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Spatio-mechanical regulation of Eph receptors at the cell-cell interface

  • Author(s): Greene, Adrienne Celeste
  • Advisor(s): Groves, Jay T
  • Drubin, David
  • et al.
Abstract

Spatial organization and movement of receptors and ligands at a cell-cell interface is emerging as a key regulatory component of signal transduction. We are particularly interested in understanding the regulation of the EphA2 receptor tyrosine kinase (RTK) movement and spatial reogranization. EphA2 is a unique receptor tyrosine kinase in that it signals in a juxtacrine geometry--EphA2's cognate ligand, ephrinA1, is expressed on the surface of an apposing cell. This unique protein arrangement not only provides a mechanism by which the receptor may experience extracellular forces, but also renders the system challenging to decode. Misregulation of EphA2 often occurs in many aggressive cancers and our lab has recently discovered that EphA2 is sensitive to spatial and mechanical aspects of the cell's microenvironment. We have developed a unique experimental platform in which we use a synthetic supported lipid membrane on a glass substrate to replace the ephrinA1 ligand-expressing cell in a cell-cell contact. This membrane is interfaced with living MDAMB231 breast cancer cells overexpressing EphA2, which effectively mimics a cell-cell junction. The advantage of this approach is the ability to study receptor-ligand interactions using high-resolution microscopy in a live-cell setting as well as modify not only the biochemical content of the membrane, but also the mobility of lipids and proteins. By introducing nanoscale barriers, such as grids patterned on the glass substrate, lipid and protein mobility can be redefined to specific micron-scale features. These diffusion barriers pattern the ligands in the synthetic membrane and allow us to study how spatial organization regulates signaling events. We have developed unique imaging assays to reveal that EphA2 signaling is sensitive to the mechanical properties of a breast cancer cell's microenvironment, which might have direct implications in physical aspects of tumor biology. We have expanded this work to study the structural contributions of Eph receptor clustering at the single molecule level and how this might regulate proper Eph signaling. Finally, we are also probing the cross-talk between membrane-bound ephrinA1 and soluble, secreted ephrinA1 signaling and how EphA2 signaling can be triggered in mechanically-dependent and mechanically-independent mechanisms. Together, these studies provide insight into the regulation of EphA2 signaling and how different aspects of this signaling might be altered in cancer progression.

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