NanoPlan - Deciphering the molecular construction rules of integrin-based cell adhesions.

• Applicant: Professor Dr. Carsten Grashoff
• Subject Area: Cell Biology
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 316712267

Cell adhesion to the extracellular matrix (ECM) is fundamental to many developmental and pathophysiological processes. It is mediated by integrin receptors that bind to a wide range of extracellular ligands and intracellularly connect to the actin cytoskeleton in macromolecular structures called focal adhesions (FAs). FAs are highly complex assemblies containing hundreds of different proteins processing chemical as well as mechanical signals during cell adhesion; however, the molecular processes that govern FA organization and mechano-chemical signaling remain incompletely understood. Furthermore, mammals express 24 distinct integrin receptors with specific ligand binding and signaling properties but the underlying mechanisms mediating integrin receptor specificity are still unclear. The major obstacle for a better understanding of integrin function has been the lack of suitable techniques that allow a systematic investigation of FAs with sufficient temporal and spatial resolution. We, the members of this French-German Consortium, have therefore developed techniques that will allow such an analysis. We have established unique cell systems to study distinct integrin receptors and their intracellular regulators at physiological expression levels and we have developed super-resolution microscopy methods to study the dynamic nanoscale organization of individual FAs proteins. In addition, we have developed mass-spectrometry approaches to study the molecular composition of FAs and engineered biosensors to quantify integrin force transduction with piconewton sensitivity in cells. We propose to combine our tools to investigate how integrins and their associated molecules regulate the nanoscale organization of FAs to modulate chemical and mechanical signaling during cell adhesion in an integrin subtype specific fashion.In the first part of the project, single-protein tracking and super-resolution microscopy will be used to determine the dynamics of distinct integrin subunits in genetically modified cells. We will combine super-resolution microscopy with nano-patterning to control the lateral spacing of integrin subtype specific ligands and apply molecular tension sensors to correlate FA nanoscale organization with integrin force transduction. In the second part, we will analyze the role of integrin regulators on FA nanoscale organization, molecular dynamics and force transduction. Finally, we will evaluate how externally applied forces are processed by distinct integrin receptors and their associated molecules. These experiments will utilize custom-built cell stretchers that allow super-resolution microscopy as well as molecular force measurements in living cells. Together, the proposed experiments should provide unprecedented insights into the molecular construction rules of FAs. The expected results should be valuable to the cell adhesion community and be of general interest to the cell biological and biophysical community.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partners: Privatdozent Dr. Grégory Giannone, Ph.D.; Dr. Olivier Rossier