Instant integrin mechanosensing

Abstract

Single-cell force spectroscopy reveals rapid, biphasic integrin activation and reinforcement of cell-matrix bonds during the initial steps of fibroblast adhesion.

Most cells in the human body attach to the extracellular matrix (ECM), a polymer network connecting tissues, in order to survive and function properly. Cellular adhesion to the ECM is mediated by transmembrane adhesion molecules such as integrins¹. These proteins bind to the ECM protein fibronectin² and are the main contributors for cellular adhesion. In the early 90’s it was shown that integrins also function as a mechanosensor that mediates force transmission to the actin cytoskeleton³, an intracellular polymer network that determines cellular stiffness. Since then several studies have identified intracellular proteins such as talin⁴ and vinculin⁵ that facilitate force sensing and response. While much is known about the force sensing process, a comprehensive understanding of the early processes that regulate adhesion and mechanical signalling at the cell surface, namely how quickly cells sense and respond to force via integrins is still not available. Now, writing in Nature Materials⁶, Daniel Müller and colleagues report that integrin mechanosensing during adhesion is fast and involves the biphasic reinforcement of cell-ECM linkage.

To determine within what time frame and how integrins respond to external forces, Müller and collaborators used atomic force microscopy (AFM)-based single cell force spectroscopy (SCFS). A fibroblast, a type of tissue cells that are essential in wound healing, was attached to an AFM cantilever covered with Concanavalin A, which was then lowered onto a fibronectin coated substrate allowing the cell to become attached via integrin binding. The authors measure the strength and dynamics of the integrin binding by retracting the AFM cantilever containing the fibroblast from the substrate at different speeds. To determine the contribution of different integrin subtypes for adhesion (24 integrin subtypes exist), the authors first knocked out all integrins in wild-type fibroblasts and transfected them with αVβ3 and αVβ1 (αV-class), α5β1 alone or αV-class together with α5β1. They studied the adhesion responses of wild type or αV-class/α5β1 fibroblasts to substrates containing fibronectin fragments specifically recognized by αV-class and α5β1 integrins or substrates with vitronection, which is recognized solely by αV-class integrins. They observed that the fibroblast adhesion force, measured as the maximum deflection of the AFM cantilever before the cell was detached from the substrate strengthens in response to retraction speeds from 1 to 5 μm s⁻¹, decreases for a retraction speed of 6 μm s⁻¹ before increasing again at retraction speeds from 6 to 20 μm s⁻¹, albeit at smaller magnitudes. This biphasic force response was absent in fibroblasts expressing only αV-class integrins and was substantially attenuated in those containing only α5β1. Moreover, fibroblasts expressing αV-class/α5β1 integrins did not respond biphasically when attaching to vitronectin-coated substrates. Overall, these findings suggest that α5β1 integrins strengthen fibroblast-substrate adhesion and that this response is subsequently potentiated by αV-class integrins. Remarkably, this process occurs as early as 2 s after the fibroblast adhered to the fibronectin-coated substrate, with the strengthening of cell adhesion in response to force occurring in less than 0.5 s. This response is much faster than the minimum 60-second requirement for integrin clustering⁷ and recruitment of intracellular partners to assemble dynamic focal complexes and stable focal adhesions, suggesting that the process of integrin mechanosensing and force strengthening is almost instantaneous.

Müller and colleagues then explored the mechanisms governing the rapid integrin biphasic mechanosensing. Talin is an intracellular protein that connects integrins with the actin cytoskeleton and is involved in integrin signalling⁸. As expected, the biphasic force response was abolished when talin was knocked out or the filamentous actin that forms the cytoskeleton was disrupted, since integrin intracellular anchoring points that balance the forces applied to integrin extracellular heads were affected. The authors then showed that at ~40-pN retraction force, the single bond between α5β1 in a live fibroblast and a fibronectin fragment switched from a catch to a slip bound, i.e. from a bound whose lifetime increases with force to a bond whose lifetime decreases with force, which had been shown for purified proteins⁹. This behaviour is dependent on fibronectin’s Arg-Gly-Asp (RGD) tripeptides, the specific sites recognized by α5β1 integrins, reinforcing the finding that integrin biphasic force response is mainly determined by α5β1 integrins. The authors also studied if intracellular partners of activated integrins were involved in this fast force response. Focal adhesion kinase (FAK) and Src kinase family are one of the first downstream molecules that interact with integrin tails for signalling. Inhibition of FAK activity or Src kinase family with specific pharmacological agents abolished the biphasic force response, suggesting that these are involved in this process.

The work of Müller and coworkers reveals that a rapid integrin mechanosensing mechanism is operative in live fibroblasts but several questions remain unanswered. Studies with recombinant integrins suggest that catch-slip bond behaviour can be observed in a single purified α5β1 molecule⁹, whereas in a living cell, FAK and Src kinase family activities seem to be also required. This difference may arise from the activation of additional integrins or the strengthening of already activated integrins. Future studies assessing whether FAK and Src activities are required for the catch-slip bond behaviour in cells expressing a single α5β1 integrin would aid our understanding of this process. It would also be informative to determine if FAK and Src are activated by force before the 0.5 s strengthening response occurs, since the exact time frame at which FAK is activated by mechanical force on fibronectin-coated substrates¹⁰ is not known and Src is activated within 0.3 s after force is applied to integrins¹¹. Furthermore, whereas it is know that that phosphatidylinositol 4, 5-biphosphate (PIP₂) regulates FAK activity¹⁰, Müller and colleagues found that PIP₂ had no effect on the force response of α5β1 integrins but the reason why remains to be resolved.

Regardless of the underlying mechanism, the finding that integrin mechanosensing is essentially instantaneous constitutes a significant step in establishing integrins as first responders in mechanosensing. The work of Müller and colleagues will stimulate researchers in the fields of mechanobiology and mechanomedicine to explore in detail how various integrin subtypes respond to force and how integrins, in crosstalk with cell-cell mechanosensor cadherins¹², regulate cell adhesion, migration and invasion in embryogenesis, physiology, and cancer.

Figure 1. Rapid integrin mechanosensing.

Upon interacting with fibronectin-coated substrates, fibroblasts respond biphasically to force. Initially (t = 0), α5β1 integrins at their cell surface (light and dark blue) bind to fibronectin becoming activated (upright conformation) and strengthening cellular adhesion to the substrate. After this fast response (t = 0.5 s), adhesion is further potentiated by αVβ3 integrins (light and dark green) that stimulate activated integrins and activates additional inactive integrins (bent conformation). These processes are dependent on integrin anchoring to the actin cytoskeleton via talin and activation of intracellular signaling proteins FAK and Src. FAK – focal adhesion kinase; Src – a non-receptor tyrosine kinase; F-actin – filamentous actin.