Measuring Diffusion and Binding Kinetics by Contact Area FRAP

Tolentino, Timothy; Wu, Jianhua; Zarnitsyna, Veronika I.; Fang, Ying; Dustin, Michael L.; Zhu, Cheng

doi:10.1529/biophysj.107.114447

Cited by 79 publications

(85 citation statements)

References 38 publications

(67 reference statements)

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“…Photobleaching measurements showed that CD4/pMHC II binding is reversible, and that the mobility of CD4 in the contact is more than 10-fold lower compared with outside the contact. Although the 2D off-rate (k off ) for the CD4/pMHC II interaction could not be determined in the present experiments, it can be estimated to be of the order of 250 s , which is comparable to that measured for protein-protein interactions of higher affinity between T cells and APCs (13,29). However, the k off is orders of magnitude larger (12,13,29).…”

Section: Discussionmentioning

confidence: 42%

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

Jönsson

Southcombe

Santos

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4-and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D K d of ∼5,000 molecules/μm 2 . This value is two to three orders of magnitude higher than previously measured 2D K d values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination. T cells with αβ T-cell receptors (TCRs) comprise functionally distinct subsets depending on which transcription factors and which of two coreceptors, CD8 or CD4, they express. CD8 + T cells respond to peptide agonists presented by major histocompatibility class I molecules (pMHC I) and are cytotoxic, whereas conventional CD4 + cells recognize peptide-MHC class II (pMHC II) and provide "help" defined by the cytokines they secrete (1). Cell adhesion assays explain this functionality insofar as CD8 and CD4 bind directly to pMHC I and pMHC II, respectively (2, 3). CD4 comprises two pairs of V-set and C2-set Ig superfamily domains, with early mutational data showing that the "top" two domains bind pMHC II (4). Crystal structures of cross-species and affinitymatured CD4/pMHC II complexes suggest that CD4 binds a pocket formed by the α2 and β2 domains of pMHC II (5, 6). The role of coreceptors in heightening T-cell responses is well established. For example, whereas CD4 + T-cells can respond to single pMHC II complexes presented by antigen-presenting cells (APCs), 30 or more complexes are required if CD4 is blocked (7).How CD4 achieves these effects, however, is incompletely understood. Coreceptors are pivotal in recruiting the kinase Lck to TCR/pMHC complexes (8, 9), but for reasons that are unclear coreceptor/pMHC interactions are extraordinarily weak. Traditionally, weak protein interactions are characterized using surface plasmon resonance (SPR) measurements, where one protein is tethered to the sensor surface and over it the other is passed at various concentrations. The SPR-based ...

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Section: Discussionmentioning

confidence: 42%

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

Jönsson

Southcombe

Santos

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…From then on, many experimental assays have been developed by coordinating the biological experiments and mechanical measurements. These include micropipette aspiration [25,31,36,51], optical tweezers [37,52,53], biological force probes [45,[54][55][56], atomic force microscopy (AFM) [38,39,43,[57][58][59][60][61], flow chamber [62][63][64][65], microcantilever needle [28], centrifugation [66], rosetting [67], cone-plate viscometer [68,69], surface force apparatus [70,71], and fluorescence recovery after photobleaching (FRAP) [72,73]. Here, two assays of micropipette aspiration and atomic force microscopy are exemplified to demonstrate how they work.…”

Section: Experimental Approaches For Quantifying 2d Kinetics and Forcmentioning

confidence: 99%

Mechanokinetics of receptor–ligand interactions in cell adhesion

Lü

Zhang

et al. 2015

Acta Mech Sin

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Receptor-ligand interactions in blood flow are crucial to initiate such biological processes as inflammatory cascade, platelet thrombosis, as well as tumor metastasis. To mediate cell adhesion, the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum, i.e., two-dimensional (2D) binding, which is different from the binding of a soluble ligand in fluid phase to a receptor, i.e., three-dimensional (3D) binding. While numerous works have been focused on 3D kinetics of receptor-ligand interactions in the immune system, 2D kinetics and its regulations have been less understood, since no theoretical framework or experimental assays were established until 1993. Not only does the molecular structure dominate 2D binding kinetics, but the shear force in blood flow also regulates cell adhesion mediated by interacting receptors and ligands. Here, we provide an overview of current progress in 2D binding and regulations, mainly from our group. Relevant issues of theoretical frameworks, experimental measurements, kinetic rates and binding affinities, and force regulations are discussed.Keywords Receptor-ligand interactions · Selectins · β 2 integrins · 2D binding kinetics B Mian Long

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“…FRAP (Fluorescence Recovery After Photobleaching) is a method for the diffusion kinetics (and also, correctly, reaction-diffusion kinetics) measurements in living cells using fluorescence microscopy (Zlatanov, 1987;Lopez, 1988;Swaisgood, 1989; Anders, 1990;Nagy, 1990; Bryers, 1998;Salomé, 1998;Tinland, 1998;Higgs, 2000;Reits, 2001;Carrero, 2003;Cha 2004;Joubert, 2004;Fukano, 2004;James, 2004;Sprague, 2005;Lele, 2006;Abbaci, 2007;Febo-Ayala, 2007;Travascio, 2007;Dushek, 2008;Kang, 2008;McNally, 2008;Tolentino, 2008;Lambert, 2009;Travascio, 2009; Hagman, 2010;Mueller, 2010;Mai, 2011;Wachsmuth, 2014;Yapp, 2016) which allows to estimate quantitatively the two dimensional lateral diffusion in molecularly thin film containing fluorescent-labeled probes, or for single cell examination (i.e. the study of lateral mobility of cellular molecules).…”

Section: Introductionmentioning

confidence: 99%

Ybridization of laser-induced spectrofluorescence analysis (lifs), matrix-assisted laser desorption / ionization mass spectrometry (maldi), fluorescence recovery after photobleaching (frap) anf fluorescence loss in photobleaching (flip) microtechnics

Orekhov¹,

Jablokov²,

Skrynnik³

2016

Journal of Biomedical Technologies

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Abstract. Novel MALDI MS + FLIP approaches for verifying continuity of membranous structures and measurements of nucleus-cytoplasm exchange rates are proposed. Novel approaches for the measurements of lateral diffusion / molecular mobility and bindings using MALDI + FRAP hybridization are proposed. FRAP (Fluorescence Recovery After Photobleaching) is a method for the diffusion kinetics measurements in living cells using fluorescence microscopy which allows to estimate quantitatively the two dimensional lateral diffusion in molecularly thin film containing fluorescent-labeled probes, or for single cell examination (i.e. the study of lateral mobility of cellular molecules). Fluorescence Loss in Photobleaching (FLIP) is a microscopic technique predominantly performed using laser scanning microscopy (e.g. for tagged protein local photobleaching by short, intensive laser excitation on CLSM platform) used for the studies on molecular mobility inside the cells and membranes. MALDI (Matrix-Assisted Laser Desorption / Ionization) is a soft ionization technique used in mass spectrometry, allowing for the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragmented when ionized by more conventional ionization methods (according to encyclopedic definition). The above laser-based technique is readily compatible with MALDI using the same laser beam for MALDI and FLIP. IntroductionFRAP (Fluorescence Recovery After Photobleaching) is a method for the diffusion kinetics (and also, correctly, reaction-diffusion kinetics) measurements in living cells using fluorescence microscopy (Zlatanov, 1987;Lopez, 1988;Swaisgood, 1989; Anders, 1990;Nagy, 1990; Bryers, 1998;Salomé, 1998;Tinland, 1998;Higgs, 2000;Reits, 2001;Carrero, 2003;Cha 2004;Joubert, 2004;Fukano, 2004;James, 2004;Sprague, 2005;Lele, 2006;Abbaci, 2007;Febo-Ayala, 2007;Travascio, 2007;Dushek, 2008;Kang, 2008;McNally, 2008;Tolentino, 2008;Lambert, 2009;Travascio, 2009; Hagman, 2010;Mueller, 2010;Mai, 2011;Wachsmuth, 2014;Yapp, 2016) which allows to estimate quantitatively the two dimensional lateral diffusion in molecularly thin film containing fluorescent-labeled probes, or for single cell examination (i.e. the study of lateral mobility of cellular molecules). Similar, though less common techniques (also referred to as FRAP) have been developed for the three dimensional diffusion and molecular binding investigation inside the cell. Modern confocal scanning laser microscopes (CSLMs) (Hardingham, 2000;Gribbon, 2001; Loerke,2005;Seiffert, 2005;Braeckmans, 2007;Campbell, 2007;Maung, 2007;Mazza, 2008;Tsibidis, 2008;Kang, 2010;Roberti, 2011; Kang, 2012;De Los Santos, 2015;Lemcke, 2016) are equipped with the system for local photobleaching in selected regions (Figure 1). But currently there are no completely satisfactory methods for molecular-chemical characterization of diffusion / binding kinetics for FRAP using MALDI (Matrix-Assiste...

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Measuring Diffusion and Binding Kinetics by Contact Area FRAP

Cited by 79 publications

References 38 publications

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

Mechanokinetics of receptor–ligand interactions in cell adhesion

Ybridization of laser-induced spectrofluorescence analysis (lifs), matrix-assisted laser desorption / ionization mass spectrometry (maldi), fluorescence recovery after photobleaching (frap) anf fluorescence loss in photobleaching (flip) microtechnics

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