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Ephrin-A5 induces rounding, blebbing and de-adhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Isobel D. Lawrenson^1,*, Sabine H. Wimmer-Kleikamp^1,*, Peter Lock¹, Simone M. Schoenwaelder², Michelle Down³, Andrew W. Boyd³, Paul F. Alewood⁴ and Martin Lackmann^1,

¹ Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
² The Australian Centre for Blood Diseases, Monash Department of Medicine, Box Hill Hospital, Victoria 3128, Australia
³ Queensland Institute for Medical Research, 300 Herston Rd, Herston, Queensland 4029, Australia
⁴ Centre for Drug Design and Development, IMB, St Lucia, Queensland 4068, Australia

View larger version (29K): [in a new window]   Fig. 1. Expression levels of EphA3 and ephrin-A on various melanoma cell lines and 293T cells. (A) LK63 human B-cell leukaemia cells, melanoma cells and 293T cells as indicated were analysed for cell-surface expression of EphA3 (a-c,e,g,h) with IIIA4 anti-EphA3 monoclonal antibody (solid lines) or an IgG1 isotype matched nonrelevant control antibody (broken lines), and of ephrin-A5 (d,f) with an EphA3-human Fc fusion protein (solid lines) or with EphA1-human Fc fusion protein (broken lines). Analysed cell populations were gated to exclude propidium iodide (PI)-positive dead cells. FITC-labelled sheep anti-mouse antibodies and anti-human Fc antibodies (both Jackson Laboratories) were used for visualisation. (B) Expression of EphA3 in melanoma cell lines was examined by northern blot analysis. LK63 human B-cell leukaemia cells were used as positive control (lane1); LiBr cells (lane 2), AO2 cells (lane 3), AO9 cells (lane 4). The 28S mRNA is shown as loading control.

View larger version (46K): [in a new window]   Fig. 2. Melanoma cell rounding, de-adhesion and membrane blebbing induced by clustered ephrin-A5 Fc is inhibited by soluble, monomeric ephrin-A5. (A) LiBr, AO9 and AO2 melanoma cells on fibronectin-coated glass coverslips were stimulated with 10 nM clustered ephrin-A5 Fc. Soluble, monomeric ephrin-A5 was added as inhibitor (+ inhibitor) to a parallel LiBr culture at 100-fold molar excess. Selected pictures of representative microscopic images taken during a 50 minute time course are shown. Arrowheads denote cells that retract their processes and round up during stimulation. Bar, 20 µm. A supplemental video of the AO9 time course is available at: http://www.ludwig.edu.au/addinfo/Figure2_AO9.mpg. (B) LiBr melanoma cells on fibronectin-coated glass coverslips before (-) or after (+) 10 minute ephrin-A5 stimulation were fixed, permeabilised and stained with rhodamine-phalloidin to visualise polymerised actin. Bar, 20 µm. (C) Melanoma cells on fibronectin-coated 96-well plates were treated as decribed in (A) and at indicated times cells were fixed and stained with crystal violet. Values represent mean A550 absorbances from quadruplicate wells ±s.d.: , LiBr cells; , AO9 cells; , LiBr cells with inhibitor; [UNK], AO2 cells. (D) Ephrin-A5 treatment does not impair cell viability in LiBr or EphA3 293T cells. Cultures of LiBr (grey bars) or EphA3 293T (black bars) cells in 24-well plates were treated with preclustered human IgG (control) or ephrin-A5 for 2 hours at 37°C, detached by EDTA treatment and cell viability in quadruplicate samples determined by trypan blue exclusion assay. To induce apoptosis, cells in parallel wells were treated for 4 hours with 1 µM staurosporin (St.sp.) or 0.5 mM H2O2. Control cells in parallel wells were treated for 30 minutes with the caspase inhibitor zVAD-fmk (0.1 mM) before addition of the apoptotic reagent. (E) Ephrin-A5 stimulation of LiBr cells does not result in increased numbers of apoptotic cells. LiBr monolayer cultures were treated for 2 hours at 37°C with (a), 1.5 µg/ml or (b), 7.5 µg/ml pre-clustered ephrin-A5 Fc; (c) 7.5 µg/ml preclustered control IgG; and (d) 1 µM staurosporin. Cells in the supernatant and detached by trypsin/versene treatment were combined, washed in Annexin V binding buffer and analysed for binding of Annexin V to the outer plasma membrane (apoptosis) and propidium iodide (PI) uptake (necrosis) by flow cytometry. Viable cells (lower left quadrant) are negative for Annexin V and PI, apoptotic cells (upper left quadrant) are positive for Annexin V, necrotic cells (upper right quadrant) are positive for Annexin V and PI.

View larger version (54K): [in a new window]   Fig. 3. Ephrin-A5-induced cell rounding involves contraction of the actin and microtubule network and loss of peripheral focal contacts. Parental (A,B) or EphA3-293T cells (C-J) were grown on fibronectin-coated glass coverslips. Following stimulation with ephrin-A5 fixed and permeabilised cells were incubated with rhodamine-phalloidin (A-D), or primary antibodies against -tubulin (E,F) and phospho-tyrosine (G-J) and Alexa-labelled secondary antibodies to visualise microtubule and focal complexes, respectively. Images were taken with a confocal fluorescence microscope; images in I and J represent vertical sections of individual cells illustrated in C and D, respectively. Bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 6. Crk rapidly binds the phopshorylated EphA3 ID and is able to distinguish between ligand-independent and ligand-mediated receptor phosphorylation. (A) Coomassie-stained SDS PAGE (top) and anti-PY western blot (bottom) illustrating the phosphorylated EphA3 ID as purified from the E. coli (1), dephosphorylated by TC45Pase (2) and rephosphorylated by the addition of ATP and Mg2+ (3). (B) Ephrin-A5-dependent association of Crk with endogenous EphA3 in LiBr melanoma cells. Adherent cultures of LiBr cells were treated for 10 minutes with preclustered ephrin-A5 Fc (+) or left untreated (-). Endogenous Crk in cell lysates was immunoprecipitated with anti-CrkII antibody and western blots probed with the same antibody to detect total CrkII or with anti-EphA3 antibodies to detect CrkII-associated EphA3. Parallel samples were immunoprecipitated with anti-EphA3 Mab and western blots probed for EphA3 and phosphotyrosine as indicated. (C) Dose-response of ephrin-A5 stimulated CrkII recruitment to EphA3. EphA3-transfected 293T cells were transferred to tissue culture wells containing surface-bound ephrin-A5 Fc at the indicated concentrations for 10 minutes, and endogenous CrkII in the cell lysates was immunoprecipitated with anti-Crk antibody. In parallel samples, the cells left in suspension were left untreated (-) or stimulated with 10 nM preclustered ephrin-A5 in solution (+). Blots of whole-cell lysates and of precipitated proteins were probed with anti-EphA3 and anti-CrkII antibodies as indicated. (D) Time course of CrkII binding to EphA3. EphA3 was (co) immunoprecipitated from lysates of EphA3 293T cells, which had been treated for the indicated times with 1.5 µg/ml of preclustered ephrin-A5 Fc, and analysed by western blot with anti- EphA3 and anti-phosphotyrosine antibodies as indicated. (E) 293T cells were left nontransfected (nil) or were transiently transfected with cDNAs encoding single, double (2x) or triple (3x) Tyr (Y)-Phe(F) mutants of EphA3. Anti-EphA3 immunoprecipitates from lysates of cells stimulated for 10 minutes with preclustered ephrin-A5 Fc (+) or left untreated (-), were analysed by western blot probed with anti-PY antibody. As loading control the total lysate was analysed by anti EphA3 western blot. Endogenously expressed Crk was immunoprecipitated from the same lysates and analysed by immunoblot for associated EphA3. (F) Crk was extracted from lysates of Crk-transfected 293T cells using Sepharose-coupled phosphopeptide Y596, 602P (Y2xP-Seph) or the nonphosphorylated peptide as control (Y2x-Seph) and analysed by western blot using the anti-Crk monoclonal antibody (left panel). The CrkII band in the crude cell lysate is shown in a parallel lane. Lysates from ephrin-A5 Fc stimulated cells were immunoprecipitated with anti-Crk antibody in the absence (nil) or presence of 50 µM of EphA3-derived phosphopeptides as indicated in the figure (right panel). Immunoprecipitates were analysed by western blots probed with antibodies to EphA3.

View larger version (70K): [in a new window]   Fig. 4. EphA3 juxtamembrane and activation loop tyrosines are required for sequential cell rounding and de-adhesion of EphA3 transfected 293T cells. 293T cells were transiently transfected either with pEGFP-actin (Clontech) alone (B, GFP-actin), or in addition to either wild-type (A, w/t) or mutant EphA3 with tyrosine to phenylalanine mutations at position 596 (Y596F), 602 (Y602F), 779 (Y779F), 596 and 602 (2xYF) or 596, 602 and 779 (3xYF) as indicated. Transfected cells on fibronectin-coated glass coverslips were stimulated with soluble clustered ephrinA5 Fc (1.5 µg/ml). Control samples were incubated with a 50-fold molar excess of soluble monomeric ephrin-A5 as inhibitor (B, w/t + Inhibitor) 30 minutes before addition of preclustered ephrin-A5. Cells were surveyed by confocal microscopy 10 minutes before and 50 minutes after the addition of ephrin-A5 Fc, and photos were taken at representative time periods selected. Bar, 20 µm.

View larger version (26K): [in a new window]   Fig. 5. Dose-dependent inhibition of cell adhesion and spreading by surface-bound ephrin-A5. (A) Capacity of Protein A-coated 96-well plates for Ephrin-A5 Fc () and of Protein A-bound ephrin-A5 for EphA3 (). Ephrin-A5 Fc was applied at indicated concentrations and bound protein in the supernatant was estimated by Biacore analysis. To confirm the competence of tethered ephrin-A5 to interact with the receptor, the nonbound fraction of EphA3 (2 µg/ml) after incubation on ephrin-A5-coated wells was determined by Biacore analysis. The amount of bound EphA3 estimated from this assay is shown (). (B) 293T cells, transiently transfected with w/t or mutant EphA3, and pEGFP-actin were plated (5x104cells/well) onto wells coated with ephrinA5-Fc at the indicated densities (ng/mm2). After 5 hours, adherent cells were fixed with 4% paraformaldehyde and examined by fluorescence microscopy. Sections of w/t EphA3-transfected cells are shown. Bar, 20 µm. (C) Following microscopy, adherent cells were quantified using crystal violet staining. Cell attachment is expressed as a percentage (mean±s.d. from three independent assays) relative to adhesion seen on non-ephrin-A5-coated wells; [UNK], w/t EphA3; , Y596F EphA3; , Y602F EphA3; , Y779F EphA3; , Y596F+Y602F EphA3.

View larger version (90K): [in a new window]   Fig. 7. Dominant-negative SH3* CrkII abrogates ephrin-A5-mediated cytoskeletal changes. EphA3 293T cells (control) were transiently transfected with myc-tagged, w/t CrkII, dominant-negative SH3* CrkII or both w/t and SH3* CrkII as indicated. Parallel cultures of stimulated (+) or nonstimulated (-) cells were fixed in 4% PFA. Cells were stained with rhodamine-phalloidin (Phalloidin) or anti-Myc antibody (anti-Myc) to monitor the actin cytoskeleton or CrkII derived proteins, respectively.

View larger version (22K): [in a new window]   Fig. 8. Ephrin-A5 stimulation of EphA3-expressing 293T cells mediates a rapid increase of active Rho A. The levels of active RhoA in parental (B) or EphA3-293T cells (A,C-E) transfected with w/t Crk (D) or SH3* Crk (E) as indicated, and which had been treated as adherent (A,B,D) or suspension (C) cultures with preclustered ephrin-A5, were analysed at the indicated times by precipitating GTP-bound RhoA with GST-RBD. Precipitated proteins and aliquots of the total lysates were analysed by immunoblotting with an anti-RhoA Mab. The intensity of bands representing active RhoA (RhoA-GTP) was normalised by comparison with Rho levels in corresponding total lysates using densitometry, and is indicated relative to baseline levels (given the arbitrary value `1') in the absence of ephrin-A5 stimulation. Each result represents the mean and s.d. from a minimum of three independent experiments.

View larger version (50K): [in a new window]   Fig. 9. Inhibition of Rho and ROCK activities abrogate ephrin-A5-mediated cell rounding and membrane blebbing. EphA3 293T cells (A-D) were treated with 10 µM C3 transferase exoenzyme overnight (C,D) and control cells left untreated (A,B). Parallel cultures of stimulated (+) or nonstimulated (-) cells were fixed in 4% PFA and stained with rhodamin-phalloidin. (E) Treatment with C3 transferease reduces RhoA available for ribosylation. Lysates fromEphA3 293T cells with (+) or without (-) overnight C3 transferase treatment were used for ADP ribosylation assays in the presence of radioactive 32P-NAD. RhoA-associated radioactivity was quantified after SDS-PAGE and autoradiography (top) by densitometry. Mean and s.d. from two independent experiments are shown (bottom). (F) Cell blebbing in EphA3-293T cells, left untreated or after pretreatment with 10 µM ROCK inhibitor Y-27632 or 10 µM PI3 kinase inhibitor LY 294002 (2 hours), was monitored by time-lapse microscopy. As control, blebbing in parental nontransfected 293T cells is shown. Representative fields containing approximately 100 cells were monitored for 10 minutes before and 50 minutes after stimulation. Total and blebbing cells were counted by an observer blinded to experimental conditions. Values represent mean percentages and s.d. (three independent experiments) of cells that commenced blebbing following addition of ephrin-A5. Supplemental videos of time courses illustrating responses of EphA3-393T cells without or with Y-27632 treatment are available at: http://www.ludwig.edu.au/addinfo/Figure9F.mpg and at: http://www.ludwig.edu.au/addinfo/Figure9Finhib.mpg, respectively.

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