measuring-tcr-pmhc-binding-in-situ-using-a-fret-based-microscopy-assay

Measuring TCR-pMHC Binding <em>In Situ</em> using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

single-molecule-fluorescence-microscopy-on-planar-supported-bilayers

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

enrichment-native-lipoprotein-particles-with-microrna-subsequent

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

T cell activation is determined by the number of presented antigens.

Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in <1 min. Strikingly, active Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species.

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.

Localization Microscopy of Actin Cytoskeleton in Human Platelets.

microRNAs (miRNAs) are post-transcriptional regulators of messenger RNA (mRNA), and transported through the whole organism by-but not limited to-lipoprotein particles. Here, we address the miRNA profile in serum and lipoprotein particles of healthy individuals in comparison with patients with uremia. Moreover, we quantitatively determined the cellular lipoprotein-particle-uptake dependence on the density of lipoprotein particle receptors and present a method for enhancement of the transfer efficiency. We observed a significant increase of the cellular miRNA level using reconstituted high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yielded no measurable effect. Thus, we conclude that no relevant effect of lipoprotein-particle-mediated miRNA-transfer exists under in vivo conditions though the miRNA profile of lipoprotein particles can be used as a diagnostic marker.

Serum and Lipoprotein Particle miRNA Profile in Uremia Patients.

The determination of pharmacologically relevant constants is crucial in order to understand the effects of compounds interacting with various membrane receptors. In this report we study a venom component of the Central American scorpion Centruroides limbatus, a short peptide termed hongotoxin(1) (HgTX(1)), which specifically binds to the voltage-gated potassium channel K(V)1.3 at a molecular stoichiometry of 1:1. A toxin analogue (HgTX(1)-A19C) was subjected to fluorescence labelling studies with Cy5. Utilising an ultrasensitive microscopic method (single-dye tracing; SDT) we were able to directly visualise HgTX(1)-A19C-Cy5 binding to the voltage-gated potassium channel K(V)1.3 on Jurkat cells at the single molecule level. For the first time, this approach allowed the determination of both the dissociation constant (K(D)) and the off-rate (k(off)) of HgTX(1)-A19C-Cy5 on living cells. In order to validate this novel approach, the data obtained with SDT were correlated to radioligand binding studies performed under identical conditions using a radioiodinated HgTX(1) analogue.

Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy.

Ultrasensitive pharmakologische Charakterisierung des spannungsgesteuerten Kaliumkanal K (V) 1.3 von Einzelmolek&#xFC;l-Fluoreszenz-Mikroskopie untersucht.

Trafficking of secretory vesicles along neurites of PC12 cells was visualized by 2D and 3D real-time imaging using fluorescence microscopy. Vesicle motion along distinct pathways was directly seen. From an overlay of individual pathways, the underlying cytoskeletal filament could be imaged at a subwavelength resolution. Continuous vesicle transport was interrupted by periods of diffusive motion with concomitant pathway changes. Statistical analysis shows that such interruptions were distributed stochastically along the filament, indicating a limited processivity of motor proteins also in a cellular context. Periods of diffusive motion facilitated the interaction with actively transported vesicles. Frequent associations and dissociations of vesicles have been observed consistently, pointing to a functional relevance of vesicle cotransport.

Visualization of vesicle transport along and between distinct pathways in neurites of living cells.

Visualisierung von Vesikeltransport entlang und zwischen verschiedenen Pfaden in Neuriten von lebenden Zellen.

We propose here an approach for the analysis of single-molecule trajectories which is based on a comprehensive comparison of an experimental data set with multiple Monte Carlo simulations of the diffusion process. It allows quantitative data analysis, particularly whenever analytical treatment of a model is infeasible. Simulations are performed on a discrete parameter space and compared with the experimental results by a nonparametric statistical test. The method provides a matrix of p-values that assess the probability for having observed the experimental data at each setting of the model parameters. We show the testing approach for three typical situations observed in the cellular plasma membrane: i), free Brownian motion of the tracer, ii), hop diffusion of the tracer in a periodic meshwork of squares, and iii), transient binding of the tracer to slowly diffusing structures. By plotting the p-value as a function of the model parameters, one can easily identify the most consistent parameter settings but also recover mutual dependencies and ambiguities which are difficult to determine by standard fitting routines. Finally, we used the test to reanalyze previous data obtained on the diffusion of the glycosylphosphatidylinositol-protein CD59 in the plasma membrane of the human T24 cell line.

Versatile analysis of single-molecule tracking data by comprehensive testing against Monte Carlo simulations.

Wir bieten hier einen Ansatz f&uuml;r die Analyse der Einzelmolek&uuml;l-Trajektorien basiert auf einen umfassenden Vergleich eines experimentellen Daten-Sets mit mehreren Monte Carlo-Simulationen von den Diffusionsprozess. Es erm&ouml;glicht quantitative Datenanalyse, besonders wenn der analytischen Behandlung eines Modells nicht durchf&uuml;hrbar ist. Simulationen sind auf eine diskrete Parameterraum durchgef&uuml;hrt und im Vergleich mit den experimentellen Ergebnissen durch eine nichtparametrische statistische Test. Die Methode stellt eine Matrix p-Werte, die die Wahrscheinlichkeit f&uuml;r die experimentellen Daten auf jede Einstellung die Modellparameter beobachtet haben beurteilen. Zeigen wir den Testansatz f&uuml;r drei typische Situationen in der Plasma-Zellmembran beobachtet: ich), kostenlose Brownsche Bewegung des Tracers, Ii), hop Verbreitung des Tracers im eine periodische Netzwerk von Quadraten und Iii), vor&uuml;bergehende Bindung des Tracers zu langsam Verbreitung Strukturen. Durch Auftragen den p-Wert als Funktion der die Modellparameter, kann man leicht identifizieren die konsistenten Parametereinstellungen aber auch erholen gegenseitigen Abh&auml;ngigkeiten und Mehrdeutigkeiten, die schwierig zu bestimmen, von Standardausstattung-Routinen sind. Schlie&szlig;lich haben wir den Test um zur&uuml;ck erhaltenen Daten &uuml;ber die Verbreitung der CD59 Glykosylphosphatidyl-Protein in der Plasmamembran von der menschlichen T24-Zelllinie zu diagnostizieren.

Vielseitige Analyse der Einzelmolek&uuml;l-Tracking-Daten durch umfassende Tests gegen Monte-Carlo-Simulationen.

The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide-major histocompatibility complex (pMHC) molecules on other cells. If productive, these interactions promote the formation of an immunological synapse. Here we show that synaptic TCR-pMHC binding dynamics differ significantly from TCR-pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCR-pMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4-12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR-pMHC complexes, indicating that the TCR binds pMHC independently of CD4.

TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.

Die Anerkennung der fremde Antigene von T-Lymphozyten ist wichtig, die adaptive Immunantworten. Es wird durch spezifische T-Zell-Antigenrezeptoren (TCRs) binden an Antigene Peptid-Major Histocompatibility complex (pMHC) Molek&uuml;le auf andere Zellen angetrieben. Wenn produktiv, f&ouml;rdern diese Interaktionen die Bildung von eine immunologische Synapse. Hier zeigen wir, dass synaptische TCR-pMHC Bindung Dynamik von TCR-pMHC Bindung in L&ouml;sung deutlich unterscheiden. Wir verwendeten Einzelmolek&uuml;l-Mikroskopie und Fluoreszenz Resonanz Energietransfer (FRET) zwischen Leuchtstoff markierte TCRs und ihrer Verwandten pMHC-Liganden, um die Kinetik der TCR-pMHC Bindung an Ort und Stelle zu messen. Im Vergleich zu L&ouml;sung Messungen der Dissoziation dieses Komplexes deutlich erh&ouml;ht (4-12-fach). Unterbrechung der Aktin-Polymere umgekehrt dieser Effekt, der darauf hinweist, dass Zytoskelett Dynamik dieser Interaktion direkt oder indirekt zu destabilisieren. Dennoch war TCR Affinit&auml;t f&uuml;r pMHC deutlich erh&ouml;ht, wie das Ergebnis einer gro&szlig;en (ca. 100-fold) zu erh&ouml;hen, in der Vereinigung Rate, eine wahrscheinlich Folge der komplement&auml;re Molekulare Orientierung und clustering. In T-Helferzellen schl&auml;gt das CD4-Molek&uuml;l, kooperativ mit TCR auf der gleichen pMHC Komplex zu binden. Jedoch CD4-Blockade hatte keine Auswirkung auf die synaptische TCR-Affinit&auml;t, noch hat es destabilisieren TCR-pMHC-komplexe, die darauf hinweist, dass TCR pMHC unabh&auml;ngig von der CD4 bindet.

TCR-Peptid-MHC-Interaktion in-situ zeigen beschleunigte Kinetik und erh&ouml;hte Affinit&auml;t.

Diffusing membrane constituents are constantly exposed to a variety of forces that influence their stochastic path. Single molecule experiments allow for resolving trajectories at extremely high spatial and temporal accuracy, thereby offering insights into en route interactions of the tracer. In this review we discuss approaches to derive information about the underlying processes, based on single molecule tracking experiments. In particular, we focus on a new versatile way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on comprehensive comparison of an experimental data set against the hypothetical outcome of multiple experiments performed on the computer. Since Monte Carlo simulations can be easily and rapidly performed even on state-of-the-art PCs, our method provides a simple way for testing various - even complicated - diffusion models. We describe the new method in detail, and show the applicability on two specific examples: firstly, kinetic rate constants can be derived for the transient interaction of mobile membrane proteins; secondly, residence time and corral size can be extracted for confined diffusion.

What can we learn from single molecule trajectories?

Diffundierenden Membrane Bestandteile sind st&auml;ndig eine Vielzahl von Kr&auml;ften ausgesetzt, die ihre stochastische Pfad zu beeinflussen. Einzelmolek&uuml;l-Experimente erm&ouml;glichen zum Aufl&ouml;sen von Flugbahnen bei extrem hoher r&auml;umlicher und zeitlicher Genauigkeit, und bietet Einblicke in unterwegs Interaktionen des Tracers. In diesen Bericht diskutieren wir Ans&auml;tze, um Informationen &uuml;ber die zugrunde liegenden Prozesse, basierend auf Einzelmolek&uuml;l Verfolgung Experimenten abgeleitet werden. Insbesondere konzentrieren wir uns auf eine neue vielseitige M&ouml;glichkeit, Einzelmolek&uuml;l-Verbreitung in Ermangelung einer vollst&auml;ndigen analytischen Behandlung zu analysieren. Die Methode basiert auf umfassenden Vergleich eines experimentellen Daten-Sets gegen das hypothetische Ergebnis mehrere Experimente, die auf dem Computer ausgef&uuml;hrt. Da Monte-Carlo-Simulationen schnell und einfach auch auf Zustand-of-the-Art-PCs durchgef&uuml;hrt werden k&ouml;nnen k&ouml;nnen, bietet unsere Methode eine einfache M&ouml;glichkeit, verschiedene - auch kompliziert - Verbreitung-Modelle zu Testzwecken. Wir beschreiben die neue Methode im Detail und zeigen die Anwendbarkeit auf zwei konkrete Beispiele: Erstens kinetischen Geschwindigkeitskonstanten abgeleitet werden k&ouml;nnen, f&uuml;r die vor&uuml;bergehende Interaktion von mobilen Membranproteinen; Zweitens kann Residenz-Zeit und Corral-Gr&ouml;&szlig;e f&uuml;r begrenzten Diffusion extrahiert werden.

Was k&ouml;nnen wir von Einzelmolek&uuml;l-Trajektorien lernen?

The binding of peptide-loaded major histocompatibility complex (pMHC) to the T cell receptor (TCR) represents the central step in T cell antigen recognition. It proceeds in the cell contact area between a T cell and an antigen-presenting cell termed the immunological synapse. An important and unresolved issue is how T cells discriminate between potentially harmful and harmless antigens. One limitation has been the difficulty to measure interaction parameters directly, that is, as they occur in the immunological synapse. Here we present a single-molecule approach to determine pMHC-TCR interaction kinetics in situ based on diffusion analysis of dye-labeled pMHC. We find synaptic off-rates >10-fold accelerated when compared to the dissociation of purified proteins measured in vitro.

ArticleTotal : 3	Year
Publication title Cited by 9	2015
Publication title Cited by 16	2015
Publication title Cited by 10	2019

Article	Year
T cell activation is determined by the number of presented antigens. Nano letters\| PubMed ID: 24117051	2013
Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nature communications\| PubMed ID: 26271728	2015
Localization Microscopy of Actin Cytoskeleton in Human Platelets. International journal of molecular sciences\| PubMed ID: 29641438	2018
Serum and Lipoprotein Particle miRNA Profile in Uremia Patients. Genes\| PubMed ID: 30400676	2018
Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy. Histochemistry and cell biology\| PubMed ID: 11914916	2002
Visualization of vesicle transport along and between distinct pathways in neurites of living cells. Microscopy research and technique\| PubMed ID: 14755603	2004
Versatile analysis of single-molecule tracking data by comprehensive testing against Monte Carlo simulations. Biophysical journal\| PubMed ID: 18805933	2008
TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity. Nature\| PubMed ID: 20164930	2010
What can we learn from single molecule trajectories? Current protein & peptide science\| PubMed ID: 22044145	2011
Determination of interaction kinetics between the T cell receptor and peptide-loaded MHC class II via single-molecule diffusion measurements. Biophysical journal\| PubMed ID: 22853916	2012

Markus Axmann

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