Integrins are transmembrane adhesion receptors that facilitate cell adhesion by binding extra-cellular ligands to provide a mechanical linkage between a cell and the extracellular matrix (ECM). Many types of integrins form micron-sized clusters, which create the foundation for various cell-matrix adhesion complexes including focal adhesions. These complexes are populated by a diverse group of membrane, structural, adaptor, and enzymatic proteins [1
], and signaling via these complexes affects many important cellular processes [2
]. Integrin clusters thus provide the platform for signal propagation as well as force transduction through focal adhesions; as a result cell signaling and adhesion depend directly on the spatial and temporal characteristics of integrin cluster formation and dispersion [3
Because integrin binding, clustering, and signaling depend on the availability of insoluble extracellular ligands [7
], the availability of integrin binding sites is a critical property of the ECM proteins to which cells adhere. There is also a growing body of experimental evidence indicating that cells sense and respond to the concentration of ECM ligands available to them. Cell migration speed reaches a maximum at intermediate ECM density [10
], and recent evidence suggests that the relationship between cell migration speed and ECM density may be mediated in part by a balance between integrin-mediated cell adhesion forces and myosin-mediated cell contractility [14
]. The spacing between integrin ligands also affects cell spreading and migration [15
], and grouping of integrin ligands in a clustered pattern has been shown to decrease the overall density of ligands necessary to support cell migration [16
], suggesting that the local density of integrin ligands is more important than the global density. Cells also exhibit a phenomenon known as haptotaxis, or cell migration in response to a concentration gradient of adhesion ligand [17
], a behavior that clearly requires the ability to direct cell migration in response to changes in ECM density.
Although it is clear that cells can sense and respond to different concentrations of ECM proteins adsorbed to a surface, it is unknown if this behavior is simply a result of differences in the number of integrin-ECM bonds and the resulting decrease in adhesion strength, or if cells can sense the availability of ECM binding sites and respond accordingly by regulating focal adhesion dynamics. Moreover, it is currently unknown what effect ECM density has on the clustering behavior of integrins. Given the important role of integrin clustering in supporting and regulating cell adhesion and migration [19
], it is essential to understand how ECM density affects integrin clustering and ensuing focal adhesion formation. In this work, we characterize how integrin clustering changes as a function of ECM density by measuring the properties of integrin clusters formed in cells adhering to different concentrations of ECM protein. By implementing a labeling, measurement, and analysis technique designed specifically to identify bound integrins accurately, we are able to quantify the differences in integrin clusters present in cells adhering to different concentrations of ECM proteins.
Cluster properties such as size, shape, and location within the cell, are intrinsically non-uniform, showing significant variability within the same cell and between cells. Any attempt to characterize such heterogeneous population properties with their respective averages, while convenient, will largely be ineffective: large variability will obscure changes to mean cluster properties, making it difficult to determine with reasonable precision, the effects of different experimental variables on integrin clustering behavior. Proper characterization of the effect of different experimental conditions on integrin properties cannot be based on mean properties only; instead, we propose the use of appropriate probability distribution models to characterize the population behavior of integrin cluster size, shape, and location within the cell. The parameters of the probability distribution models used to describe integrin clusters in cells adherent to different concentrations of ECM are then used to quantify how cells alter integrin clustering behavior in response to adhesion on different concentrations of immobilized ECM proteins.