Patterned EGF Reveals Differential Distributions of Activated Kinase and Integrin Activities

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This confocal fluorescence image on the cover of Biophysical Journal volume 107, issue 11, shows a single cell attached to micron-sized patterns labeled with red fluorescence (immobilized streptavidin). These patterns present irreversibly attached epidermal growth factor (EGF), which engages EGF receptors expressed at the cell surface. A consequence of this engagement is stimulated phosphorylation of tyrosine residues at the patterned features, detected as blue fluorescence with a monoclonal anti-phosphotyrosine antibody. Concentration of phosphotyrosine labeling at the patterned sites can be seen more clearly in the single color images of this cell (not overlayed) in Figure 4 of our paper in this issue of the Journal.

Most interesting in this image is the concentration of the beta1 subunit of an integrin in green. The striated labeling around the more peripheral EGF features that are engaging the cell suggests that focal adhesion complexes are formed at these sites. As quantified in our paper using a novel radial analysis method, we find that this integrin is more concentrated at patterned features in contact with the cell periphery, rather than at the cell center, where the tyrosine phosphorylation is just as abundant as it is in the periphery. Together, these results suggest that the earliest signaling steps mediated by EGF receptors are activated similarly wherever the patterned EGF engages the cells, while more downstream signaling that recruits integrin-containing focal adhesion complexes is preferentially activated at the cell periphery. Possibly tension forces at these peripheral sights are greater, and inside-out activation of integrin signaling complexes may result.

The experiment represented by this cover image was carried out by Devin Wakefield, a graduate student in our laboratory. We were prompted to submit a cover image by Dr. Kirsten Bryant, a former graduate student in our laboratory and a co-author in this study. The image represents the multidisciplinary efforts and interests in our laboratory, that range from efforts to understand molecular mechanisms of store-operated calcium entry in mast cells to super resolution imaging of receptor clustering dynamics. These efforts provide new tools and methodologies for application to a broad range of cell signaling questions. Our laboratory website summarizes our research activities: http://baird.chem.cornell.edu

– Amit Singhai, Devin L. Wakefield, Kirsten L. Bryant, Stephen R. Hammes,
David Holowka, and Barbara Baird

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