Spatial organization of EphA2 at the cell-cell interface modulates trans-endocytosis of ephrinA1.
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.
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California; Department of Molecular and Cell Biology, University of California, Berkeley, California.
Adrienne C
AC
Greene
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California.
Samuel J
SJ
Lord
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California.
Aiwei
A
Tian
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California; Department of Mechanical Engineering, University of California, Berkeley, California.
Christopher
C
Rhodes
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California.
Hiroyuki
H
Kai
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California; Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California; Mechanobiology Institute, National University of Singapore, Singapore. Electronic address: jtgroves@lbl.gov.
Jay T
JT
Groves
D051722:ADAM Proteins / Q000378:metabolism; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D036382:Ephrin-A1 / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D036104:Receptor, EphA2 / Q000737:chemistry* / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera
D002966:Clathrin; D036382:Ephrin-A1; D008024:Ligands; D008565:Membrane Proteins; D036104:Receptor, EphA2; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D034281:Dynamins