Stephan Wilmes

Triple‐Color Super‐Resolution Imaging of Live Cells: Resolving Submicroscopic Receptor Organization in the Plasma Membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.

Self-Controlled Monofunctionalization of Quantum Dots for Multiplexed Protein Tracking in Live Cells†

Tracking the motion of individual proteins on the surface of live cells has contributed considerably towards unveiling the functional organization of proteins in the plasma membrane. Individual proteins labeled with quantum dots (QDs) can be imaged over long time periods with ultrahigh spatial and temporal resolution, yielding powerful information on the spatiotemporal dynamics of proteins at the plasma membrane in live cells. A key challenge for the application of QDs is to site-specifically attach proteins to the surface of these nanoparticles in a stoichiometric manner without affecting protein function. Several procedures for rendering surfaces of QDs biocompatible have been described, thus reducing non-specific binding and protein denaturation on the QD surface. However, functionalized biocompatible QDs used to target cell surface proteins generally result in multipoint attachment to the target proteins because the number of functional groups on the QDs is very difficult to control. Such multiply functionalized QDs induce clustering of target proteins on the cell surface, biasing not only lateral diffusion but also the functional properties of these proteins. As a consequence, increased endocytosis has been observed upon binding of QDs functionalized with multiple epidermal grow factor (EGF) molecules to cell-surface EGF receptors. Stochastic functionalization of multiple reactive sites on the QD offers the choice of obtaining only a minor fraction of the QDs with a single functional group, or a significant fraction of QDs with multiple functional groups. Preparation of homogeneous, monofunctional QDs currently relies on electrophoretic purification, which has been achieved only for very small QDs. These compact QDs are designed with very thin surface coatings, which have the disadvantage of showing relatively strong non-specific interactions. Most approaches for targeting proteins using QDs in live cells are based on biotin–streptavidin interactions, which form quasi-irreversible complexes. For multiplexed, generic labeling of proteins on the cell surface, further targeting strategies are required. We have recently described tris(hydroxymethyl)methylamine–nitrilotriacetic acid (Tris-NTA) moieties for highly specific and stable attachment of fluorophores and other functional units to histidine-tagged proteins in vitro and on the surface of live cells. The lifetime of Tris-NTA complexes with His-tagged proteins is in the order of several hours, which is well-suited to medium-term single molecule tracking applications. Herein, we have attempted to control the functionalization degree of QDs with Tris-NTA by means of electrostatic repulsion. We devised a bottom-up coupling chemistry based on a novel Tris-NTA derivative (1; Figure 1a), which comprises a thiol-terminated hepta(ethylene glycol) linker. This compound was generated in situ by reduction of the disulfide-linked dimer (1a ; see the Supporting Information, Scheme S1) and coupled to commercially available polymer-coated and amine-functionalized QDs by means of a hetero-bifunctional cross-linker (Figure 1b). Covalently attachment of 1 to surfaces modified with maleimide-functionalized polyethylene glycol (PEG) polymer brush and specific immobilization of His-tagged proteins was confirmed by label-free detection (Supporting Information, Figure S1). To control the degree of functionalization with Tris-NTA on the QD surface, the reaction of 1 with surface maleimide groups was performed at low ionic strength. Under these conditions, all QDs were reacted with Tris-NTA, as confirmed by an increase in negative charges detected by anion exchange chromatography and agarose gel electrophoreses (Figure 1c,d). These assays indicated relatively monodisperse electrostatic properties after coupling of 1, despite the fact that it was reacted at a large excess (660 mm of compound 1 to 1 mm QD). Coupling of 1 at higher ionic strength yielded QDs with a substantially higher degree of functionalization, as confirmed by a further shift of the signals both in anion exchange chromatography and agarose gel electrophoresis (Figure 1c,d). To characterize the functional properties of Tris-NTAcoupled QDs, binding to immobilized hexahistidine (H6) [*] Dr. C. You, S. Wilmes, O. Beutel, S. L chte, Y. Podoplelowa, F. Roder, C. Richter, T. Seine, D. Schaible, Prof. Dr. J. Piehler Division of Biophysics, Universit t Osnabr ck Barbarstrasse 11, 49076 Osnabr ck (Germany) Fax: (+49)541-969-2262 E-mail: piehler@uos.de Homepage: http://www.biologie.uni-osnabrueck.de/Biophysik/ Piehler/

Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling

Quantitative single-molecule receptor dimerization assays show dimerization of IFNAR1 and IFNAR2 upon IFN treatment, and reveal the limiting role of IFNAR1 binding affinity in complex assembly and the regulatory role of USP18.

STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK–STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.

Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency.

Mathematical modeling of the cellular responses to cytokine variants of differing binding affinities may help design better therapies. Modeling cytokine behavior The use of cytokines, such as interleukin-2 (IL-2) or IL-13, as therapies has been hampered by the fact that many cytokines share receptor subunits on different cell types. Moraga et al. generated recombinant variants of IL-13 with a wide range of binding affinities for the IL-13 receptor. Mathematical modeling of the correlation between the receptor binding affinities of the variants and the extents to which they differentially stimulated early and late cellular responses highlighted aspects of receptor-ligand binding properties that should aid in the development of more effective cytokine therapies. Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

Electrostatically controlled quantum dot monofunctionalization for interrogating the dynamics of protein complexes in living cells.

Quantum dots (QD) are powerful labels for probing diffusion and interaction dynamics of proteins on the single molecule level in living cells. Protein cross-linking due to multifunctional QD strongly affects these properties. This becomes particularly critical when labeling interaction partners with QDs for interrogating the dynamics of complexes. We have here implemented a generic method for QD monofunctionalization based on electrostatic repulsion of a highly negatively charged peptide carrier. On the basis of this method, monobiotinylated QDs were prepared with high yield as confirmed by single molecule assays. These QDs were successfully employed for probing the assembly and diffusion dynamics of binary and ternary cytokine-receptor complexes on the surface of living cells by dual color single QD tracking. Thus, sequential and dynamic recruitment of the type I interferon receptor subunits by the ligand could be observed.

Dynamic submicroscopic signaling zones revealed by pair correlation tracking and localization microscopy.

Unraveling the spatiotemporal organization of signaling complexes within the context of plasma membrane nanodomains has remained a highly challenging task. Here, we have applied super-resolution image correlation based on tracking and localization microscopy (TALM) for probing transient confinement as well as ligand binding and intracellular effector recruitment of the type I interferon (IFN) receptor in the plasma membrane of live cells. Ligand and receptor were labeled with monofunctional quantum dots, thus allowing long-term tracking with very high spatial and temporal resolution without an artificial receptor cross-linking at the cell surface. Dual-color TALM was employed for visualizing protein-protein interactions involved in IFN signaling at both sides of the plasma membrane with high spatial and temporal resolution. By pair correlation analyses based on time-lapse TALM images (pcTALM), complex assembly within dynamic submicroscopic zones was identified. Strikingly, recruitment of the IFN effector protein signal transducer and activator of transcription 2 (STAT2) into these dynamic signaling zones could be observed. The results suggest that confined diffusion zones in the plasma membrane are employed as transient platforms for the assembly of signaling complexes.

Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.

Rapid Transfer of Transmembrane Proteins for Single Molecule Dimerization Assays in Polymer-Supported Membranes

Dimerization of transmembrane receptors is a key regulatory factor in cellular communication, which has remained challenging to study under well-defined conditions in vitro. We developed a novel strategy to explore membrane protein interactions in a controlled lipid environment requiring minute sample quantities. By rapid transfer of transmembrane proteins from mammalian cells into polymer-supported membranes, membrane proteins could be efficiently fluorescence labeled and reconstituted with very low background. Thus, differential ligand-induced dimerization of the type I interferon (IFN) receptor subunits IFNAR1 and IFNAR2 could be probed quantitatively at physiologically relevant concentrations by single molecule imaging. These measurements clearly support a regulatory role of the affinity of IFNs toward IFNAR1 for controlling the level of receptor dimerization.

Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

Single-molecule tracking and spatial stochastic modeling reveal receptor dimer stabilization by nanoscale confinement zones. The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.