fluorescence-biomembrane-force-probe-concurrent-quantitation-receptor

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

The University of Sydney

MOTIVATION: Abrupt reduction/resumption of thermal fluctuations of a force probe has been used to identify association/dissociation events of protein-ligand bonds. We show that off-rate of molecular dissociation can be estimated by the analysis of the bond lifetime while the on-rate of molecular association can be estimated by the analysis of the waiting time between two neighboring bond events. However, the analysis relies heavily on subjective judgments and is time-consuming. To automate the process of mapping out bond events from thermal fluctuation data, we develop a hidden Markov model (HMM)-based method. RESULTS: The HMM method represents the bond state by a hidden variable with two values: bound and unbound. The bond association/dissociation is visualized and pinpointed. We apply the method to analyze a key receptor-ligand interaction in the early-stage of hemostasis and thrombosis: the von Willebrand factor (VWF) binding to platelet glycoprotein IbÎ± (GPIbÎ±). The numbers of bond lifetime and waiting time estimated by the HMM are much more than those estimated by a descriptive statistical method from the same set of raw data. The kinetic parameters estimated by the HMM are in excellent agreement with those by a descriptive statistical analysis, but have much smaller errors for both wild-type and two mutant VWF-A1 domains. Thus, the computerized analysis allows us to speed up the analysis and improve the quality of estimates of receptor-ligand binding kinetics.

An HMM-Based Algorithm for Evaluating Rates of Receptor-Ligand Binding Kinetics from Thermal Fluctuation Data.

MOTIVATION : Brusque r&eacute;duction/reprise des fluctuations thermiques d&#39;une sonde de force a &eacute;t&eacute; utilis&eacute; pour identifier des &eacute;v&eacute;nements d&#39;association/dissociation des liaisons prot&eacute;ine-ligand. Nous montrons que hors-taux de mol&eacute;culaire dissociation peut &ecirc;tre estim&eacute;e par l&#39;analyse de la dur&eacute;e de vie de liaison, tandis que la sur-taux de liaison mol&eacute;culaire peut &ecirc;tre estim&eacute;e par l&#39;analyse du temps d&#39;attente entre deux &eacute;v&eacute;nements de liaison de voisins. Cependant, l&#39;analyse s&#39;appuie fortement sur des jugements subjectifs et prend du temps. Pour automatiser le processus de mappage d&#39;&eacute;v&eacute;nements de liaison de donn&eacute;es de fluctuation thermique, nous d&eacute;veloppons un mod&egrave;le de Markov cach&eacute; (HMM)-m&eacute;thode de base. R&Eacute;SULTATS : La m&eacute;thode HMM repr&eacute;sente l&#39;&eacute;tat de la liaison par une variable masqu&eacute;e avec deux valeurs : li&eacute; et non consolid&eacute;. L&#39;association/dissociation de la liaison est visualis&eacute;e et mis en &eacute;vidence. Nous appliquons la m&eacute;thode pour analyser une interaction ligand-r&eacute;cepteur cl&eacute; dans le stade pr&eacute;coce de l&#39;h&eacute;mostase et la thrombose : la liaison de facteur de von Willebrand glycoprot&eacute;ine plaquettaire Ib&alpha; (GPIb&alpha;). Le nombre de liaisons dur&eacute;e de vie et les temps d&#39;attente estim&eacute; par le HMM est bien plus que celles estim&eacute;es par une m&eacute;thode statistique descriptive le m&ecirc;me ensemble de donn&eacute;es brutes. Les param&egrave;tres cin&eacute;tiques, estim&eacute;es par le HMM sont en excellent accord avec ceux qui, par une analyse statistique descriptive, mais sont beaucoup plus petites erreurs pour deux domaines de VWF-A1 mutants et sauvage. Ainsi, l&#39;analyse informatis&eacute; nous permet d&#39;acc&eacute;l&eacute;rer l&#39;analyse et d&#39;am&eacute;liorer la qualit&eacute; des estimations de la cin&eacute;tique de liaison r&eacute;cepteur-ligand.

Un algorithme bas&eacute; sur HMM pour &eacute;valuer les tarifs de liaison r&eacute;cepteur-Ligand cin&eacute;tique de Fluctuation thermique donn&eacute;es.

MOTIVAZIONE: Brusca riduzione/ripresa delle fluttuazioni termiche di una sonda di forza &egrave; stato utilizzato per identificare gli eventi di associazione/dissociazione dei legami proteina-ligando. Mostriamo che off-rate di molecolare dissociazione pu&ograve; essere stimata mediante l&#39;analisi della durata dell&#39;obbligazione, mentre il tasso di associazione molecolare pu&ograve; essere stimato mediante l&#39;analisi del tempo d&#39;attesa tra due vicini eventi bond. Tuttavia, l&#39;analisi si basa pesantemente su giudizi soggettivi e richiede molto tempo. Per automatizzare il processo di mappatura dei eventi bond dai dati di fluttuazione termica, sviluppiamo un modello di Markov nascosto (HMM)-metodo di base. RISULTATI: Il metodo HMM rappresenta lo stato di legame di una variabile nascosta con due valori: associati e non associati. L&#39;associazione/dissociazione di legame &egrave; visualizzata e individuata. Applichiamo il metodo per analizzare un&#39;interazione recettore-ligando chiave nella fase iniziale di emostasi e trombosi: l&#39;associazione von Willebrand factor (VWF) glicoproteina piastrinica Ib&alpha; (GPIb&alpha;). I numeri di bond durata e tempo di attesa stimato dall&#39;HMM sono molto di pi&ugrave; rispetto a quelli stimati da un metodo statistico descrittivo dallo stesso insieme di dati grezzi. I parametri cinetici stimati dall&#39;HMM sono in ottimo accordo con quelli di un&#39;analisi statistica descrittiva, ma sono molto pi&ugrave; piccoli errori per wild-type e mutanti VWF-A1 due domini. Cos&igrave;, l&#39;analisi computerizzata ci permette di velocizzare l&#39;analisi e migliorare la qualit&agrave; delle stime della cinetica di legame recettore-ligando.

Un algoritmo per la valutazione delle tariffe di cinetica del ricevitore-Ligand Binding da fluttuazione termica dati basato su HMM.

Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS(-) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS(-), megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.

Loss of the F-BAR protein CIP4 reduces platelet production by impairing membrane-cytoskeleton remodeling.

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln(1238)-Glu(1260) (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln(1238)-Glu(1260) peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln(1238)-Glu(1260) reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.

The N-terminal flanking region of the A1 domain regulates the force-dependent binding of von Willebrand factor to platelet glycoprotein Ibα.

Plexins and semaphorins comprise a large family of receptor-ligand pairs controlling cell guidance in nervous, immune, and vascular systems. How plexin regulation of neurite outgrowth, lymphoid trafficking, and vascular endothelial cell branching is linked to integrin function, central to most directed movement, remains unclear. Here we show that on developing thymocytes, plexinD1 controls surface topology of nanometer-scaled β1 integrin adhesion domains in cis, whereas its ligation by sema3E in trans regulates individual β1 integrin catch bonds. Loss of plexinD1 expression reduces β1 integrin clustering, thereby diminishing avidity, whereas sema3E ligation shortens individual integrin bond lifetimes under force to reduce stability. Consequently, both decreased expression of plexinD1 during developmental progression and a thymic medulla-emanating sema3E gradient enhance thymocyte movement toward the medulla, thus enforcing the orchestrated lymphoid trafficking required for effective immune repertoire selection. Our results demonstrate plexin-tunable molecular features of integrin adhesion with broad implications for many cellular processes.

Dynamic control of β1 integrin adhesion by the plexinD1-sema3E axis.

Cancer cell adhesion to the vascular endothelium is a critical step of tumour metastasis. Endothelial surface molecule Thy-1 (CD90) is implicated in the metastatic process through its interactions with integrins and syndecans. However, how Thy-1 supports cell-cell adhesion in a dynamic mechanical environment is not known. Here we show that Thy-1 supports β1 integrin- and syndecan-4 (Syn4)-mediated contractility-dependent mechanosignalling of melanoma cells. At the single-molecule level, Thy-1 is capable of independently binding α5β1 integrin and syndecan-4 (Syn4) receptors. However, in the presence of both α5β1 and Syn4, the two receptors bind cooperatively to Thy-1, to form a trimolecular complex. This trimolecular complex displays a unique phenomenon we coin 'dynamic catch', characterized by abrupt bond stiffening followed by the formation of catch bonds, where force prolongs the bond lifetime. Thus, we reveal a new class of trimolecular interactions where force strengthens the synergistic binding of two co-receptors and modulates downstream mechanosignalling.

Dynamic catch of a Thy-1-α5β1+syndecan-4 trimolecular complex.

The impact of flow disturbances on platelet adhesion is complex and incompletely understood. At the molecular scale, platelet glycoprotein Ibα (GPIbα) must associate with the von Willebrand factor A1 domain (VWF-A1) with a rapid on-rate under high hemodynamic forces, as occurs in arterial thrombosis, where various transport mechanisms are at work. Here, we theoretically modeled the coupled transport-reaction process of the two-dimensional (2D) receptor-ligand association kinetics in a biomembrane force probe to explicitly account for the effects of molecular length, confinement stiffness, medium viscosity, surface curvature, and separation distance. We experimentally verified the theoretical approach by visualizing association and dissociation of individual VWF-A1-GPIbα bonds in a real-time thermal fluctuation assay. The apparent on-rate, reciprocal of the average time intervals between sequential bonds, decreased with the increasing gap distance between A1- and GPIbα-bearing surfaces with an 80-nm threshold (beyond which bond formation became prohibitive) identified as the combined contour length of the receptor and ligand molecules. The biomembrane force probe spring constant and diffusivity of the protein-bearing beads also significantly influenced the apparent on-rate, in accordance with the proposed transport mechanisms. The global agreement between the experimental data and the model predictions supports the hypothesis that receptor-ligand association behaves distinctly in the transport- and reaction-limited scenarios. To our knowledge, our results represent the first detailed quantification of physical regulation of the 2D on-rate that allows platelets to sense and respond to local changes in their hemodynamic environment. In addition, they provide an approach for determining the intrinsic kinetic parameters that employs simultaneous experimental measurements and theoretical modeling of bond association in a single assay. The 2D intrinsic forward rate for VWF-A1-GPIbα association was determined from the measurements to be (3.5 ± 0.67) × 10(-4)μm(2) s(-1).

Transport regulation of two-dimensional receptor-ligand association.

Circulating von Willebrand factor (VWF) adopts a closed conformation that shields the platelet glycoprotein Ibα (GPIbα) binding site in the VWF-A1 domain. Immobilized at sites of vascular injury, VWF is activated by its interaction with collagen and the exertion of increased hemodynamic forces. Studies on native VWF strings and isolated A1 domains suggest the existence of multiple A1 binding states in different biophysical contexts. In this single-molecule study, we have used a biomembrane force probe (BFP) and a flow chamber to identify and characterize a collagen binding induced conformation with a higher affinity to platelet GPIbα. As force increases, our results show that collagen binding increases the stability of GPIbα bond with both VWF and isolated A1 domain. However, the collagen 2D binding affinity for VWF-A3 domain is 10 times of that for A1 domain, suggesting the initial VWF capture is mediated by A3-collagen interaction while A1-collagen regulates the subsequent VWF activation. Our results reveal the molecular mechanism of collagen-regulated, A1-mediated platelet adhesion enhancement. Characterization of different A1 states provides insights into binding heterogeneity of VWF in different scenarios of inflammation and thrombosis.

Article	Year
An HMM-Based Algorithm for Evaluating Rates of Receptor-Ligand Binding Kinetics from Thermal Fluctuation Data. Bioinformatics (Oxford, England)\| PubMed ID: 23599504	2013
Loss of the F-BAR protein CIP4 reduces platelet production by impairing membrane-cytoskeleton remodeling. Blood\| PubMed ID: 23881916	2013
The N-terminal flanking region of the A1 domain regulates the force-dependent binding of von Willebrand factor to platelet glycoprotein Ibα. The Journal of biological chemistry\| PubMed ID: 24062306	2013
Dynamic control of β1 integrin adhesion by the plexinD1-sema3E axis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24344262	2014
Dynamic catch of a Thy-1-α5β1+syndecan-4 trimolecular complex. Nature communications\| PubMed ID: 25216363	2014
Transport regulation of two-dimensional receptor-ligand association. Biophysical journal\| PubMed ID: 25863068	2015
Von Willebrand factor-A1 domain binds platelet glycoprotein Ibα in multiple states with distinctive force-dependent dissociation kinetics. Thrombosis research\| PubMed ID: 26213126	2015

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