Using force to visualize conformational activation of integrins

The development of biophysical approaches to analyze integrin–ligand binding allows us to visualize in real time the conformational changes that shift the bond affinity between low- and high-affinity states. In this issue, Chen et al. (2012. J. Cell Biol. http://dx.doi.org/jcb.201201091) use these approaches to validate some aspects of the classical integrin regulation model; however, their data suggest that much of the regulation occurs after ligand binding rather than in preparation for ligand binding to occur.Cell adhesion is a critical development that spurred the evolution of metazoans and is integrated into virtually all physiological functions, from energy and metabolism to movement and defense against invasive organisms. Adhesion receptors share with other cell surface receptors, such as the tyrosine kinase growth factor or G-protein–coupled receptors, the ability to transmit extracellular signals into cells (Menko and Boettiger, 1987). However, their primary function is mechanical and their signaling function appears to devolve from their adhesive function (Friedland et al., 2009). The mechanical function of adhesion receptors involves both the number of bound receptors and their spatial distribution on the cells. The strength of adhesion is determined primarily by the number of adhesive bonds (bonds between cell surface adhesion receptors and cell or extracellular matrix–bound ligands). Because cells need to move and change shape, they need to vary the number and positions of their adhesive bonds. This requires the cells to control the binding and unbinding of adhesion receptors. To accomplish this regulation, it is necessary to modulate the affinity of the binding reaction. The classical way to modulate binding affinity is through allosteric regulation in which the binding of a ligand to one domain on the receptor changes its conformation and modulates the binding of another ligand to another domain. This is the basis of the classical model for the regulation of the best understood of the adhesion receptor families, the integrins (Ye et al., 2010). More recently, another way to change the affinity of integrin–ligand bonds has been discovered. Because integrins that are physically bound to the substrate are also bound, through focal complexes inside the cell, to the actin cytoskeleton (Pavalko et al., 1991), intracellular actin-myosin contraction can exert tension on the integrin–ligand bond (Friedland et al., 2009). Tension will change the integrin conformation (by force) and change the integrin–ligand binding affinity (Kong et al., 2009). For most chemical bonds, tension reduces bond lifetime and increases the dissociation rate (these bonds are called “slip bonds”); but for integrin–ligand bonds, tension stabilizes the bond and increases the bond lifetime (these bonds are called “catch bonds”). In this issue of JCB, Chen et al. present a novel approach that allows us to visualize both the conformational switching of integrins and switching between short and long bond lifetimes. Their analysis brings together the classical and the catch bond models of regulation and may change our perception of how adhesive bonds are regulated.The classical model for integrin regulation is a three-state model: inactive, active, and active/bound to ligand. Integrin activation is based on the interconversion between the inactive and the active state (Frelinger et al., 1991; Ye et al., 2010). The regulation is fundamentally allosteric, in which the final common step involves the binding of talin and/or kindlin to the cytoplasmic domain of the β subunit of integrin, causing a separation of the α and β subunit cytoplasmic domains. This generates an allosteric change that is propagated to the extracellular domain, resulting in a conversion from the low- to the high-affinity state that is primed to bind to ligand. In the x-ray diffraction structure of integrin extracellular domains, the overall structure is bent but can be converted by reasonable calculations to an extended form (Xiong et al., 2001). It was proposed that the bent form represented the inactive and the extended form represented the active form of integrin (Takagi et al., 2002). Thus, integrin activation would generate a 15–20-nm shift in the ligand-binding domain (αA domain) away from the plasma membrane (Fig. 1). Over the past 20 or more years, the classical model has been developed in significant molecular detail. However, these analyses have generally followed a biochemical bias and have been relatively blind both to the analysis of integrin dissociation (which is difficult to analyze biochemically in cells with many adhesive bonds) and to the role of mechanics and forces in the regulation of integrin function.Open in a separate windowFigure 1.Measuring integrin conformational transitions using the Bioforce probe. Bonds between the αA domain (purple) of integrin α_Lβ2 and its ligand I-CAM-1 attached to a bead are formed by bringing the two into contact. Bonds can form with either the bent conformation (left) or the extended conformation (right). Bonds formed in the bent conformation can switch to the extended conformation without dissociation. This would increase bond stability (and hence affinity by slowing the dissociation rate). Bonds formed in the extended form can switch to the bent form without dissociation, but this will reduce their stability and increase the dissociation rate. The conformational switches are followed by the position of the bead. Lines A and B mark the displacement between the two conformations. The RBC (top) and the cell (bottom) would be attached to the Bioforce probe micropipettes.To understand how Chen et al. (2012) visualized and analyzed the binding properties of integrin using biophysical approaches, it is necessary to describe their basic experimental strategy. The authors used a Bioforce probe that consists of two micropipettes, one holding the cell expressing the integrin, the other holding a red blood cell (RBC) to which is attached a bead coated with the ligand (see Video 1 in Chen et al., 2012). A video camera monitors the position of the bead with high precision (3 nm). A micromanipulator moves the cell micropipette until the cell touches the bead (with a force of 20 pN for 100 ms) and then is retracted a set distance and held. The objective of this is to allow a single integrin–ligand bond to form; the retraction prevents additional bonds from forming. If a bond forms, the bead will follow the retraction because it is attached through a ligand to a cell surface integrin. The RBC, which acts as a spring, will be stretched. After a time, the bond will dissociate and the RBC will retract the bead. This allows the measurement of the lifetime of single integrin–ligand bonds. The new insight comes when the movement of the bead is followed during the lifetime of the bond. The force tracings show two distinct events: a displacement away from the cell membrane and a reciprocal displacement toward the cell membrane. The mean magnitude of these displacements was similar to that predicted from the x-ray diffraction data for the bent and extended forms of the integrin (∼17 nm). This interpretation was reinforced through the measurement of bond stiffness. More variation in the displacement of the bead indicated a weaker bond when it was in the bent state and a shorter bond lifetime for the bond in the bent state, which also indicates a weaker bond. Thus, force differences (the displacement of the bead held by the RBC/spring) allow us to see integrin conformational shifts in real time.The Bioforce probe has allowed us to observe movements of single molecular domains, which, remarkably, correspond to movements predicted in the classical model for integrin activation. Because those experiments were performed using intact cells, they provide strong evidence for the existence of both the extended and bent conformations on the cell surface and for the generation of increased affinity by integrin extension. In the classical model for integrin activation, the focus has been on the observed conformational shift between extended and bent forms that can occur with purified integrins (Ye et al., 2010). This switch is generally observed with the integrin in an unbound state. The Bioforce probe sees the other side of the coin. Binding to the ligand occurred to either the bent (inactive) or extended (active) form, and the switching between the two states occurred while the ligand was bound. This distinction is important because each model points to different control mechanisms. The classical model points to a regulation of the binding rate to the ligand, which is governed by the energy of activation, the collision frequency, and the frequency in which collisions lead to bond formation. The Bioforce probe analysis points to mechanisms that affect the rate of dissociation, which involves stability of the bond and can be modulated by force as well as chemistry. The biochemical bias of methods that support the classical model are not adept at analyzing the postbinding changes in bond stability. In contrast, Bioforce probe experiments use direct physical manipulation to form the bond, and hence the natural events of bond formation are not observable. The analysis contains the elements that affect bond dissociation but are missing elements of bond association events. In each case, the experimental analysis biases the conclusion. Because the classical and the Bioforce probe approaches complement each other, we have a better basis for generating a more accurate model of integrin regulation.     

Integrin activation and matrix binding mediate cellular responses to mechanical stretch

Mechanical tension is a critical determinant of cell growth, differentiation, apoptosis, migration, and development. Integrins have been implicated in sensing force but little is known about how forces are transduced to biochemical signals. We now show that mechanical strain stimulates conformational activation of integrin alphavbeta3 in NIH3T3 cells. Integrin activation is mediated by phosphoinositol 3-kinase and is followed by an increase in integrin binding to extracellular matrix proteins. Mechanical stretch stimulation of JNK was dependent on new integrin binding to extracellular matrix. These data define a molecular mechanism for the role of integrins in mechanotransduction.     

Antimicrobial peptides: Modes of mechanism,modulation of defense responses

Complicated schemes of classical breeding and their drawbacks, environmental risks imposed by agrochemicals, decrease of arable land, and coincident escalating damages of pests and pathogens have accentuated the necessity for highly efficient measures to improve crop protection. During co-evolution of host-microbe interactions, antimicrobial peptides (AMPs) have exhibited a brilliant history in protecting host organisms against devastation by invading pathogens. Since the 1980s, a plethora of AMPs has been isolated from and characterized in different organisms. Nevertheless the AMPs expressed in plants render them more resistant to diverse pathogens, a more orchestrated approach based on knowledge of their mechanisms of action and cellular targets, structural toxic principle, and possible impact on immune system of corresponding transgenic plants will considerably improve crop protection strategies against harmful plant diseases. This review outlines the current knowledge on different modes of action of AMPs and then argues the waves of AMPs'' ectopic expression on transgenic plants'' immune system.Key words: antimicrobial peptides, durable resistance, gene technology, immune system, metchnikowin, sustainable agriculture     

A theoretical approach to G-protein modulation of cellular responsiveness

 Structure and function of cells often depend critically on molecular signals arriving at their surface. There are universal mechanisms of signal transduction and signal processing across cell membranes. In this paper the mechanisms involving guanine-nucleotide regulatory proteins (“G-proteins”) and certain receptor-kinases are considered. On the basis of recent findings in molecular biology a mathematical model is developed taking into account all essential components in the biochemical network between first and second messenger. There are two coupled feedback loops inherent in this process. The model finally consists of three nonlinear equations, which are obtained from a system of originally ten equations by using conservation laws and quasi-steady state conditions. The second part of the paper contains a mathematical analysis of the model. Solutions describing the temporal development of the involved biochemical species are shown to be bounded, more specifically to remain, independent of the size of the input signal, in a bounded domain of the state space. For the situation of stationary input signals existence, uniqueness and asymptotic stability of steady states are derived. We also demonstrate biologically relevant stimulus-response properties like monotonicity and saturation effects. For temporally non-constant input signals we show numerically that the model is able to produce phenomena of hypersensitivity and desensitization which are important characteristics of cellular responsiveness. Received 18 March 1996; received in revised form 15 April 1996     

贮脂细胞激活的分子细胞学机制

贮脂细胞的激活是启动纤维化的关键病理事件,其激活的分子细胞学机制包括：贮脂细胞（肌成纤维细胞）分泌的ＴＧＦ－β对其自身分泌作用,是贮脂细胞激活的永久刺激因素;内皮细胞通过释放ＰＤＧＦ和ＦＧＦ刺激肌成纤维细胞增殖;Ｋｕｐｆ－ｆｅｒ细胞、单核细胞等通过释放免疫介质影响贮脂细胞增殖及改变细胞合成基质能力,而促进贮脂细胞激活;肝细胞可以通过其释放的介质和直接的膜接触而启动贮脂细胞的活化。激活的贮脂细胞产生     

Oligonucleotide NX1838 inhibits VEGF165-mediated cellular responses in vitro

Summary VEGF (vascular endothelial growth factor) overproduction has been identified as a major factor underlying pathological angiogenesis in vivo, including such conditions as psoriasis, macular degeneration, and tumor proliferation. Endothelial cell tyrosine kinase receptors, KDR and Flt-1, have been implicated in VEGF responses including cellular migration, proliferation, and modulation of vascular permeability. Therefore, agents that limit VEGF-cellular interaction are likely therapeutic candidates for VEGF-mediated disease states (particularly agents blocking activity of VEGF₁₆₅, the most frequently occurring VEGF isoform). To that end, a nuclease-resistant, VEGF₁₆₅-specific aptamer NX1838 (2′-fluoropyrimidine, RNA-based oligonucleotide/40-kDa-PEG) was developed. We have assessed NX1838 inhibition of a variety of cellular events associated with VEGF, including cellular binding, signal transduction, calcium mobilization, and induction of cellular proliferation. Our data indicate that NX1838 inhibits binding of VEGF to HUVECs (human umbilical vein endothelial cells) and dose-dependently prevents VEGF-mediated phosphorylation of KDR and PLCγ, calcium flux, and ultimately VEGF-induced cell proliferation. NX1838-inhibition of VEGF-mediated cellular events was comparable to that observed with anti-VEGF monoclonal antibody, but was ineffective as an inhibitor of VEGF₁₂₁-induced HUVEC proliferation. These findings, coupled with nuclease stability of the molecule, suggest that NX1838 may provide therapeutic utility in vivo.     

A cellular mechanism for prepulse inhibition

In prepulse inhibition (PPI), startle responses to sudden, unexpected stimuli are markedly attenuated if immediately preceded by a weak stimulus of almost any modality. This experimental paradigm exposes a potent inhibitory process, present in nervous systems from invertebrates to humans, that is widely considered to play an important role in reducing distraction during the processing of sensory input. The neural mechanisms mediating PPI are of considerable interest given evidence linking PPI deficits with some of the cognitive disorders of schizophrenia. Here, in the marine mollusk Tritonia diomedea, we describe a detailed cellular mechanism for PPI--a combination of presynaptic inhibition of startle afferent neurons together with distributed postsynaptic inhibition of several downstream interneuronal sites in the startle circuit.     

Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades

The non-receptor tyrosine kinase PYK2 appears to function at a point of convergence of integrins and certain G protein-coupled receptor (GPCR) signaling cascades. In this study, we provide evidence that translocation of PYK2 to focal adhesions is triggered both by cell adhesion to extracellular matrix proteins and by activation of the histamine GPCR. By using different mutants of PYK2 as green fluorescent fusion proteins, we show that the translocation of PYK2 to focal adhesions is not dependent on its catalytic activity but rather is mediated by its carboxyl-terminal domain. Translocation of PYK2 to focal adhesions was attributed to enhanced tyrosine phosphorylation of PYK2 and its association with the focal adhesion proteins paxillin and p130(Cas). Translocation of PYK2 to focal adhesions, as well as its tyrosine phosphorylation in response to histamine treatment, was abolished in the presence of protein kinase C inhibitors or cytochalasin D treatment, whereas activation of protein kinase C by phorbol ester resulted in focal adhesion targeting of PYK2 and its tyrosine phosphorylation in an integrin-clustering dependent manner. Overexpression of a wild-type PYK2 enhanced ERK activation in response to histamine, whereas a kinase-deficient mutant substantially inhibited this response. Furthermore, inhibition of PYK2 translocation to focal adhesions abolished ERK activation in response to histamine treatment. These results suggest that PYK2 apparently links between GPCRs and focal adhesion-dependent ERK activation and can provide the molecular basis underlying PYK2 function at a point of convergence between signaling pathways triggered by extracellular matrix proteins and certain GPCR agonists.     

Multiscale models of integrins and cellular adhesions

Computational models of integrin-based adhesion complexes have revealed important insights into the mechanisms by which cells establish connections with their external environment. However, how changes in conformation and function of individual adhesion proteins regulate the dynamics of whole adhesion complexes remains largely elusive. This is because of the large separation in time and length scales between the dynamics of individual adhesion proteins (nanoseconds and nanometers) and the emergent dynamics of the whole adhesion complex (seconds and micrometers), and the limitations of molecular simulation approaches in extracting accurate free energies, conformational transitions, reaction mechanisms, and kinetic rates, that can inform mechanisms at the larger scales. In this review, we discuss models of integrin-based adhesion complexes and highlight their main findings regarding: (i) the conformational transitions of integrins at the molecular and macromolecular scales and (ii) the molecular clutch mechanism at the mesoscale. Lastly, we present unanswered questions in the field of modeling adhesions and propose new ideas for future exciting modeling opportunities.     

Variant glycosylation: an underappreciated regulatory mechanism for beta1 integrins

Although it has been known for many years that beta1 integrins undergo differential glycosylation in accordance with changes in cell phenotype, the potential role of N-glycosylation as a modulator of integrin function has received little attention. One reason for the relatively limited interest in this topic likely relates to the fact that much of the prior research was correlative in nature. However, new results now bolster the hypothesis that there is a causal relationship between variant glycosylation and altered integrin activity. In this review, the evidence for variant glycosylation as a regulatory mechanism for beta1 integrins are summarized, with particular emphasis on: (1). outlining the instances in which cell phenotypic variation is associated with differential beta1 glycosylation, (2). describing the specific alterations in glycan structure that accompany phenotypic changes and (3). presenting potential mechanisms by which variant glycosylation might regulate integrin function.     

Spy1 expression prevents normal cellular responses to DNA damage: inhibition of apoptosis and checkpoint activation

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.     

内质网应激的细胞效应分子机制

内质网应激（endoplasmic reticulum stress,ERs）是内质网腔内错误折叠蛋白聚积的一种适应性反应,适度ERs通过激活未折叠蛋白反应起适应性的细胞保护作用,而过高和持久的ERs则通过诱导转录因子CHOP表达、激活caspase-12和c—Jun氨基末端激酶（JNK）等导致细胞凋亡。近年来,越来越多的研究提示内质网应激是神经退行性病变、2型糖尿病以及肥胖等疾病发生过程中的重要环节。对内质网应激的细胞效应分子机制进行综述。随着对ERs机制理解的深入,有可能会发现新的分子标志物或新的诊疗策略。     

Cyclophosphamide teratogenesis: evidence for compensatory responses to induced cellular toxicity

Cyclophosphamide (CP) administered ip to pregnant mice on day 10 of gestation (day of plug = day 0) is teratogenic (exencephaly, cleft palate, and limb malformations) at 20 mg/kg and embryolethal at higher doses. In the present study, CP was administered at 1, 5, 10, or 20 mg/kg on day 10 of gestation. Embryos were removed at 8 and 28 hr postdosing, and two embryos from each litter were immediately stained with Nile blue sulfate (NBS) to identify areas of cell death. The remaining embryos were frozen and forelimb buds subsequently removed for flow cytometric (FCM) analysis of the cellular DNA synthetic cycle. Additional litters were examined near term (day 17) for morphological abnormalities; these data were correlated with embryonic toxicity as detected by NBS staining and FCM analysis. Only the highest dose produced malformations. In marked contrast, a dose-related increase in the percentage of limb bud cells in the S (DNA synthetic) phase of the cell cycle was detectable at all doses. Inhibition of DNA synthesis was detected at all doses 8 hr post exposure and persisted through 28 hr for doses greater than or equal to 10 mg/kg. NBS staining indicated increased cell death in the alar plate of the neural tube 28 hr after exposure to 10 mg/kg CP and generally increased cell death in areas of rapid cell proliferation throughout the embryo at 20 mg/kg. The absence of an overt teratogenic response at dose levels that produced significant perturbation of the cell cycle indicates that a measure of embryonic damage can be compensated for or repaired. The implications of these findings for the existence of thresholds in developmental toxicity are discussed.