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First published online 19 December 2006
doi: 10.1242/jcs.03333

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Ena/VASP proteins mediate repulsion from ephrin ligands

¹ Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK
² Institute for Clinical Biochemistry and Pathobiochemistry, University of Würtzburg, Würtzburg, Germany
³ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
⁴ Department of Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK

View larger version (54K): [in this window] [in a new window]   Fig. 1. Neural crest cells avoid substrate-bound ephrin stripes. Neural crest cells avoid stripes of substrate-bound ephrin-B2-Fc, undergoing repeated cycles of lamellipodial protrusion and collapse at sites of contact with ephrin-B2-Fc. (A) Cells distal to the neural tube explant avoided the ephrin-B2-Fc stripes and exhibited a striped pattern on the coverslip. (B) Neural crest cells on negative control stripes failed to discriminate between the sets of stripes. (C) Neural crest cells that contact substrate-bound ephrin-B2-Fc (lighter broader stripe) undergo a dramatic loss of lamellipodia (black arrow). (D) Neural crest cells undergo cycles of protrusion (red arrows) and retraction (black arrows) along the edges of substrate-bound ephrin-B2-Fc stripes. (E) Retraction from ephrin-B2-Fc (pink overlay) is associated with membrane ruffling (black arrows) and the internalisation of phase bright vesicles (red arrows). A red asterisk denotes the cell to which timepoints refer. Bars, 130 µm; ephrin stripes are 40 µm wide.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Soluble ephrins destabilise neural crest cell lamellipodia. (A) Neural crest cells were examined using timelapse videomicroscopy for 30 minutes and then stimulated with 2 µg/ml pre-clustered ephrin-B2-Fc for 45-60 minutes. The withdrawal of neural crest cell lamellipodia (red arrow) was associated with membrane ruffling (red arrowhead) following contact with the ephrin-B2-Fc stripe. (B) Membrane ruffling led to the internalisation of phase bright vesicles. (C) Lamellipodial dynamics were analysed by kymography prior to stimulation (15.5±1.2 peaks/hour) and in the presence of 2 µg/ml pre-clustered ephrin-B2-Fc (25.2±1.1 peaks/hour); in the presence of ephrin-B2-Fc the lifetime of protrusions was decreased (P<0.05, n=9). A representative kymograph from a total of nine neural crest cells examined is shown (three separate timelapse movies). (D) A trace of the kymograph shown in C. Bars, 10 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 3. Wild-type MEFs express Eph receptors and respond repulsively to substrate-bound ephrin stripes. Wild-type MEFs were stimulated for 0, 5, 15 and 30 minutes with 1 µg/ml pre-clustered ephrin-A5-Fc and then lysed in immunoprecipitation buffer. Endogenous EphA4 was immunoprecipitated using mouse anti-EphA4. (A) Samples were western blotted for phospho-tyrosine (p-Y) and EphA4 to demonstrate EphA4 activation and loading, respectively. Bars, 130 µm. (B-D) 6x103 wild-type MEFs (green; F-actin staining) were plated to human Fc (negative control; B), ephrin-A5-Fc (C), or ephrin-B2-Fc (D) stripes and incubated for 18 hours. (E) Graph shows the average percentage of cells (+ 1 s.e.m. error bars, n=4) avoiding ephrin stripes and the equivalent stripe in the matched negative controls for those experiments. The difference between these values represents the repulsive response to a particular ephrin ligand (black arrow).

View larger version (36K): [in this window] [in a new window]   Fig. 4. Ena/VASP proteins are required for repulsion from substrate-bound ephrin stripes. (A-D) Wild-type MEFs, VASP–/– MEFs and MVD7 cells bind pre-clustered ephrin-A5-Fc, demonstrating Eph receptor expression (A,C,D); pre-clustered human Fc, applied as a negative control, was not bound by these cells (wild-type MEFs shown in B). (E) The loss of VASP and/or Mena does not prevent the activation of endogenous Eph receptors: EphA4 immunoprecipitations showed an increase in phospho-tyrosine (p-Y) following stimulation with 1 µg/ml pre-clustered ephrin-A5-Fc for 15 minutes, compared to unstimulated cells (0 minute) for each cell type. (F) The repulsive responses of VASP–/– MEFs and MVD7 cells were reduced compared to those of wild-type MEFs. Results are the means and s.e.m. from three or four experiments, with 600-800 cells counted for each condition. Means are significantly different from Wt MEF controls (denoted by *), according to Student's t-test [P<0.05, n=3 (VASP–/– cells) or 4 (MVD7 cells)]. Bars, 20 µm (A-D).

View larger version (59K): [in this window] [in a new window]   Fig. 5. Ena/VASP proteins localise to sites of Eph receptor activation. (A) Schematic diagram of the neighbouring cell injection assay (see Marston et al., 2003): EphB4-expressing cells (green) make p-Eph-positive protrusions (red) under ephrin-B2-expressing cells (blue), which subsequently collapse rearwards as membrane ruffles. This leads to the internalisation of p-Eph and cell-cell separation around 3 hours post-injection. (B,C) Non-injected, starved Swiss 3T3 cells were stained for VASP (B) and Mena (C) to demonstrate localisation in the absence of Eph receptor activation. (D-G) Swiss 3T3 fibroblasts were injected with pCIneo-EphB4 (B4) or pRK5-ephrin-B2 (*) and left to express for 2-3.5 hours, then fixed and co-stained for activated Eph receptors using anti-phospho-tyrosine (p-Y) or anti-p-Eph antibodies (red in merged images) and EphB4 (D,E), VASP (F) or Mena (G; green in merged images). EphB4 and p-Eph staining overlap at sites of EphB4-ephrin-B2 contact, including in some internalised vesicles (D,E). VASP (F) and Mena (G) localise to the EphB4/ephrin-B2 interface at sites of Eph receptor activation and are often found at the margins of protrusions made by EphB4-expressing cells at these sites. Bars, 20 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Ena/VASP proteins are necessary for the internalisation of p-Eph in response to cell surface-bound ephrin-B2. (A-D) Swiss 3T3 fibroblasts in a starved, confluent monolayer were injected with 200 µg/ml pRK5-ephrin-B2 and 100 µg/ml biotin-dextran adjacent to cells co-injected with 100 µg/ml pCIneo-EphB4 and either 100 µg/ml pcDNA3-EGFP-APPPP-mito (A,B) or pcDNA-EGFP-FPPPP-mito (C,D) and left to express for 3.5 hours. Cells were then fixed and stained for biotin-dextran (blue) and p-Eph (red) to detect ephrin-B2- and EphB4-expressing cells, respectively; EGFP fluorescence (green) revealed expression of FPPPP-mito or APPPP-mito (A and C also show F-actin staining in green). The APPPP-mito-expressing cells showed internalisation of p-Eph (A,B). By contrast, p-Eph internalisation was inhibited in FPPPP-mito-expressing cells, with p-Eph staining instead remaining at sites of EphB4-ephrin-B2 contact (C,D). (E) Cells that expressed EphB4 and FPPPP/APPPP-mito, contacting ephrin-B2-expressing cells were scored for internalisation of p-Eph and the presence of large, invasive protrusions. FPPPP-mito expression significantly reduced the percentage of cells that internalised activated Eph receptors compared to APPPP-mito (P<0.05, n=5), with 193 (APPPP-mito) and 119 (FPPPP-mito) cells counted for each condition (five separate experiments). No difference was observed in the proportion of cells that displayed large, invasive protrusions. Bars, 10 µm.