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C Wofsy

Publications and source records attributed to C Wofsy.

At least 19 recordsLinked to original sources
PubMed2001

Kinetic proofreading models for cell signaling predict ways to escape kinetic proofreading.

In the context of cell signaling, kinetic proofreading was introduced to explain how cells can discriminate among ligands based on a kinetic parameter, the ligand-receptor dissociation rate constant. In the kinetic proofreading model of cell signaling, responses occur only when a bound receptor undergoes a complete series of modifications. If the ligand dissociates prematurely, the receptor returns to its basal state and signaling is frustrated. We extend the model to deal with systems where aggregation of receptors is essential to signal transduction, and present a version of the model for systems where signaling depends on an extrinsic kinase. We also investigate the kinetics of signaling molecules, "messengers," that are generated by aggregated receptors but do not remain associated with the receptor complex. We show that the extended model predicts modes of signaling that exhibit kinetic discrimination for some range of parameters but for other parameter values show little or no discrimination and thus escape kinetic proofreading. We compare model predictions with experimental data.

Animals↗
PubMed2001

Calculations show substantial serial engagement of T cell receptors.

The serial engagement model provides an attractive and plausible explanation for how a typical antigen presenting cell, exhibiting a low density of peptides recognized by a T cell, can initiate T cell responses. If a single peptide displayed by a major histocompatibility complex (MHC) can bind, sequentially, to different T cell receptors (TCR), then a few peptides can activate many receptors. To date, arguments supporting and questioning the prevalence of serial engagement have centered on the down-regulation of TCR after contact of T cells with antigen presenting cells. Recently, the existence of serial engagement has been challenged by the demonstration that engagement of TCR can down-regulate nonengaged bystander TCR. Here we show that for binding and dissociation rates that characterize interactions between T cell receptors and peptide-MHC, substantial serial engagement occurs. The result is independent of mechanisms and measurements of receptor down-regulation. The conclusion that single peptide-MHC engage many TCR, before diffusing out of the contact region between the antigen-presenting cell and the T cell, is based on a general first passage time calculation for a particle alternating between states in which different diffusion coefficients govern its transport.

Antigen-Presenting Cells↗
PubMed2001

Expression and modulation of FcepsilonRIalpha and FcepsilonRIbeta in human blood basophils.

BACKGROUND: The IgE receptor (FcepsilonRI) may exist as a tetramer (alphabetagamma2) or a trimer (alphagamma2) because FcepsilonRIbeta is dispensable for membrane expression of FcepsilonRIalpha. FcepsilonRIbeta amplifies signaling of FcepsilonRI so that regulation of FcepsilonRIalpha:beta stoichiometry would affect cellular responsiveness. OBJECTIVE: We examined basophils from a variety of donors for differences in their expression of FcepsilonRIalpha and FcepsilonRIbeta protein. METHODS: Enriched blood basophils were assessed at baseline and after IL-3 culture for FcepsilonRIalpha and FcepsilonRIbeta protein by Western blotting, surface FcepsilonRIalpha by flow cytometry, and FcepsilonRIbeta mRNA by real-time PCR. Basophil functional response was measured by allergen-triggered histamine release. RESULTS: For the FcepsilonRIalpha subunit, 2 protein bands with molecular weights of 50 kd and 60 kd were identified by Western blots. The 60-kd band correlated to surface-expressed FcepsilonRIalpha detected by flow cytometry (Spearman R = 0.78, P <.01). Surface FcepsilonRIalpha also correlated with FcepsilonRIbeta protein (Spearman R = 0.92, P <.01). FcepsilonRIbeta protein levels increased disproportionately with higher surface FcepsilonRIalpha expression. The ratio of FcepsilonRIbeta to FcepsilonRIalpha varied 10-fold among donors and correlated with surface FcepsilonRIalpha. Basophil 50-kd alpha protein levels were similar despite a 10-fold range in surface FcepsilonRIalpha expression, implying stores of this protein such as those found in eosinophils. Unlike eosinophils, the basophil 50-kd protein was lost with culture and was absent from supernatants. Levels of beta protein and mRNA were enhanced by IL-3 culture, whereas FcepsilonRIalpha expression (by flow cytometry and 60 kd) was not. CONCLUSION: These findings demonstrate variable stoichiometry of FcepsilonRIalpha:beta in whole cells and that this stoichiometry can be altered by IL-3 culture. With the assumption that all detected beta protein is surface expressed, these findings suggest a variable stoichiometry for FcepsilonRIalpha:beta that is also related to FcepsilonRIalpha surface expression.

Adult↗
PubMed1999

Dissociation of HIV-1 from follicular dendritic cells during HAART: mathematical analysis.

Follicular dendritic cells (FDC) provide a reservoir for HIV type 1 (HIV-1) that may reignite infection if highly active antiretroviral therapy (HAART) is withdrawn before virus on FDC is cleared. To estimate the treatment time required to eliminate HIV-1 on FDC, we develop deterministic and stochastic models for the reversible binding of HIV-1 to FDC via ligand-receptor interactions and examine the consequences of reducing the virus available for binding to FDC. Analysis of these models shows that the rate at which HIV-1 dissociates from FDC during HAART is biphasic, with an initial period of rapid decay followed by a period of slower exponential decay. The speed of the slower second stage of dissociation and the treatment time required to eradicate the FDC reservoir of HIV-1 are insensitive to the number of virions bound and their degree of attachment to FDC before treatment. In contrast, the expected time required for dissociation of an individual virion from FDC varies sensitively with the number of ligands attached to the virion that are available to interact with receptors on FDC. Although most virions may dissociate from FDC on the time scale of days to weeks, virions coupled to a higher-than-average number of ligands may persist on FDC for years. This result suggests that HAART may not be able to clear all HIV-1 trapped on FDC and that, even if clearance is possible, years of treatment will be required.

Anti-HIV Agents↗
PubMed1999

One lyn molecule is sufficient to initiate phosphorylation of aggregated high-affinity IgE receptors.

In response to antigenic stimuli, the multisubunit immune recognition receptors become aggregated and then phosphorylated on their cytoplasmic tyrosines. For the clonotypic receptors of B and T cells and for Fc receptors such as the high-affinity receptor for IgE (FcepsilonRI), a Src family kinase initiates this phosphorylation. We ask whether aggregation of the initiating kinase itself is required for signal transduction or whether, alternatively, a single associated kinase molecule can phosphorylate the receptors in an aggregate. We formulate the alternative molecular mechanisms mathematically and compare predictions with experimental findings on FcepsilonRI-bearing cells expressing varying amounts of the transfected Src family kinase Lyn. The data are consistent with the requirement of only a single Lyn molecule per FcepsilonRI aggregate to initiate signaling and are inconsistent with a mechanism requiring more than one Lyn molecule.

Animals↗
PubMed1999

A quantitative approach to signal transduction.

The high affinity receptor for IgE (FcepsilonRI), is one of a family of immunoreceptors whose antigen-induced clustering leads to a variety of cellular responses. The signaling pathways are enormously complex but by focusing on only the most initial steps, it is now possible to sketch plausible molecular models that relate the interaction of multivalent antigens with the receptor-bound IgE to the earliest cellular events. In this paper, we describe how we have combined quantitative experimentation and mathematical modeling to probe this system further. We also discuss some of the formidable challenges that remain before we can claim reasonably complete understanding of even these early events.

Animals↗
PubMed1999

Analysis of vertical fluorescence resonance energy transfer from the surface of a small-diameter sphere.

Fluorescence resonance energy transfer (FRET) measurements have been used to analyze fluorophore separations in a number of varying geometries, including small particles and extended surfaces. This study focuses on the geometry created by a donor extended above the surface of a small sphere (radius < R0), where the acceptors are integrated into the sphere surface. The model of this geometry was based on an amphipathic molecule with its lipophilic region integrated into a detergent micelle and its hydrophilic region extending outward from the micelle surface, where the donor fluorophore is attached to the hydrophilic region of the molecule. Based on random acceptor incorporation into the micelle, a Poisson distribution was used to calculate the distribution of acceptor probes across the micelle population. The model converges to RET on a flat surface when the radius of the micelle exceeds 0.8 R0. The model was also used to simulate FRET data showing that the positions of donors above the micelle surface could be uniquely resolved. Experimental verification of the model was achieved in a sulfobetaine palmitate micelle with fluorescein isothiocyanate donors attached to detergent-solubilized lipopolysaccharide (LPS) and lipophilic Fast-DiI acceptors. The use of steady-state analysis allowed resolution of cases in which donors were located at different distances from the surface. Combining steady-state with excited-state lifetime analysis allowed resolution of cases where there was a combination of distances. Given the large number of biomolecules that interact with lipids, this approach may prove generally useful for defining molecular conformation.

Biophysical Phenomena↗
PubMed1999

Effective rate models for the analysis of transport-dependent biosensor data.

Optical biosensors, including the BIACORE, provide an increasingly popular method for determining reaction rates of biomolecules. In a flow chamber, with one reactant immobilized on a chip on the sensor surface, a solution containing the other reactant (the analyte) flows through the chamber. The time course of binding of the reactants is monitored. Scientists using the BIACORE to understand biomolecular reactions need to be able to separate intrinsic reaction rates from the effects of transport in the biosensor. For a model to provide a useful basis for such an analysis, it must reflect transport accurately, while remaining simple enough to couple with a routine for estimating reaction rates from BIACORE data. Models have been proposed previously for this purpose, consisting of an ordinary differential equation with 'effective rate coefficients' incorporating reaction and transport parameters. In this paper we investigate both the theoretical basis and numerical accuracy of these and related models.

Biological Transport↗
PubMed1997

Exploiting the difference between intrinsic and extrinsic kinases: implications for regulation of signaling by immunoreceptors.

When receptors must interact with an extrinsic kinase to initiate signaling, the kinase can play a regulatory role that is not available to intrinsic receptor kinases. Whether control is exercised at this level depends critically on the amount of kinase available to the receptors and on the potential for redistribution of the kinase during signaling. This study demonstrates that the high affinity receptor for IgE (Fc epsilonRI) on rat basophilic leukemia cells is regulated by its initiating kinase. We present a mathematical model that allows for the reversible recruitment of extrinsic kinases to phosphorylated immunoreceptor tyrosine-based activation motifs. By comparing model predictions to experimental time courses of phosphorylation, we infer that Lyn is limiting, that redistribution occurs after receptors are aggregated, and that the redistribution makes the relationship between tyrosine phosphorylation and receptor aggregation nonlinear.

Animals↗
PubMed1997

Shuttling of initiating kinase between discrete aggregates of the high affinity receptor for IgE regulates the cellular response.

Using defined oligomers of IgE, our group previously studied the quantitative relationship between the aggregation of the high affinity receptors for IgE (Fc epsilonRI) and the earliest signals initiated by such aggregation: the phosphorylation of tyrosines on the receptor. Notably, at certain doses of the oligomers such phosphorylation reached a plateau level well before the aggregation of the receptors had reached a maximum. These findings and others led us to propose that aggregates of the receptor were competing for a limited amount of the critical kinase-thought to be Lyn in this system. This paper describes a test of this proposal. We incubated cells with two distinguishable IgEs and examined the effect of aggregating one or the other or both types on the phosphorylation. When receptors binding antigen-specific IgE were aggregated with polyvalent antigen, they became rapidly phosphorylated as expected. Remarkably, however, Fc epsilonRI that had already been phosphorylated by the binding of dimers of IgE, became dephosphorylated simultaneously. Furthermore, when the antigen-driven aggregates were dissociated with hapten, the phosphorylation pattern reverted to that seen prior to the addition of antigen: as the antigen-driven aggregates became dephosphorylated, the receptors stably aggregated by the bound oligomers became rapidly rephosphorylated. Dephosphorylation of oligomer-driven aggregates was also partially reversed during the "spontaneous" dephosphorylation of the antigen-driven receptors seen at longer times after addition of antigen. Thus signal transduction in this system is in part regulated by the shuttling of limited amounts of the kinase that initiates the cascade of phosphorylations.

Animals↗
PubMed1996

Why is it so hard to dissociate multivalent antigens from cell-surface antibodies?

It is often difficult to induce dissociation of multivalent antigens that are bound to cells by surface-associated immunoglobulin (sIg), even when high concentrations of monovalent hapten are used to fill all free binding sites. Here, Byron Goldstein and Carla Wofsy discuss the evidence that this appears to be due to a cellular response triggered by sIg crosslinking and propose that a receptor-desensitization process is responsible for the transition of multivalent antigens to a dissociation-resistant state.

Animals↗
PubMed1995

Kinetics of tyrosine phosphorylation when IgE dimers bind to FC epsilon receptors on rat basophilic leukemia cells.

Previously, we demonstrated that aggregates of the high affinity receptor for IgE (Fc epsilon RI), formed by the binding of chemically cross-linked oligomers of IgE, continue to signal early and late cellular responses long after the formation of new aggregates is blocked. In the present work, we explore quantitatively the relationship between aggregation of the receptors and one of the earliest biochemical changes this initiates. We compare the time course of aggregate formation, inferred from studies of the binding of dimers of IgE, and the time course of phosphorylation of tyrosines on receptor subunits when the receptors are aggregated. A simple model does not fit the data. It appears that aggregates formed late in the response are less effective signaling units than those formed initially. We propose new explanations for the persistence of the response and the unusual kinetics.

Animals↗
PubMed1995

The kinetics of bivalent ligand-bivalent receptor aggregation: ring formation and the breakdown of the equivalent site approximation.

When bivalent ligands capable of bridging binding sites on two different receptors interact with bivalent receptors, aggregates form. The aggregates can be of two types: chains (open structures containing n receptors, n-1 doubly bound ligands and 0, 1, or 2 singly bound ligands) and rings (closed structures containing n receptors and n doubly bound ligands). Both types of aggregates have been detected experimentally. In general, to determine the time dependence of the concentration of any particular aggregate requires solving an infinite set of coupled ordinary differential equations (ODEs). Perelson and DeLisi [19] showed that great simplification results if all receptor binding sites are equivalent, i.e., the binding properties of a site on a receptor are independent of the size of the aggregate the receptor is in. If only chains form, the problem reduces to solving two coupled ODEs for the concentrations of singly and doubly bound ligands. From the solutions to these ODEs, the time dependence of the entire aggregate size distribution can be determined. We show that the equivalent site approximation as formulated by Perelson and DeLisi [19] is incompatible with ring formation. We then present a modified equivalent site approximation that is useful if chains of any size can form but rings above a certain size (k) cannot. We show how to reduce the resulting infinite set of coupled ODEs to a closed system of at most 4k + 2 ODEs for the ligand concentrations, the ring concentrations, and the concentrations of all chains up to size k. Although we can only predict the kinetics of aggregate formation for aggregates of size k or less, at equilibrium the modified equivalent site approximation yields the complete aggregate size distribution.

Animals↗
PubMed1994

Dynamics of signal transduction after aggregation of cell-surface receptors: studies on the type I receptor for IgE.

Many ligands stimulate cellular responses by aggregating the cell-surface receptors to which they are bound. We investigated several mechanistic questions related to aggregation of receptors by using the high-affinity receptor for IgE (Fc epsilon RI) on mast cells as a model system. We briefly exposed cells to covalently cross-linked oligomers of IgE and then added excess monomeric IgE to prevent further aggregation. Early events were examined by monitoring the phosphorylation of protein tyrosines; later events were examined by monitoring secretion. We found that aggregated receptors continue to signal both late and early events in the absence of formation of new aggregates. Additional experiments suggested that the clustered receptors undergo a dynamic process of phosphorylation and dephosphorylation. Our findings suggest that for these and related receptors that function by aggregation, the persistence of signal transduction is directly related to the intrinsic affinity of the ligand for the individual receptor.

Animals↗
PubMed1992

Interpretation of Scatchard plots for aggregating receptor systems.

Aggregation of cell surface receptors, with each other or with other membrane proteins, occurs in a variety of experimental systems. The list of systems where receptor aggregation appears to be important in understanding ligand binding and cellular responses is growing rapidly. In this paper we explore the interpretation of equilibrium binding data for aggregating receptor systems. The Scatchard plot is a widely used tool for analyzing equilibrium binding data. The shape of the Scatchard plot is often interpreted in terms of multiple noninteracting receptor populations. Such an analysis does not provide a framework for investigating the role of receptor aggregation and will be misleading if there is a relation between receptor aggregation and ligand binding. We present a general model for the equilibrium binding of a ligand with any number of aggregating receptor populations and derive theoretical expressions for observable Scatchard plot features. These can be used to test particular models and estimate model parameters. We develop particular models and apply the general results in the cases of six aggregating receptor systems where ligand binding and receptor aggregation are related: cross-linking of monovalent cell surface proteins by monoclonal antibodies, cross-linking of cell surface antibodies by bivalent ligand, antibody-induced co-cross-linking of cell surface antibodies and Fc gamma receptors, ligand-enhanced aggregation of identical epidermal growth factor receptors, aggregation of heterologous receptors for interleukin 2 to form a high-affinity receptor, and association of receptors, including those for interleukins 5 and 6, with nonbinding accessory proteins that influence receptor affinity or effector function.

Animals↗
PubMed1992

Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are also important in shaping the binding curves. Because we have controlled experimentally for many sources of receptor heterogeneity, we have limited the potential explanations for residual Scatchard plot curvature.

Animals↗
PubMed1991

Relationship of IgE receptor topography to secretion in RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking IgE-receptor complexes with anti-IgE antibody leads to degranulation. Receptor cross-linking also stimulates the redistribution of receptors on the cell surface, a process observed here by labeling the anti-IgE with 15 nm protein A-gold particles that are visible by back-scattered electron imaging in the scanning electron microscope. We report that anti-IgE binding stimulates the redistribution of IgE-receptor complexes at 37 degrees C from a dispersed topography to distributions dominated sequentially by short chains, small clusters, and large aggregates of cross-linked receptors. Cells incubated with 1 microgram/ml anti-IgE, a concentration that stimulates maximum net secretion, redistribute receptors into chains and small clusters during a 15 min incubation period. At 3 and 10 micrograms/ml anti-IgE, net secretion is reduced and the majority of receptors redistribute rapidly into clusters and large aggregates. The addition of Fab fragments with the high anti-IgE concentrations, to reduce cross-linking, delays receptor aggregation and enhances secretion. The progression of receptors from small clusters to large aggregates is prevented in cells treated with dihydrocytochalasin B to prevent F-actin assembly. These results establish that characteristic patterns of receptor topography are correlated with receptor activity. In particular, they link the formation of large receptor aggregates to reduced signalling activity. Cytoskeleton-membrane interaction is implicated in the formation or stabilization of the large receptor clusters.

Actins↗