Micropatterned Binary Nanoarrays as a Tool to Determine the Spatial Requirement of Heterotypic Integrin Clusters
Final Report Abstract
The cell with its hierarchical network of nanomachines uses specific transmembrane receptors, called integrins, to bind extracellular matrices (ECM). Cell adhesion to the ECM is one of the fundamental processes that are central to many developmental as well as pathophysiological processes. The integrin family in mammalian cells consists of 24 distinct heterodimeric receptors often classified according to their binding specificity to extracellular ligands, like arginine-glycine-aspartate (RGD) with a high binding affinity for 8 of the 24 integrins. Upon binding to extracellular ligands, integrins cluster and intracellularly connect to the actin cytoskeleton via macromolecular structures called focal adhesions (FAs). FAs are highly complex, dynamic interfaces processing chemical as well as mechanical information during cell adhesion, and in turn controls cell migration, proliferation, survival and differentiation. However, it remains unclear due to the binding redundancies of ECM proteins how cellular functions are regulated by a single or a mixture of integrin sub-types. To address this, our groups showed that fibronectin-binding αv-class and α5β1 integrins feature different structural and mechanical roles to assemble FAs. Moreover, these integrins are recruited with different stoichiometries and exhibit distinct spatial dynamics within FAs. Although defining these parameters is crucial to understand integrin-mediated mechano-chemical signaling output, further characterization of the integrin properties is needed to dissect their individual and cooperative functions. Therefore, we proposed in the frame of this project to identify the nano-partitioning and tension requirements of αv-class and α5β1 integrins to mediate cell adhesion. To achieve this, we tested if variations in ligand density would differently regulate the role of these two integrin classes. To do so, we used surfaces made of quasi-hexagonally arranged gold particles, acting as an ordered scaffold to immobilize integrin-binding ligands, like a fibronectin fragment. By investigating fibroblasts that either express αV-class (αVβ3 and αVβ5), α5β1, or both integrin classes, we found out that the lateral spacing between integrins differentially regulates cell adhesion and FA maturation depending on the integrin diversity. The critical ligand spacing required to mature FAs lies below 78 nm for cells expressing one integrin sub-type. In contrast, a larger number of cells expressing both integrins adhere to 78 nm-spaced ligands and only undergo a slight cell area reduction while incompletely maturing FAs. However, αv- and αv/α5β1-expressing cells cannot adhere to surfaces with a ligand interdistance of 92 nm, whereas α5β1-cells look similar on both 78 and 92 nm-spaced ligands by displaying elongated morphologies, typical of higher protrusive activity. On substrates functionalized with ligands spaced every 125 nm, cells float without attaching. To rescue cell adhesion, we found that multimers of fibronectin fragment are sufficient for the three cell lines to adhere by forming dense integrin clusters. However, we are still investigating if cells are capable to mix different integrin subtypes to form adhesive nano-clusters. Integrins mediate adhesion but also mechano-transduction, the conversion of mechanical forces into biochemical signals, both involved in cellular functions. Many techniques, like single-molecule force-spectroscopy and traction force microscopy, are used to characterize mechano-sensitive processes, but most of them cannot resolve single molecule forces generated by a defined integrin sub-type because they measure the collective contribution of many integrins. Therefore, we chose a method based on the use of surface immobilized ligands linked to a tether that ruptures at a critical force (tension tolerance) applied by single integrins. Here, we used hybridized DNA strands as rupturable tethers conjugated with ligands that selectively bind to α5β1 or αv-class integrins and with a tunable tension tolerance ranging from 23 to >100 pN. In doing this, we found that the tension applied by a single integrin to its ligand features different requirements to initiate and mediate cell adhesion. Not many cells attach and poorly adhere to α5β1-ligands with a tension tolerance of 43 pN and αvclass ligands of 33 pN. By increasing the ligand tension tolerance, the number of spread cells continuously increases from 43 to >100 pN on α5β1 ligands and from 33 to 55 pN on αv-class ligands. However, the use of rupturable tethers with a unique tension tolerance does not allow defining if all integrins upon ligand engagement require being under the same tension during FA maturation and cell migration. Therefore, the engineering of substrates modified with multiplexed tension-sensitive tethers will be needed to learn more about the mechanism controlling FA reinforcement and integrin turnover, and ultimately define the building plan followed by integrins to assemble functional FAs.
Publications
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A Molecular Toolkit to Functionalize Ti-Based Biomaterials that Selectively Control Integrin-Mediated Cell Adhesion. Chem. Eur. J., 19 (2013), 9218-9223
Rechenmacher, F., Neubauer, S., Mas-Moruno, C., Dorfner, P.M., Polleux, J., Guasch, J., Conings, B., Boyen, H.G., Bochen, A., Sobahi, T.R., Burgkart, 1 R., Spatz, J.P., Fässler, R., Kessler, H.
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Mechanosensitive Feedback Regulation of Myosin-II in Fibroblasts Requires a Cooperation of β1- and αv-Class Integrins. Nature Cell Biology, 15 (2013), 625-636
Schiller, H.B., Hermann, M., Polleux, J., Vignaud, T., Zanivan, S., Sun, Z., Friedel, Raducanu, A., Gottschalk, K., Théry, M., Mann, M., Fässler, R.
