Restriction of Receptor Movement Alters Cellular Response: Physical Force Sensing by EphA2

Abstract

Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.Mammalian cells exhibit marked sensitivity to physical aspects of their environment, such as compliance (1), texture (2), and geometry (3). Tensional homeostasis between and within cells contributes to proper cell differentiation, development, and, ultimately, survival (4). Because most cellular decision making occurs via chemical processes, understanding the coupling between physical forces and chemical signaling networks is of fundamental importance. Focal adhesions, which consist of protein assemblies organized at sites where cell-surface integrin receptors bind extracellular matrix ligands, are the most widely studied interface for tensile force transduction (5). However, the majority of membrane receptors are not associated with focal adhesions. The mechanisms (and even existence) of chemomechanical regulatory coupling in these systems remain largely unknown.It is becoming clear that spatial organization of cell surface receptors can regulate associated signal transduction pathways (6-9). An important corollary is that mechanical forces acting on ligands can influence receptor spatial organization and, correspondingly, signaling (10-12). Juxtacrine signaling, in which receptor and ligand reside in apposed cell membranes, represents an important class of intercellular communication where physical restriction of ligand spatial organization and movement is evident (6,13). Here, we reconstitute the juxtacrine signaling geometry between live cells expressing the EphA2 receptor tyrosine kinase and supported membranes displaying laterally mobile ephrin-A1 ligand.EphA2 is implicated and functionally altered in a number of cancers. In particular, 40% of human breast cancers overexpress the receptor (14). Upon binding to natively membraneanchored ephrin-A1, EphA2 undergoes dimerization, transphosphorylation of the cytoplasmic domains, recruitment of a molecular complex with SHC and GRB2 adaptor proteins, and subsequent activation (15). EphA2 activation stimul...

Lipid-nanostructure hybrids and their applications in nanobiotechnology

“…In addition, the EphA2 signaling pathway of cancer cells was regulated spatially and mechanically by a Cr metal line nanofabricated substrate created via electron-beam lithography (Figure 8b). 87 The EphA2, a receptor tyrosine kinase, is overexpressed in B40% of The average radial distribution function was found to exhibit a strong correlation (r ¼ 0.91, P ¼ 7 Â 10 À8 ) with invasion potentials that were determined with the modified Boyden chamber analysis. Bottom: images of fluorescently labeled ephrin-A1 ligands of EphA2-expressing mammary epithelial cells on a supported membrane.…”

“…Bottom: images of fluorescently labeled ephrin-A1 ligands of EphA2-expressing mammary epithelial cells on a supported membrane. 87 (c) Schematic illustration of the binary ligand system that presents immobilized FN-Cy3 and laterally mobile EGF-647. Representative brightfield, RICM, and fluorescence microscopy images of three cells that are engaged with a nanopatterned supported membrane.…”

Molecular mechanisms in signal transduction at the membrane

“…This system has recently been reconstituted between live EphA2-expressing cells and supported membranes displaying ephrin-A1 (ref 86). Ligand binding, receptor clustering and phosphorylation are observed.…”

Eph receptor and ephrin function in breast, gut, and skin epithelia

“…6,[28][29][30] The size of the receptor signaling clusters can vary depending on the mobility of ephrins on adjacent membranes and the cell type in question. 31 Eph receptor can also form hetero-oligomer clusters with members of a different receptor subclass. 32,33 Taken together, this allows for a considerable degree of complexity in the molecular organization of Eph receptor signaling complexes in epithelial tissues where multiple family members are expressed.…”