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Chemical research has established a technique using ruthenated cytochrome c protein fragments to create homogenous photoactive electron transfer systems. The model electron transfer system uses microperoxidase-11 and an attached photoactive ruthenium tris(bipyridyl) donor. Rapid reversible electron transfer is established using the novel system.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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-2 R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn 2+ , which induces integrin extension, enhances cell entry of SARS-CoV-2 R18 . 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. RGD-integrin antagonists inhibited SARS-CoV-2 R18 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 Gα 13 . Using cell-permeable peptide inhibitors of talin and Gα 13 binding to the cytoplasmic tail of an integrin's β subunit, we demonstrate that talin-mediated signaling is essential for productive infection., (© 2021. The Author(s).)

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.

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.

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 SNV R18 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.

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 P2Y 2 receptor (P2Y 2 R) mediates SNV infectivity by interacting in cis with β 3 integrins, which activates the latter during infection. P2Y 2 R 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 P2Y 2 R is prone to upregulation under conditions of inflammation, we compared the expression level of P2Y 2 R in formalin fixed tissues of HCPS decedents using a TaqMan assay and immunohistochemistry. Our TaqMan results show that the expression of P2Y 2 R 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 P2Y 2 R, uPA, PAI-1, and TF antigens. We also observed increased staining for SNV antigens in the same tissue segments where P2Y 2 R expression was upregulated. Conversely, sections of low P2Y 2 R expression showed weak manifestations of macrophages, SNV, PAI-1, and TF. Coincident localization of P2Y 2 R and PAI-1 on macrophage deposits suggests an inflammation-dependent mechanism of increasing pro-coagulant activity in HCPS in the absence of tissue injury.

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.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β 3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β 5 /β 3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y 2 R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant α IIb β 3 integrins and (RGD)P2Y 2 R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y 2 R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y 2 R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing α IIb β 3 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 Gα 13 protein from binding to the cytoplasmic domain of the β 3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y 2 R 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., (© 2017 Bondu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

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.

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.

Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-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., (© 2015 Chigaev et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell-matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82's contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion., (© 2014 Termini et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Decay accelerating factor (DAF/CD55) is targeted by many pathogens for cell entry. It has been implicated as a co-receptor for hantaviruses. To examine the binding of hantaviruses to DAF, we describe the use of Protein G beads for binding human IgG Fc domain-functionalized DAF ((DAF)₂-Fc). When mixed with Protein G beads the resulting DAF beads can be used as a generalizable platform for measuring kinetic and equilibrium binding constants of DAF binding targets. The hantavirus interaction has high affinity (24-30 nM; k(on) ~ 10⁵ M⁻¹ s⁻¹, k(off) ~ 0.0045 s⁻¹). The bivalent (DAF)₂-Fc/SNV data agree with hantavirus binding to DAF expressed on Tanoue B cells (K(d) = 14.0 nM). Monovalent affinity interaction between SNV and recombinant DAF of 58.0 nM is determined from competition binding. This study serves a dual purpose of presenting a convenient and quantitative approach of measuring binding affinities between DAF and the many cognate viral and bacterial ligands and providing new data on the binding constant of DAF and Sin Nombre hantavirus. Knowledge of the equilibrium binding constant allows for the determination of the relative fractions of bound and free virus particles in cell entry assays. This is important for drug discovery assays for cell entry inhibitors.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.

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.

In flow cytometry, the quantitation of fluorophore-tagged ligands and receptors on cells or at particulate surfaces is achieved by the use of standard beads of known calibration. To the best of our knowledge, only those calibration beads based on fluorescein, EGFP, phycoerythyrin and allophycocyanine are readily available from commercial sources. Because fluorophore-based standards are specific to the selected fluorophore tag, their applicability is limited to the spectral region of resonance. Since quantum dots can be photo-excited over a continuous and broad spectral range governed by their size, it is possible to match the spectral range and width (absorbance and emission) of a wide range of fluorophores with appropriate quantum dots. Accordingly, quantitation of site coverage of the target fluorophores can be readily achieved using quantum dots whose emission spectra overlaps with the target fluorophore.This chapter focuses on the relevant spectroscopic concepts and molecular assembly of quantum dot fluorescence calibration beads. We first examine the measurement and applicability of spectroscopic parameters, ε, φ, and %T to fluorescence calibration standards, where ε is the absorption coefficient of the fluorophore, φ is the quantum yield of the fluorophore, and %T is the percent fraction of emitted light that is transmitted by the bandpass filter at the detector PMT. The modular construction of beads decorated with discrete quantities of quantum dots with defined spectroscopic parameters is presented in the context of a generalizable approach to calibrated measurements of fluorescence in flow cytometry.

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., (2010 Elsevier Inc. All rights reserved.)

Quantitative analysis of the staining of cell membranes with the cationic amphiphile, octadecyl rhodamine B (R18), is confounded by probe aggregation and changes to the probes' absorption cross section and emission quantum yield. In this paper, flow cytometry, quantum-dot-based fluorescence calibration beads, and FRET were used to examine real-time transfer of R18 from water to two limiting models of the cellular plasma membrane, namely, a single-component disordered membrane, dioleoyl-L-alpha-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol, and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration was raised from 0 to 10 mol % in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that were reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parametrized the propagation of concentration-dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum-albumin-mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions where the critical aggregation concentration (CAC) of the probe is defined as 14 nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to that for DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally, this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.

The binding of full and partial agonist ligands (L) to G-protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G-proteins (LRG complexes). We describe the assembly of detergent-solubilized LRG complexes on beads. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide-mediated ternary complex disassembly. Ternary complexes were assembled with three formyl peptide receptor constructs (wild type, FPR-Galpha(i2) fusion, and FPR-GFP fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(i)(1)gamma(2) and beta(4)gamma(2)). Experimental evidence suggests that thermodynamic stability of ternary complexes depends on subunit isotype. Comparison of assemblies derived from the three constructs of FPR and G-protein heterotrimers composed of the available subunit isotypes demonstrate that the fast step is associated with the separation of receptor and G-protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G-protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.

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.

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.

The authors describe a biosensing concept based on the release of compounds, which are encapsulated within lipid-coated porous silica microspheres, by detergents and toxins that disrupt supported lipid bilayers (SLBs) on the microspheres. Suspension and microfluidic based methods have been developed to monitor the release of the encapsulated compounds in response to membrane disruption. The authors established that the SLBs on porous microspheres can endure experimental conditions necessary for their incorporation into packed microchannels while maintaining the bilayer integrity and functionality. Model compounds including a nonionic detergent (Triton X-100), a membrane active protein (alpha-hemolysin), and a membrane lytic antimicrobial peptide (melittin) were successfully utilized to interact with different formulations of SLBs on porous silica microspheres. The results demonstrate the stability of the SLBs on the microspheres for several weeks, and the feasibility of using this system to detect the release of fluorescent dyes as well as other molecular reporters. The latter were detected by their involvement in subsequent biospecific interactions that were detected by fluorescence. This study exemplifies proof of concept for developing new chemical and biochemical sensors and drug delivery systems based on the disruption of lipid membranes coating porous silica microspheres that encapsulate dyes or bioactive compounds.

We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.

Rapid activation of integrins in response to chemokine-induced signaling serves as a basis for leukocyte arrest on inflamed endothelium. Current models of integrin activation include increased affinity for ligand, molecular extension, and others. In this study, using real-time fluorescence resonance energy transfer to assess alpha(4)beta(1) integrin conformational unbending and fluorescent ligand binding to assess affinity, we report at least four receptor states with independent regulation of affinity and unbending. Moreover, kinetic analysis of chemokine-induced integrin conformational unbending and ligand-binding affinity revealed conditions under which the affinity change was transient whereas the unbending was sustained. In a VLA-4/VCAM-1-specific myeloid cell adhesion model system, changes in the affinity of the VLA-4-binding pocket were reflected in rapid cell aggregation and disaggregation. However, the initial rate of cell aggregation increased 9-fold upon activation, of which only 2.5-fold was attributable to the increased affinity of the binding pocket. These data show that independent regulation of affinity and conformational unbending represents a novel and fundamental mechanism for regulation of integrin-dependent adhesion in which the increased affinity appears to account primarily for the increasing lifetime of the alpha(4)beta(1) integrin/VCAM-1 bond, whereas the unbending accounts for the increased capture efficiency.

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work, we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot 1005-0045, and QD585, lot 0905-0031, from Invitrogen). This is the first step in the development of calibration beads to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited state lifetime measurements, to characterize their spectroscopic behavior, concentrating on the 400- to 500-nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480 nm excitation up to 0.4 at 450 nm and down again to 0.15 at 350 nm. For QD585, QY values were constant at 0.2 between 500 and 400 nm, but they dropped to 0.1 at 350 nm. We attribute the wavelength dependencies to heterogeneity in size and surface defects in the QD525, consistent with characteristics described previously in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots and the quantitative needs of assay development in biological applications.

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.

Elevated expression or activity of the epidermal growth factor (EGF) receptor is common in ovarian cancer and is associated with poor patient prognosis. Our previous studies demonstrated that expression of the constitutively active mutant form of the EGF receptor (EGFRvIII) in ovarian cancer cells led to reduction in integrin alpha2 surface expression, defects in cell spreading, and disruption of focal adhesions. Inhibition of EGFRvIII catalytic activity reversed the response, suggesting that EGF receptor activation regulates integrin alpha2. In this study we found that EGF treatment resulted in a transient loss of integrin alpha2 from the cell surface. Before EGF stimulation, integrin alpha2 and EGF receptors were associated based on biochemical and immuno-colocalization approaches. After EGF treatment, EGF receptor and integrin alpha2 were internalized and segregated into different compartments. Integrin alpha2, but not EGF receptor, was associated with caveolin-1 and GM1 (Gal&#95;1,3GalNAc&#95;1,4(Neu5Ac-&#95; 2,3)Gal&#95;1,4Glc&#95;1,1-ceramide) gangliosides, suggesting caveolae-mediated endocytosis. Moreover, integrin alpha2 was subsequently targeted to the Golgi apparatus and the endoplasmic reticulum. Together, these findings demonstrate that activated EGF receptor transiently modulates integrin alpha2 cell surface expression and stimulates integrin alpha2 trafficking via caveolae/raft-mediated endocytosis, representing a novel mechanism by which the EGF receptor may regulate integrin-mediated cell behavior.

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.

We describe a process for covalently linking proteins to glass microscope slides and microbeads in a manner that optimizes the reactivity of the immobilized proteins and that is suitable for high-throughput microarray and flow cytometry analysis. The method involves the diazo coupling of proteins onto activated self-assembled monolayers formed from p-aminophenyl trimethoxysilane. Proteins immobilized by this method maintained bioactivity and produced enhanced levels of protein-protein interaction, low background fluorescence, and high selectivity. The binding of immobilized proteins to their specific binding partner was analyzed quantitatively and successfully correlated with solution concentrations. Diazotized surfaces bound more efficiently to proteins containing a hexahistidine tag than those without a his-tag. Moreover, significantly higher reactivity of the immobilized his-tagged proteins was observed on diazotized surfaces than on amine-terminated surfaces. Results suggest that his-tagged proteins are immobilized by reaction of the his-tag with the diazotized surface, thus offering the possibility for preferential orientation of covalently bound proteins.