Abstract
ABSTRACTVisualizing fluorescent proteins is essential for understanding cellular function. While advances in microscopy can now resolve individual molecules, determining whether the labeled molecules report native behaviors and how the measured behaviors can be coupled to cellular outputs remains challenging. Here, we used integrin alpha-beta heterodimers – which connect extracellular matrix (ECM) and the cytoskeleton – to quantify the mobility and conformation of labeled integrins. We found that while unlabeled and labeled integrins all localized to adhesions and support anchorage-dependent cell function, integrin mobility decreased when the beta rather than the alpha subunit was labeled. In contrast to unlabeled and alpha labeled subunits, beta labeled subunits changed cellular behavior; decreasing protrusive activity and increasing adhesion size and the extent of cell spreading. Labeling the beta subunit changed the integrin conformation, extending the molecule and exposing an epitope that is revealed by activation with Mn2+ treatment. Our findings indicate labeling induced changes in dynamic integrin behavior alter molecular conformation as well as cellular adhesion-dependent function to demonstrate a coupling between molecular inputs and distinct cellular outputs.This article has an associated First Person interview with the first author of the paper.
Highlights
Integrins are bi-directional signaling molecules that form attachments between the extracellular matrix (ECM) and the cytoskeleton
Single-molecule mobility of integrins depends on which subunit is fluorescently labeled labeled integrins localize to adhesion complexes, localization is a population level assessment of behavior (Ballestrem et al, 2001; Laukaitis et al, 2001) and we wanted to evaluate the behavior of labeled integrins at the single-molecule level
One of the subunits was labeled with the photoactivatible fluorophore mEos2, which was attached to the integrin cytoplasmic tails with optimized linkers (Jaqaman et al, 2016; Kanchanawong et al, 2010) and these plasmids are available from Addgene
Summary
Integrins are bi-directional signaling molecules that form attachments between the extracellular matrix (ECM) and the cytoskeleton. Of whether the alpha or the beta subunit is labeled, labeled integrins have been shown to express on the cell surface and be capable of concentrating at adhesion complexes where they bind ECM and establish connections with the cytoskeleton (Laukaitis et al, 2001; Tsuruta et al, 2002).
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