Analyzing the spatiotemporal regulation of intracellular force transduction in living cells
Final Report Abstract
The objective of this research project has been the molecular analysis of integrin-dependent force transduction in living cells using our previously published tension sensor technique. The experiments were expected to increase our molecular understanding of how mechanical signals are translated into a biochemical response during cell adhesion. We have reached these research goals by developing two novel, single-molecule calibrated biosensors and by applying them to the evolutionary conserved cell adhesion protein talin. The newly developed biosensors present a significant technological advance as they allow the analysis of a previously inaccessible force regime in cells. While our previous method was sensitive to very low forces of only 1‒6 piconewton (pN), the two biosensors developed in this project allow efficient measurements at 6‒8 pN and 9–11 pN, a force regime that appears to be physiologically highly relevant. In addition, our new biosensors benefit from improved dynamic range and a sharp forceresponse threshold that greatly facilitates data interpretation. Application of the new biosensors to talin revealed that this integrin activator establishes a mechanical linkage upon cell adhesion, which bears forces of 7–10 pN and is mechanically regulated by actin and vinculin association. Our experiments demonstrate that the observed mechanical engagement of talin is crucial for cell adhesion-dependent mechanotransduction and we found that, unexpectedly, talin force transduction is isoform-specific such that extracellular rigidity sensing is modulated by differential expression of talin-1 and talin-2. Together, these findings provide a molecular explanation for the inherent coupling of cell adhesion with mechanosensing, a phenomenon that has been puzzling cell biologists for decades. In summary, we have improved our tension sensing technique to analyse mechanical forces in cells that could not be measured previously. Similar to our original technique, which has been applied to study a whole range of biomechanical questions, we expect our improved probes to be widely used by the research community. Our cell biological experiments identified a molecular mechanism that explains how cells feel their mechanical environment, in particular extracellular rigidity. As tissue stiffness changes during development or with the onset of disease states such as cancer, atherosclerosis or tissue scarring, it will be important to investigate the role of the individual talinisoforms during these processes in more detail. The here developed biosensors will be highly useful for these kind of studies and should allow the analysis of force transduction in a wide range of cell types and even whole organisms.
