FRET Detection of Cellular α4-Integrin Conformational Activation

Chigaev, Alexandre; Buranda, Tione; Dwyer, Denise C.; Prossnitz, Eric R.; Sklar, Larry A.

doi:10.1016/s0006-3495(03)74809-7

Cited by 120 publications

(182 citation statements)

References 74 publications

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“…These VLA-4 structural and functional changes appear to parallel the global conformational rearrangement of the extracellular domains induced by ligands and divalent cation (74) and the switchblade model for the ␣ v ␤ 3 integrin based on electron microscopy, NMR, and epitope exposure data (75). Using fluorescence resonance energy transfer and the LDV-FITC probe (21), we found a striking correlation between the degree of VLA-4 extension and its affinity (76). The prediction that force could increase adhesion bond strengths, catch bond (54), was verified by atomic force microscopy of P-selectin binding to P-selectin glycoprotein ligand-1 (77).…”

Section: Vla-4 Affinity Modulation By Shearmentioning

confidence: 79%

See 1 more Smart Citation

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Zwartz¹,

Chigaev²,

Dwyer

et al. 2004

Journal of Biological Chemistry

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Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown. We have shown previously that the affinity of the ␣ 4 ␤ 1 integrin, very late antigen-4 (VLA-4), measured with an LDV-containing small molecule, varies with cellular avidity, measured from cell disaggregation rates. In this study, we measured in real time affinity changes of VLA-4 in response to shear. The resulting affinity was comparable with the state mediated by receptor signaling and corresponded in time with intracellular Ca 2؉ responses. Ca Leukocytes are recruited to endothelial cells in a multistep process using selectin and integrin adhesion molecules (1, 2). These molecules allow a cell to tether, roll, adhere, and transmigrate along and across an endothelial layer. Selectin and some integrin molecules and their associated ligands mediate tethering and rolling interactions. Firm adhesion is mediated by vascular ligands of the immunoglobulin superfamily such as vascular cell adhesion molecule 1 (VCAM-1) 1 and their associated integrins (1, 2). The adhesive strength or avidity (3) of cells expressing integrins can be rapidly modulated by chemokines and chemoattractants, which also regulate leukocyte recruitment and migration across vascular endothelium. The rapid changes in avidity have been attributed to changes in the number of interacting molecules or valency due to molecular redistribution or clustering and to changes in the affinity of the individual receptor-ligand bonds (3-10).Physiological shear can also regulate leukocyte traffic by stimulating mechanosensors on neutrophils, monocytes, lymphocytes, erythrocytes, and platelets (see Ref. 11 and references therein). Shear arises from bifurcating blood vessels or rapid changes in blood vessel diameters. Shear acting on leukocytes, bound to endothelial cells, produces mechanical stress on the cells or their receptors, regulating cell growth and proliferation, protein synthesis, gene expression, and blood cell recruitment (12, 13). Integrins (such as ␣ v ␤ 3 , ␣ 5 ␤ 1 , ␣ 4 ␤ 1 , and ␣ 2 ␤ 1 ) on endothelial cells can act as mechanosensors to changes in blood flow (13,14) and trigger an intracellular signaling pathway involving focal adhesion kinase and mitogen-activated protein kinase cascades. How shear specifically induces blood cell adhesiveness or recruitment through mechanosensors is unknown. Indirect evidence shows that increased integrin binding to the extracellular matrix occurs when shear acts on cells or their mechanosensors to induce intracellular signaling. For example, intracellular signaling leads to conformational changes and activation of ␣ v ␤ 3 on endothelial cells and ␣ 4 ␤ 1 and ␣ 5 ␤ 1 integrins on monocytic cells (15, 16). Shear acting on endothelial cells affects the GTPase Rho signaling pathway and in monocyti...

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Section: Vla-4 Affinity Modulation By Shearmentioning

confidence: 79%

“…Possible mechanisms of VLA-4 activation by fluid flow and other mechanical factors. The bent and extended conformation of VLA-4 is shown; a more extended conformation has higher affinity for ligand (76). I, mechanical extension of VLA-4 can be accomplished by pulling a counterstructure ligand (VCAM-1).…”

Section: Discussionmentioning

confidence: 99%

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Zwartz¹,

Chigaev²,

Dwyer

et al. 2004

Journal of Biological Chemistry

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“…If a dynamic region of a membrane protein were labeled with a fluorophore and a transition metal were bound to the membrane, tmFRET could be used to report movement of the membrane protein relative to the membrane. Although classical FRET has been used to measure movements of  4 integrin relative to the membrane, because of the long R 0 's of these fluorophores (FITC and rhodamine B), the experiments were only sensitive to distance changes with large separations of the fluorophores (Chigaev et al, 2003).…”

Section: Cell Unroofingmentioning

confidence: 99%

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

Gordon¹,

Senning²,

Aman³

et al. 2016

J Cell Biol

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The plasma membrane is a major site of signal detection, transduction, and propagation in cells. Understanding cell signaling dynamics at the plasma membrane requires approaches that can detect changes in membrane architecture over molecular distances (10-100 Å) and biological time scales (milliseconds to seconds). Methods that provide access to dynamics at the intracellular surface of the plasma membrane would be especially powerful. Fluorescence resonance energy transfer (FRET) was first applied to studies of membrane dynamics by Keller et al. (1977), who recognized that fluorescent probes incorporated into membranes could be used as an indicator of mixing of the two membranes during membrane fusion. FRET occurs when the emission spectrum of a donor fluorophore overlaps with the absorption spectrum of an acceptor, and the donor and acceptor are in close proximity (Lakowicz, 2006;Taraska and Zagotta, 2010). FRET is steeply distance dependent, and each donor-acceptor pair has a characteristic distance at which the FRET efficiency is 50% (R 0 ), where the amount of FRET is optimally sensitive to changes in distance. The R 0 value for fluorescein and rhodamine, for example, is 60 Å (Delgadillo and Parkhurst, 2010), making them an ideal FRET pair for quantifying membrane fusion (Keller et al., 1977).Transition metal ion FRET (tmFRET) is a method to probe the shorter distances typical of the conformational landscape of proteins (Taraska and Zagotta, 2010). tmFRET is a form of FRET that uses a fluorophore as a Correspondence to Sharona E. Gordon: s e g @ u w . e d u ; or William N. Zagotta: z a g o t t a @ u w . e d u Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; PCF, patch-clamp fluorometry; TIRF, total internal reflection fluorescence; tmFRET, transition metal ion FRET.donor and a nonfluorescent transition metal ion as an acceptor (Latt et al., 1970(Latt et al., , 1972Horrocks et al., 1975;Richmond et al., 2000;Sandtner et al., 2007; Taraska et al., 2009a,b;Yu et al., 2013). Transition metal ions such as Ni 2+ , Co 2+ , and Cu 2+ have broad absorption spectra that overlap the emission spectra of visible light fluorophores (see Miessler and Tarr [1999] pages 361-380 for a discussion of electronic spectra of coordination compounds). Transition metals can therefore accept energy transfer from a nearby donor fluorophore and quench the donor's fluorescence. The degree of quenching is a direct measure of the FRET efficiency (Lakowicz, 2006;Taraska et al., 2009a). The FRET efficiency is steeply dependent on distance between the donor and acceptor, allowing tmFRET to serve as a molecular ruler for distances in the membrane. The main advantages of tmFRET over classical FRET methods are (a) R 0 is very short (10-20 Å), allowing short-range interactions to be studied; (b) the transition metals have multiple transition dipoles, reducing the orientation dependence of FRET (Horrocks et al., 1975); (c) the metals can be reversibly bound to native and introduced binding sites; and (d) different...

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“…sine (referred to as LDV-containing small molecule) (31)(32)(33) and its FITC-conjugated analog (LDV-FITC-containing small molecule) were synthesized at Commonwealth Biotechnologies, Inc. (Richmond, VA). Octadecyl rhodamine B chloride (R18) was from Molecular Probes, Inc. (Eugene, OR).…”

Section: Materials-thementioning

confidence: 99%

“…Using a VLA-4-specific fluorescent probe based on an LDV-containing compound, we were able to monitor changes in integrin affinity and conformation in real time in live cells in response to cell activation by inside-out signaling (31,32). We have also demonstrated a strong correlation between VLA-4 affinity and cell adhesion avidity (33).…”

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confidence: 95%

Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents

Chigaev¹,

Zwartz²,

Buranda³

et al. 2004

Journal of Biological Chemistry

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The ␣ 4 ␤ 1 -integrin (very late antigen-4 (VLA-4), CD49d/ CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDVcontaining small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670 -48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G proteincoupled receptors or Mn 2؉ in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.The ␣ 4 ␤ 1 -integrin (very late antigen-4 (VLA-4), 1 CD49d/ CD29) is a heterodimeric protein and a member of the family of adhesion receptors that is broadly expressed in lymphocytes and dendritic cells (1) and stem cells (2). VLA-4 has a flexible molecular structure that allows initial capture, tethering, rolling, and firm attachment of cells using the same counterstructure (3, 4). These properties appear to result from regulation of affinity and conformation by "inside-out" signaling (5, 6). Although the precise molecular mechanism of integrin conformational activation is unknown, significant understanding of this mechanism has been achieved in the last several years.One model involves the "mechano-conformational" regulation of integrin affinity and conformation. It is based on the idea that the separation of the intracellular ␣-and ␤-subunit tails may initiate "a piston-like or scissors-like motion" of the transmembrane domains (7). This motion results in a large conformational rearrangement of the integrin, accompanied by a "switchblade-like" opening of the molecule (8) and its conformational activation (5,...

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FRET Detection of Cellular α4-Integrin Conformational Activation

Cited by 120 publications

References 74 publications

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents

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