@article {Galbraith158618, author = {Catherine G. Galbraith and Michael W. Davidson and James A. Galbraith}, title = {A Cautionary Tail: Changes in Integrin Behavior with Labeling}, elocation-id = {158618}, year = {2017}, doi = {10.1101/158618}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Integrins are heterodimer transmembrane proteins essential for cell adhesion and signaling during development, immunological responses, wound healing, and metastasis. Critical to their function is their unique ability to dynamically modulate their adhesive and signaling properties through changes in conformation, protein-protein interactions, and cellular distribution. While molecular, biochemical, and structural studies have uncovered ligand and regulatory protein interactions, unraveling the dynamic modulation of integrin adhesivity and signaling has relied on live-cell imaging. Fluorescence microscopy and specialized applications (FRET, FRAP, FLIP) have been critical in advancing our understanding of integrin behaviors. However, recent progress in super-resolution live cell imaging reveals integrin behaviors visible at the single molecule level that challenge our current knowledge of integrin behaviors derived from fluorescence microscopy. We found that the adhesivity and signaling of integrin heterodimers depends upon the whether the alpha or the beta subunit of the heterodimer is labeled. While imaging with conventional microscopy did not reveal any differences in unbound integrin behavior or the ability to localize to adhesions, single molecule super-resolution imaging uncovered significant differences in dynamic behavior of unbound integrins. These differences are manifested at the cellular level by alterations in protrusive activity, adhesion size, and cell spreading. Comparison with behaviors of untagged, integrins in normal and elevated affinity states indicates that fluorescently tagging the beta, but not the alpha, subunit elevates the integrin affinity state. Thus, our dense-field, live-cell single molecule super-resolution microscopy approach resolves molecular behaviors and interactions that are not visible with ensemble imaging but are functionally significant for prescribing cell behavior.}, URL = {https://www.biorxiv.org/content/early/2017/07/02/158618}, eprint = {https://www.biorxiv.org/content/early/2017/07/02/158618.full.pdf}, journal = {bioRxiv} }