Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.

Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.

Rapid parallel flow cytometry assays of active GTPases using effector beads.

We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

Integrin activation is an essential component of SARS-CoV-2 infection.

SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's ß subunit, we demonstrate that talin-mediated signaling is essential for productive infection.

Integrin activation is an essential component of SARS-CoV-2 infection.

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2 R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn 2+ , which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2 R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2 R18 with basal state integrins, but is ineffective against Mn 2+ -activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2 R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand binding function of integrins via a talin dependent mechanism and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα 13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα 13 binding to the cytoplasmic tail of an integrin's ß subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.

Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays.

Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and alpha(v)beta(3) integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K(d) approximately 26pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral-cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.

Assessing the Dynamics and Complexity of Disease Pathogenicity Using 4-Dimensional Immunological Data.

Investigating disease pathogenesis and personalized prognostics are major biomedical needs. Because patients sharing the same diagnosis can experience different outcomes, such as survival or death, physicians need new personalized tools, including those that rapidly differentiate several inflammatory phases. To address these topics, a pattern recognition-based method (PRM) that follows an inverse problem approach was designed to assess, in <10 min, eight concepts: synergy, pleiotropy, complexity, dynamics, ambiguity, circularity, personalized outcomes, and explanatory prognostics (pathogenesis). By creating thousands of secondary combinations derived from blood leukocyte data, the PRM measures synergic, pleiotropic, complex and dynamic data interactions, which provide personalized prognostics while some undesirable features-such as false results and the ambiguity associated with data circularity-are prevented. Here, this method is compared to Principal Component Analysis (PCA) and evaluated with data collected from hantavirus-infected humans and birds that appeared to be healthy. When human data were examined, the PRM predicted 96.9 % of all surviving patients while PCA did not distinguish outcomes. Demonstrating applications in personalized prognosis, eight PRM data structures sufficed to identify all but one of the survivors. Dynamic data patterns also distinguished survivors from non-survivors, as well as one subset of non-survivors, which exhibited chronic inflammation. When the PRM explored avian data, it differentiated immune profiles consistent with no, early, or late inflammation. Yet, PCA did not recognize patterns in avian data. Findings support the notion that immune responses, while variable, are rather deterministic: a low number of complex and dynamic data combinations may be enough to, rapidly, unmask conditions that are neither directly observable nor reliably forecasted.

Some mechanistic insights into GPCR activation from detergent-solubilized ternary complexes on beads.

The binding of full and partial agonist ligands (L) to G protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G proteins [ligand-receptor-G protein (LRG) complexes]. Cyclic ternary complex models are required to account for the thermodynamically plausible complexes. It has recently become possible to assemble solubilized formyl peptide receptor (FPR) and beta(2)-adrenergic receptor (beta(2)AR) ternary complexes for flow cytometric bead-based assays. In these systems, soluble ternary complex formation of the receptors with G proteins allows direct quantitative measurements which can be analyzed in terms of three-dimensional concentrations (molarity). In contrast to the difficulty of analyzing comparable measurements in two-dimensional membrane systems, the output of these flow cytometric experiments can be analyzed via ternary complex simulations in which all of the parameters can be estimated. An outcome from such analysis yielded lower affinity for soluble ternary complex assembly by partial agonists compared with full agonists for the beta(2)AR. In the four-sided ternary complex model, this behavior is consistent with distinct ligand-induced conformational states for full and partial agonists. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide-mediated ternary complex disassembly. The modular breakup of ternary complex components is highlighted by the finding that the fastest step involves the departure of the ligand-activated GPCR from the intact G protein heterotrimer. The data also show that, under these experimental conditions, G protein subunit dissociation does not occur within the time frame relevant to signaling. The data and concepts are discussed in the context of a review of current literature on signaling mechanism based on structural and spectroscopic (FRET) studies of ternary complex components.

Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases.

Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.

Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity.

A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.

Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases.

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700 nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then be attached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borne GTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells.

A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry.

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), which infects more than 200,000 people worldwide. Sin Nombre virus (SNV) and Andes virus (ANDV) cause the most severe form of HCPS, with case fatality ratios of 30%-40%. There are no specific therapies or vaccines for SNV. Using high-throughput flow cytometry, we screened the Prestwick Chemical Library for small-molecule inhibitors of the binding interaction between UV-inactivated and fluorescently labeled SNVR18 particles, and decay-accelerating factor (DAF) expressed on Tanoue B cells. Eight confirmed hit compounds from the primary screen were investigated further in secondary screens that included infection inhibition, cytotoxicity, and probe interference. Antimycin emerged as a bona fide hit compound that inhibited cellular infection of the major HCPS (SNV)- and HCPS (Hantaan)-causing viruses. Confirming our assay's ability to detect active compounds, orthogonal testing of the hit compound showed that antimycin binds directly to the virus particle and blocks recapitulation of physiologic integrin activation caused by SNV binding to the integrin PSI domain.

Upregulation of P2Y2R, Active uPA, and PAI-1 Are Essential Components of Hantavirus Cardiopulmonary Syndrome.

Sin Nombre virus (SNV) causes hantavirus cardiopulmonary pulmonary syndrome (HCPS) with the loss of pulmonary vascular endothelial integrity, and pulmonary edema without causing cytopathic effects on the vascular endothelium. HCPS is associated primarily with a dysregulated immune response. We previously found occult signs of hemostatic imbalance in the form of a sharp >30-100 fold increase in the expression of plasminogen activator inhibitor type 1 (PAI-1), in serial blood plasma draws of terminal stage-patients. However, the mechanism of the increase in PAI-1 remains unclear. PAI-1 is a primary inhibitor of fibrinolysis caused by tissue plasminogen activator (tPA) and urokinase plasminogen activator plasma (uPA). Here, we investigate factors that contribute to PAI-1 upregulation during HCPS. Using zymography, we found evidence of PAI-1-refractory uPA activity and no tPA activity in plasma samples drawn from HCPS patients. The sole prevalence of uPA activity suggested that severe inflammation drove PAI-1 activity. We have recently reported that the P2Y2 receptor (P2Y2R) mediates SNV infectivity by interacting in cis with ß3 integrins, which activates the latter during infection. P2Y2R is a known effector for several biological processes relevant to HCPS pathogenesis, such as upregulation of tissue factor (TF), a primary initiator of the coagulation cascade, stimulating vascular permeability and leukocyte homing to sites of infection. As P2Y2R is prone to upregulation under conditions of inflammation, we compared the expression level of P2Y2R in formalin fixed tissues of HCPS decedents using a TaqMan assay and immunohistochemistry. Our TaqMan results show that the expression of P2Y2R is upregulated significantly in HCPS cases compared to non- HCPS controls (P < 0.001). Immunohistochemistry showed that lung macrophages were the primary reservoir of high and coincident localization of P2Y2R, uPA, PAI-1, and TF antigens. We also observed increased staining for SNV antigens in the same tissue segments where P2Y2R expression was upregulated. Conversely, sections of low P2Y2R expression showed weak manifestations of macrophages, SNV, PAI-1, and TF. Coincident localization of P2Y2R and PAI-1 on macrophage deposits suggests an inflammation-dependent mechanism of increasing pro-coagulant activity in HCPS in the absence of tissue injury.

Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, ß3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation ß5/ß3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbß3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbß3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the ß3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.

Elevated cytokines, thrombin and PAI-1 in severe HCPS patients due to Sin Nombre virus.

Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus) is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS) with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS) measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry) of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ≥30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1) which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients.

Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal protein (RILP) effector protein.

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein-dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug-target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7-RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead 'locks' the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein-protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein-protein interactions, which can be relevant to drug discovery and development.

FRET detection of lymphocyte function-associated antigen-1 conformational extension.

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLß2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.