Analysis of Single Integrin Behavior in Living Cells

Abstract

Integrins are trans-plasma membrane receptors that mediate linkages between the extracellular matrix (ECM) and the actin cytoskeleton. In migrating cells, integrins cluster into focal adhesions (FAs). Integrin binding to ECM involves induced conformational changes that increase the affinity of the extracellular domains to ECM ligands, while indirect protein-protein interactions in FAs mediate cytoskeletal linkages. How the motion of individual integrin molecules in the plasma membrane relates to integrin activation, ECM or cytoskeletal binding, and FA formation is unknown. To address these questions, we analyzed the dynamics of single integrin molecules in migrating U2OS osteosarcoma cells. Cells expressing integrin αv or a farnesylated peptide (CAAX) fused to the photoconvertible fluorescent protein, tdEos, were analyzed by single particle tracking photoactivation localization microscopy (SPT-PALM). We imaged a high density of single photoconverted tdEos molecules by total internal reflection fluorescence microscopy (TIRFM) and tracked their trajectories using a globally-optimal SPT algorithm. Analysis of CAAX trajectories revealed that a majority of molecules (84%) exhibited Brownian diffusive behavior with 7% showing confined diffusion (D = 2.5-3×10−13 m2/s). In contrast, a majority of integrin αv trajectories exhibited confined diffusive behavior (74% confined, 21% Brownian; D = 2-5×10−14 m2/s). Image segmentation allowed classification of trajectories inside and outside FAs. Surprisingly, this showed that the proportion of confined or Brownian diffusive behavior was independent of whether molecules were inside or outside FAs. Analysis of integrin αv molecules specifically within FA showed that treatment with Mn2+ to induce integrin activation reduced the confinement radius of diffusion, whereas disruption of the actin cytoskeleton with latrunculin-A significantly increased the confinement radius. These results indicate that integrins can be immobilized without clustering, but can also remain mobile while clustered within FAs. In addition, both activation and cytoskeletal connection contribute to reduced integrin mobility within FAs.