. However, nonjunctional actin fibers were not particularly affected by EPLIN depletion. As EPLIN is known to have the ability to suppress actin depolymerization, our results suggest that EPLIN functions to link the cadherin-catenin complex to F-actin and simultaneously stabilizes this population of actin fibers, resulting in the establishment of the adhesion belt.     

Impact of corticotropin-releasing hormone on gastrointestinal motility and adrenocorticotropic hormone in normal controls and patients with irritable bowel syndrome

^a Department of Psychosomatic Medicine, ^b Department of Comprehensive Medicine, Tohoku University School of Medicine, Sendai, Japan

Correspondence to: Dr S Fukudo, Department of Psychosomatic Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-Ku, Sendai 980-8574, Japan.

Accepted for publication 19 January 1998

Background—Corticotropin-releasing hormone (CRH) plays a key role in modulating intestinal motility in stressed animals.
Aims—To evaluate the effect of CRH on intestinal motility in humans and to determine whether patients with irritable bowel syndrome (IBS) have an exaggerated response to CRH.
Subjects—Ten IBS patients diagnosed by Rome criteria and 10 healthy controls.
Methods—CRH (2 µg/kg) was intravenously administered during duodenal and colonic manometry and plasma adrenocorticotropic hormone (ACTH) was measured by radioimmunoassay.
Results—CRH induced motility of the descending colon in both groups (p<0.001) and induced greater motility indexes in IBS patients than in controls (p<0.05). CRH produced duodenal phase III motor activity in 80% of the subjects and duodenal dysmotility in 40% of IBS patients. Abdominal symptoms evoked by CRH in IBS patients lasted significantly longer than those in controls (p<0.05). CRH induced significant increases in plasma ACTH levels in both groups (p<0.001) and produced significantly higher plasma ACTH levels in IBS patients than in controls (p<0.001).
Conclusion—Human intestinal motility is probably modulated by exogenous CRH. The brain-gut in IBS patients may have an exaggerated response to CRH.
(GUT 1998;:845-849)

Keywords: irritable bowel syndrome;  corticotropin releasing factor;  adrenocorticotropic hormone;  colonic motility;  duodenal motility

     

Structures and mechanisms of actin ATP hydrolysis

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) P^G–O^3B bond cleavage; 3) four concomitant events: W1–PO₃⁻ formation, OH⁻ and proton cleavage, nucleophilic attack by the OH⁻ against P^G, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Actin, a major cytoskeletal protein, is an ATP-binding protein that exists in monomeric (G-actin) and filamentous (F-actin) forms. A large variety of cellular functions are driven by the cyclic processes of actin molecule assembly (polymerization) and disassembly (depolymerization). Actin ATP hydrolysis, which was originally discovered without knowing its biological significance (1), energetically drives the cyclic assembly–disassembly (2). Therefore, the elucidation of the mechanism of ATP hydrolysis is crucially important for our understanding of the cellular functions of actin.The actin molecule comprises two major domains, the outer domain (OD) and the inner domain (ID). An ATP molecule with a divalent cation binds in the cleft between the OD and ID (3). Upon incorporation into the actin filament, the actin molecule undergoes a conformational transition (4). In the monomeric G-form, the OD is twisted by about 20° relative to the ID, whereas in the filamentous F-form the two domains are almost flat.The entire cycle of actin assembly–disassembly proceeds in five sequential processes (below, each nucleotide tightly binds Mg²⁺, which is not explicitly indicated for simplicity. -G and -F indicate G-form and F-form actin, respectively):

• 1)ATP-G → ATP-F: The conformational transition, which is associated with the polymerization of the ATP-bound G-form actin.
• 2)ATP-F → ADP-Pi-F: The ATP hydrolysis reaction, triggered by process 1 with a rate constant of 0.3 s⁻¹ (5), which is considerably slower than other ATPases [e.g., myosin (20 to 200 s⁻¹) (6)]. The slow rate which allows further elongation, and the hydrolysis reaction occurs in the interior of the filament. The reaction is irreversible (7).
• 3)ADP-Pi-F → ADP-F: The Pi release, which occurs at an extremely slow rate with actin molecules in the interior of the filament [0.003 s⁻¹ (8) or 0.007 s⁻¹ (9)] but is much faster with molecules around barbed ends [>2 s⁻¹ (10) or 1.8 s⁻¹ (9)]. The Pi release is reversible. ADP-F has a modest affinity for Pi with a dissociation constant of 1 mM (9, 10), which is far below the cytosolic concentration, whereas ADP-G hardly binds Pi (∼60 mM) (10).
• 4)ADP-F → ADP-G: The depolymerization releases actin subunits at the ends of the actin filament.
• 5)ADP-G → ATP-G: The bound nucleotide exchanges from ADP for ATP, which occurs only in G-actin.

The cyclic reaction of barbed end assembly and the pointed end disassembly, occurring in actin alone at the critical concentration of monomers using ATP as an energy source, is called tread-milling (11). In the cytosol, where the actin concentration is far beyond the critical concentration for polymerization, cooperative behaviors of regulatory proteins accelerate the spontaneous tread-milling and/or facilitate filament turnover by promoting multiple reactions involving nucleation, capping, and severing, to drive fast cell migration (12, 13). Recent progress, particularly in kinetic analyses of single actin filaments, has revealed the contribution of the barbed end disassembly to monomer recycling (14, 15). Pi release dramatically alters the assembly properties of actin filaments in two ways: 1) by promoting spontaneous depolymerization and, more importantly, 2) by making the ADP-actin subunits more attractive to proteins that facilitate filament disassembly such as ADF/cofilin. Therefore, for comprehensively understanding of the actin cycle of assembly–disassembly, the mechanism and significance of the Pi release are of crucially important. This should be based upon our knowledge of the actin ATP hydrolysis process and the properties of ADP-Pi-F.Studies of the actin ATP hydrolysis mechanism have been hampered, mainly due to the lack of F-form actin structures at a sufficient resolution for identifying water molecules, which are the keys to the reaction. The crystal structure of the G-form actin is not suitable (3, 16), since the G-form actin is not ATP hydrolysis–competent (17). The cryogenic electron microscopy (cryo-EM) structures of the actin filament at over 3.1-Å resolutions (18–20) are also insufficient, because the details of the water molecules in the nucleoside binding cleft, particularly those surrounding the phosphate moiety, are missing. The actin filament has not been crystallized, because filaments with a fixed length have never been prepared. Previously, the ATP hydrolysis mechanism of F-form actin was studied by employing metadynamics simulations (21). However, the details of the reaction mechanism have remained obscure, since the simulations were based upon structural data that lacked water molecules (4).We now show that, in a 1:1 complex of the actin-binding protein fragmin domain-1 (F1) and actin (referred to as the F1A complex), the actin molecule is trapped in the F-form. This is the first monomeric actin structure in the F-form among the more than 260 actin structures deposited to date in the Protein Data Bank (PDB) (22). Fragmin is a member of the villin-gelsolin protein superfamily (23) and was first isolated from the slime mold Physarum polycephalum (24). Like gelsolin, fragmin severs F-actin and caps the barbed end in a Ca²⁺-dependent manner (24–26). Fragmin consists of three tandemly linked domains (F1, F2, F3) that are homologous to the N-terminal half of gelsolin (G1, G2, G3). G1 also forms a 1:1 complex with actin. However, in the G1–actin complex, the actin is in the G-form (27).The F1A complex provided 1.15-Å resolution structures of F-form actin binding AMPPNP, ADP-Pi, or ADP, with a magnesium ion at the catalytic site. Based on the high precision of the pre- and posthydrolysis structures, we performed quantum mechanics/molecular mechanics (QM/MM) calculations (28), which revealed the comprehensive reaction mechanism of the actin ATP hydrolysis. The results provide mechanistic answers to key questions regarding the cycle of actin state transitions: how the G-to-F conformational transition triggers ATP hydrolysis, why the ATP hydrolysis rate is much slower than those of other ATPases, why the hydrolysis is irreversible, why the Pi release is so slow, and why F-form actin is destabilized after the Pi-release.Our results demonstrate that, while some properties are quantitatively different, the overall catalytic strategy of actin ATP hydrolysis (up until the formation of ADP-Pi-F) is almost identical to those of P-loop type-motor proteins, such as F₁-ATPase (29), kinesin (30), and myosin (31–33), despite the substantial differences in the catalytic site structures. In contrast to the common ATP hydrolysis strategies, the processes after the hydrolysis are distinct in actin: The hydrolysis does not cause any overall conformational changes; therefore, the Pi is not promptly released and the ADP-Pi F-form actin structure is readily available. These observations have led us to propose hypotheses about how ATP hydrolysis and Pi release are related in general, and how Pi is released by actin in particular.     

Hepatocyte growth factor effects on motility of stone-diseased and stone-free human gallbladders

Hepatocyte growth factor (HGF) has unique morphogenic activity for several cell types. Besides its major effect upon liver regeneration, its motogenic activity to enhance motility has not been verified for smooth muscles. Therefore we evaluated the impact of HGF in an in-vitro model of human gallbladder motility. Twelve stone-diseased and eight stone-free muscle strips were preincubated with HGF (100 ng/ml, 200 ng/ml). For the analysis of motility, cholecystokinin (CCK) was added (0.1 nM, 0.5 nM, 2 nM, 10 nM, and 100 nM). Twelve stone-diseased and eight stone-free strips without HGF incubation served as the control group. The tone of healthy (tone/100 nM CCK: control group, 12.4 ± 3.6 mN; HGF group, 19.5 ± 4.5 mN) and stone-diseased (tone/100 nM CCK: control group, 10.8 ± 3.8 mN; HGF group, 17.3 ± 4.8 mN) muscle strips, preincubated with HGF, was increased, with a higher sensitivity to CCK. Our results suggest that there is a clear motogenic response of stone-diseased human gallbladders to HGF. Received: October 26, 1998 / Accepted: April 16, 1999     

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.     

非小细胞肺癌中自分泌运动因子受体的过表达及对患者预后的影响

目的探讨自分泌运动因子受体（AMFR）在非小细胞肺癌（NSCLC）肿瘤细胞胞膜中的表达与患者吸烟状况、肿瘤分化等临床病理特征及疾病预后的关系。方法选自2006年5月至2009年4月于我院接受手术治疗80例NSCLC患者肺癌标本,应用免疫组织化学方法检测胞膜中AMFR的表达,分析AMFR过表达与患者的吸烟状况、肿瘤分化及无瘤生存期的相关性。结果80例患者中,52例AMFR过表达,阳性率65％,吸烟患者AMFR过表达多于无吸烟患者,分别为75．9％和58．8％（P〈0．05）,肿瘤组高分化与低／未分化患者AMFR阳性表达率分别为42．3％和74．19％,AMFR过表达与肺癌的分化明显相关（P〈0．05）。生存分析显示：AMFR阴性患者无瘤生存期为28．2个月,AMFR弱阳性、阳性及强阳性患者无瘤生存期分别为33．3、26．4和7．8个月（P〈0．05）。结论肿瘤细胞胞膜中AMFR过表达与NSCLC组织分化和患者无瘤生存期呈正相关。     

白细胞介素-6在人白血病细胞中的转录与自分泌

目的 探讨白细胞介素６（ＩＬ－６）基因在不同类型白血病细胞中的构成表达以及白血病细胞是否自泌ＩＬ－６,为更好地利用ＩＬ－６／ＩＬ－６Ｒ系统介导重组白细胞介素６－假单胞菌外毒素融合蛋白（ＩＬ－６－ＰＥ４０）靶向治疗白血病提供可靠依据。方法 采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ）半定量技术、序列分析及ＥＬＩＳＡ方法检测白血病细胞系Ｕ９３７、ＨＬ６０、ＫＧ１、ＨｕＴ２８,ＣＥＭ及Ｒａｊｉ中ＩＬ－６ｍＲ     

脑源性神经营养因子在肠道中的作用

脑源性神经营养因子(BDNF),作为神经营养物质的一员,广泛分布于神经系统,在神经元分化、发育、存活中发挥重要作用。肠道各层结构中均大量表达BDNF及其受体。BDNF不仅在肠神经系统发育过程中起着重要作用,还与神经元可塑性调节相关。BDNF参与肠道感染和胃肠动力调节,其机制可能是与其调节肠道感觉和运动神经元的可塑性,并与5-羟色胺、降钙素基因相关肽等因子的相互作用相关。