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Address correspondence to Martin Lackmann, Dept. of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, Clayton, Victoria 3800, Australia. Tel.: 61 3 9905 3738. Fax: 61 3 9905 3726. email: Martin.Lackmann{at}med.monash.edu.au
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Abstract |
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Key Words: fluorescence resonance energy transfer microscopy; EphA3 receptor; receptor protein tyrosine kinase; receptor aggregation; signal transduction
S.H. Wimmer-Kleikamp and M. Lackmann's present address is Dept. of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Abbreviations used in this paper: 3YF EphA3, [Tyr596-Phe, Tyr602-Phe, Tyr779-Phe] EphA3; Ephs, Eph receptor tyrosine kinases; FLIM, fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; HEK293, human epithelial kidney 293; nb-EphA3, [Phe152-Leu, Val133-Glu] EphA3; w/t, wild-type.
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Introduction |
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Important structural and mechanistic insights into the initiation of Eph/ephrin signaling are provided by the X-ray structure of the complexed EphB2ephrin-B2 interaction domains (Himanen et al., 2001). Ephs and ephrins combine into a circular 2:2 heterotetramer, held together by Ephephrin heterodimerization and heterotetramerization interfaces (Himanen et al., 2001; Himanen and Nikolov, 2003). In the model structure, the COOH termini of Eph and ephrin domains are positioned to opposite sites of the crystal plane, in agreement with an orientation of receptors and ligands on juxtaposed cells to enable bidirectional signaling. Recently, we confirmed through analysis of EphA3 point mutants with impaired ephrin-A5 binding that the protein interfaces predicted from the EphB2ephrin-B2 crystal structure are also essential for the assembly of signaling-competent EphA3ephrin-A5 complexes (Smith et al., 2003).
We have now used GFP fusion proteins of wild-type (w/t) EphA3 and ephrin bindingimpaired mutants, together with signaling-compromised EphA3 receptors harboring mutations in their cytoplasmic domains, to examine ephrin-induced Eph receptor clustering by confocal time-lapse and fluorescence resonance energy transfer (FRET) microscopy. FRET microscopy, based on measurable energy transfer between closely located donor and acceptor fluorophores (Bastiaens and Pepperkok, 2000; Wouters et al., 2001), monitors proteinprotein interactions in situ and recently allowed elucidation of lateral signal transduction mechanisms of the EGF receptor (Reynolds et al., 2003). Here, we elucidate the mechanism leading to the assembly of extensive Eph receptor signaling clusters to cell membrane areas of ephrin contact. Our discovery of ephrin-independent recruitment of Ephs, including ephrin-binding or signaling compromised receptors, into nascent Ephephrin clusters has important consequences for the regulation of Eph/ephrin functions and for understanding intercellular communication mechanisms that are based on cell contactmediated receptor clustering.
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Results and discussion |
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EphA3 signaling clusters extend beyond direct receptorligand contacts
To examine the extent of direct EphA3ephrin contacts, we monitored, by FRET microscopy, interactions between a chimeric GFP/ephrin-A2 protein (Hattori et al., 2000), as a fluorescence donor, and Alexa546-labeled EphA3Fc, as a fluorescence acceptor. We, and others, demonstrated previously that ephrin-A2 and ephrin-A5 bind EphA3 with very similar affinities (Lackmann et al., 1997; Flanagan and Vanderhaeghen, 1998), and it is well documented that both ephrins fulfill similar, if not identical, cell biological functions in vivo (Klein, 1999). Stimulation of GFP/Ephrin-A2expressing cells with Alexa EphA3coated beads resulted within minutes in a distinct reduction in GFP lifetime, strictly confined to the beadcell interface (Fig. 3 A and Fig. S1, arrowheads depict the location of the bead, available at http://www.jcb.org/cgi/content/full/jcb.200312001/DC1).
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A comparison of the clustering events observed either by monitoring Ephephrin complexes on the cell membrane (Fig. 3 A) or by imaging phosphorylated receptors on its cytoplasmic side (Fig. 3 B) suggested that binding of ephrin-A5-Fc beads triggers formation and propagation of activated (phosphorylated) EphA3 clusters that dramatically exceed the size of ligand and receptor populations in direct contact.
Recruitment of ephrin-binding impaired receptors into signaling clusters
To examine this apparent recruitment of new Eph receptors into existing Ephephrin signaling clusters we used ephrin-binding impaired mutants of EphA3GFP, containing amino acid substitutions in both the ephrin heterodimerization and heterotetramerization (Himanen et al., 2001) sites, [Phe152-Leu, Val133-Glu] EphA3 (nb-EphA3-GFP), or in the heterodimerization site alone, [Phe152-Leu] EphA3GFP (Fig. 4 A). Recently, we demonstrated that these point mutations severely affect ephrin-A5 binding and abrogate EphA3 signaling (Smith et al., 2003). As expected, stimulation with ephrin-A5-Fc beads of cells expressing these EphA3 mutants did not yield a measurable change in GFP fluorescence lifetimes (Fig. 4 B). Intriguingly, coexpression of non-GFPtagged w/t EphA3 with either nb-EphA3-GFP or [Phe152-Leu] EphA3GFP effectively restored phosphorylation of nb-EphA3-GFP and its recruitment into large EphA3 clusters. The extensive FRET-positive area surrounding ephrin-A5-Fc beads demonstrates that cluster propagation does not rely on direct ephrin contact (Fig. 4 B). Intuitively, the role of preclustered ephrin lies in its ability to recruit neighboring receptors, which engage available Eph-binding sites according to the avidity of the ephrin aggregate. Although transgenic mouse studies (Brown et al., 2000) have provided in vivo support for this concept, the assembly mechanism of Ephephrin signaling clusters has remained unexplored. Our experiments indicate that, initiated by oligomeric ephrin, the propagation of Eph signaling clusters does not depend on ephrin contact but instead is governed by the receptors themselves.
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To explore the possible involvement of kinase and SH2 domain-independent functions of the EphA3 cytoplasmic domain, we performed the coprecipitation with EphA3cyto, a transmembrane mutant lacking the cytoplasmic domain (Fig. 4 D). Intriguingly, in EphA3
cyto coexpressing cells, nb-EphA3-GFP was effectively recruited and coprecipitated, suggesting that indeed extracellular receptorreceptor interactions facilitate the ephrin-independent propagation of Eph signaling clusters. This notion concurs with an earlier observation that a truncated EphA3 exodomain lacking the ephrin-binding domain facilitates EphA3 phosphorylation in vitro and acts as a dominant-negative inhibitor of EphA3 function in vivo (Lackmann et al., 1998).
Our findings have important implications for the understanding of Eph-mediated cell guidance. Eph-mediated but kinase-independent cell positioning has been described in several developmental programs (Boyd and Lackmann, 2001); EphA7 splice variants lacking kinase activity provide essential ephrin-A5 adhesion contacts during mouse neural fold closure, and coexpression of w/t and kinase-dead EphA7 switches cell contact repulsion to adhesion (Holmberg et al., 2000). Our data now reveal a mechanism that can recruit not only signaling-impaired but ephrin-binding compromised Ephs into the same signaling cluster. Therefore, Eph receptor clustering provides a simple mechanism to dynamically modulate a cellular response according to overall composition and abundance of receptor variants within a given cell. It will be interesting to examine if this mechanism also allows for the recruitment of different Eph RTK family members into the same cluster, and experiments to address this notion are ongoing.
The organization of transmembrane receptors into higher order signaling clusters is likely to be a universal biological communication mechanism and accounts for the finely graded responses of prokaryotic chemotaxis receptors (Gestwicki et al., 2000; Kim et al., 2002) and for ligand selectivity of T cell receptor complexes (Krummel and Davis, 2002). For ephrin-directed pathfinding, the current model suggests that migration of Eph-bearing cells into a gradient of ephrin expression is controlled by Eph receptor affinity and abundance and by competition for ephrin targets (Monschau et al., 1997; Brown et al., 2000; Feldheim et al., 2000). The clustering mechanism revealed by our findings is suited to exert this control, whereby nucleating oligomeric Ephephrin complexes will recruit a proportion of receptors into a signaling cluster that would represent the overall receptor abundance of the cell. Similar to bacterial chemotaxis mechanisms, this concept allows for precisely adjusted cellular responses controlled by graded changes in ligand abundance and receptor/ligand occupancy, a characteristic feature of Ephephrin communication.
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Materials and methods |
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Anti-EphA3 mAb, IIIA4, and affinity-purified rabbit polyclonal antibodies were described previously (Boyd et al., 1992; Lackmann et al., 1997). Other antibodies and reagents used were anti-GFP (Transduction Laboratories), 4G10 (Upstate Biotechnology), PY72 (Cancer Research UK), P-Tyr100 (New England Biolabs, Inc.), HRP-conjugated antimouse antibodies (Jackson ImmunoResearch Laboratories), HRP-conjugated antirabbit (Bio-Rad Laboratories), and rhodamine phalloidin and Lysotracker green (Molecular Probes).
Alexa Fluor 546 conjugates and polystyrene beads
Recombinant, purified ephrin-A5-Fc and EphA3-Fc were labeled with Alexa Fluor 546 (Molecular Probes). Coupling of the Alexa dye and its effect on the biological integrity of ephrin and Eph proteins were monitored by spectral (HPLC diode array detection) and BIAcore binding analysis. Binding to sensor chip-coupled EphA3 or ephrin-A5 (Lackmann et al., 1997, 1998) was used to monitor biological integrity. Alexa546 conjugates of EphA3 Fc and ephrin-A5-Fc (Alexa EphA3-Fc and Alexa ephrinA5-Fc) or unlabeled ephrin-A5-Fc were immobilized onto protein Acoated 5.6-µm polystyrene beads (Bangs Laboratories) according to the manufacturer's instructions. Ephrin-A5-Fc or Alexa ephrin-A5-Fc were preclustered (20 min) at a 1:10 molar ratio with antihuman Fc antibody (Jackson ImmunoResearch Laboratories) before experiments.
Cell culture
HEK293 (American Type Culture Collection) cells were maintained in DME, 10% FCS, and transfection of HEK293 cells was performed using Fugene 6 transfection reagent (Roche Biochemicals). Before each experiment, cells were serum starved in culture medium containing 0.5% FCS for at least 4 h. For live cell FRET microscopy experiments, the culture medium was replaced with CO2-independent imaging medium.
Microscopy and immunocytochemistry
Immunocytochemistry and time-lapse confocal microscopy done on a microscope (model 1024; Bio-Rad Laboratories) using 60x, 1.4 NA oil (fixed cells, and 60x, 1.2 NA water (live cells) immersion lenses were performed as described previously (Lawrenson et al., 2002). Lysosomal cell compartments were stained with Lysotracker green (Molecular Probes). Images of green (Lysotracker CMFDA) and Alexa546 fluorescence were collected sequentially to minimize "bleed-through" from spectral overlap. Lysotracker was excited with the 488-nm line of a 100-mW argon ion laser (Ion Laser Technology) attenuated to 3% with a neutral density filter. Alexa546 was excited with the 514-nm argon laser line attenuated to 3% with a neutral density filter. For detection, 527 long pass primary and 565 long pass secondary dichroic mirrors, separating red and green fluorescence to separate detectors, and a narrow band barrier filter (522/35) were used.
GFP EphA3 (w/t or mutant) expressing cells were stimulated, fixed, permeabilized, and stained with Cy3-conjugated antiphosphotyrosine mAb PY72 before mounting onto glass slides using Mowiol (Calbiochem). Fluorescence lifetime imaging microscopy (FLIM) sequences were obtained at 80 MHz with a microscope (model IX70; Olympus; 100 /1.4 NA oil immersion lens) and analyzed as described previously (Reynolds et al., 2003). A 476-nm argon laser line and narrow-band emission filter (model HQ510/20; Chroma Technology Corp.) were used for GFP, a 100-W mercury arc lamp with high Q Cy3 filter set (excite, model HQ545/30; dicroic, model Q580LP; and emitter, model HQ610/75) was used for Cy3 and Alexa546. GFP Fluorescence was detected with a dichroic beamsplitter (model Q495 LP; Chroma Technology Corp.) and narrow band emission filter. FRET was measured in live 293 cells between transiently expressed ephrin-A2GFP and Alexa546 EphA3-Fc.
Immunoprecipitation and Western blotting
Serum-starved cells stimulated for 10 min with 1.5 µg/ml of preclustered ephrin-A5 were lysed as described previously (Lawrenson et al., 2002). Ephrin-A5bound receptor clusters were precipitated with protein ASepharose (Amersham Biosciences) for at least 1 h at 4°C. Lysates and washed immunoprecipitates were analyzed by Western blot with appropriate antibodies and visualized using an ECL substrate (Pierce Chemical Co.).
Online supplemental material
The dynamics of ephrin-A5binding to cell surface EphA3 and subsequent assembly of Eph-A3ephrin-A5 signalling clusters was recorded in real-time by time-lapse confocal microscopy and is illustrated in Video 1. Fig. S1 illustrates the formation of Ephephrin contacts that were monitored by fluorescence lifetime imaging using GFP ephrin-A2expressing cells exposed to Alexa EphA3-Fccoated beads and micrographs of salient time points. We examined the authenticity of FRET in our experiments by monitoring GFP fluorescence lifetimes in EphA3-GFPexpressing cells after CY3 photobleaching (Fig. S2, A and B) or in the absence of the fluorescence acceptor CY3-PY72 (Fig. S2 C). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200312001/DC1.
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Acknowledgments |
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This work was supported by Melbourne University International Research, Abroad Traveling and Journal of Cell Science Traveling Fellowships (to S.H. Wimmer-Kleikamp), and National Health and Medical Research Council (grant 234707) and Anti-Cancer Counsel of Victoria (grant 62/2002) funding (to M. Lackmann).
Submitted: 1 December 2003
Accepted: 12 January 2004
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