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. 2014 May 20;106(10):2196-205.
doi: 10.1016/j.bpj.2014.03.043.

Spatial Organization of EphA2 at the Cell-Cell Interface Modulates Trans-Endocytosis of ephrinA1

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Free PMC article

Spatial Organization of EphA2 at the Cell-Cell Interface Modulates Trans-Endocytosis of ephrinA1

Adrienne C Greene et al. Biophys J. .
Free PMC article

Abstract

EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell's microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2's cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell's microenvironment and may have implications in physical aspects of tumor biology.

Figures

Figure 1
Figure 1
Schematic of a cell expressing EphA2 interacting with a supported membrane displaying ephrinA1. (A) When on a fluid membrane cells coalesce ephrinA1 into large regions of high concentration and recruit endocytosis molecules. (B) When membrane-cell contact sites are physically perturbed using chromium diffusion barriers, endocytosis is altered. (C) Bright-field and TIRF images of ephrinA1 at the interface between the cell and supported membrane on an unrestrained substrate and on 10, 5, 3, and 1 μm gridded substrates. Scale bar is 10 μm.
Figure 2
Figure 2
Molecular physiology of the EphA2-ephrinA1 contact sites. The ratio of fluorescence intensity within and outside regions of ephrinA1 enrichment is a measure of whether the cellular component is recruited to (values >1) EphA2-ephrinA1 or excluded from (values <1) those sites; values near 1 indicate homogeneous distribution throughout the cell membrane. Clathrin and dynamin are colocalized with ephrinA1, caveolin is antilocalized with ephrinA1, and actin forms a ring around the large EphA2-ephrinA1 contact site. Insets are TIRF microscopy images showing MDAMB231 cells at the membrane-cell interface. The images are false color overlays of ephrinA1 labeled with Alexa Fluor 647 (magenta) and cell components expressed as GFP fusions (green). See Fig. S3 for further details. Scale bar is 10 μm.
Figure 3
Figure 3
Time-lapse 3D reconstructions of confocal stacks of a single living MDAMB231 cell as it lands on a supported membrane displaying ephrinA1. The cell coalesces EphA2-ephrinA1 into large contact regions at the membrane-cell interface, and then internalizes the receptor and ligand over time. Only the ephrinA1 protein is fluorescently labeled (with Alexa Fluor 647), but the images are pseudocolored to encode height above coverslip (blue/green for near the coverslip and magenta for > ∼1 μm above). The gray dome approximates the cell outline, which is not fluorescent in this assay. (See Fig. S4 for a simple grayscale rendering.) An automated object-identification program counted the number of puncta within the cells to measure endocytosis of the EphA2-ephrinA1 complex; only fluorescent spots well above the coverslip and membrane-cell interface (typically 3 μm) were included in the analysis. For experiments quantifying endocytosis in hundreds of cells, samples were fixed at 45 min. Scale bar is 10 μm.
Figure 4
Figure 4
Spatiomechanical inhibition of EphA2-ephrinA1 endocytosis. (A) Fixed MDAMB231 cells on supported membranes that are mechanically restricted by 1 μm (left) or 10 μm (right) grids. On small grid pitches, cells generally exhibited fewer internal ephrinA1 puncta, indicating less endocytosis from the interface. Images are 3D renderings of confocal fluorescence data of ephrinA1 labeled using Alexa Fluor 647 (pseudocolored as in Fig. 3) and a gray dome approximating the cell outline. Scale bar is 10 μm. See also Movie S2. (B) Column scatter graph showing the amount of internalized ephrinA1 in each cell for one representative sample, which contained all grid patterns (e.g., 1, 3, 5, 10, and 20 μm or off grid). Bars are mean ± standard error of the mean. n >50 cells on each grid pitch. Note that for 1 and 3 μm, >40% of the cells contain zero puncta (see Fig. S6). (C) The result of multiple independent repeats of the representative sample shown in B. Values were first normalized to 20 μm in each sample, the normalized values at each grid pitch were then averaged across all samples. Error bars are standard error of the mean, n = 6 samples, each with hundreds of cells. P < 0.05 between 1 and 10 μm grid pitch using ratio paired t-test.
Figure 5
Figure 5
Drug inhibition of ephrinA1 trans-endocytosis. (A) Blocking the clathrin terminal domain with the small molecule Pitstop2 reduces overall ephrinA1 endocytosis. Bars are mean ± SE, n = 3 samples each condition, with >1000 cells per sample. P < 0.05 using the unpaired t-test. (B) Inhibiting ADAM10 and ADAM17 metalloprotease activity using the small molecule INCB003619 reduces overall ephrinA1 endocytosis. Bars are mean ± range, n = 2 samples each condition, with ∼1000 cells per sample. P < 0.05 using the unpaired t-test.

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