A Cautionary Tail: Changes in Integrin Behavior with Labeling

Abstract

Genetic expression of fluorescently labeled proteins is essential to visualizing dynamic behavior within live cells. Recent advances in microscopy have increased resolution to the level where it is now possible to capture individual molecules interacting. However, the criteria for determining whether a fluorescent label perturbs protein function have not undergone a corresponding increase in resolution. The effects of protein labeling on cell function are still judged by whether populations of protein localize and interact with known binding partners. Here we use integrins, bidirectional signal adhesion molecules that regulate interactions between the extracellular matrix and the cytoskeleton through a well-defined series of conformational changes to show that not all labeling strategies are the same. We found that labeling the beta subunit decreased the mobility of individual integrin molecules and the protrusive activity of the entire cell. While integrins with labeled alpha subunits behaved similarly to unlabeled integrins, labeling the beta subunit increased the size of adhesions by elevating integrin affinity and exposing the ligand induced binding domain to change the molecule conformation. Thus, our single molecule and cellular data indicate that the ability of labeled proteins to localize and interact with known binding partners does not guarantee it does not alter protein function. We propose that the behaviors of individual molecules rather than the ensemble behavior of populations need to be considered as criteria to determine if a probe is non-perturbative.