Mechanotransduction at podosomes

• Applicant: Professor Dr. Stefan Linder
• Subject Area: Cell Biology
• Term: from 2017 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 336942737

This project investigates the molecular mechanisms that control mechanotransduction at podosomes in primary human macrophages. Podosomes are multifunctional organelles that combine several key functions of invasive cells, including adhesion, mechanosensing and extracellular matrix degradation. Here, we focus on the co-regulation of mechanosensing and matrix degradation, by exploring the regulatory links between actomyosin-based contractility of podosomes, tension-dependent stretching of adhesion plaque proteins, and microtubule-based transport of proteinases.Podosomes feature an intricate architecture, with a core of branched actin surrounded by a layer of unbranched actin cables that link the top of the podosome to the ring of adhesion plaque proteins. In addition, a cap structure was found to cover the podosome. We have identified three cap proteins, LSP1, supervillin, and INF2, all of which play crucial roles in the regulation of actomyosin-based contractility. Podosomes are indeed contractile structures, and current models propose that growth of the actin core leads to forces on the lateral cables and stretching of attached ring proteins. Cycles of growth and contractility would thus lead to the demonstrated oscillatory protrusion of podosomes into the underlying matrix, which is essential for their mechanosensing ability. This proposal investigates the molecular pathways that are involved in mechanotransduction at podosomes, especially in regard to i) actomyosin regulation by cap proteins such as LSP1, supervillin and INF2, ii) the role of tension-sensitive adhesion plaque proteins such as talin and vinculin and iii) the coupling between mechanotransduction and microtubule-dependent trafficking of proteases.Together with national and international experts we will use a combination of i) molecular biological techniques including expression of fusion and mutant proteins, siRNA-mediated knockdown, immunoprecipitation and GST pull down, ii) microscopic techniques such as confocal live cell imaging, tension sensing by FRET probes, protrusion force microscopy (PTM), and the newly developed Elastic Resonator Interference Stress Microscopy (ERISM), and iii) advanced cell-based assays including software-based analysis of podosome parameters, analysis of extracellular matrix degradation, and proximity ligation assays (PLA) to detect subcellular interaction of proteins.Collectively, these interdigitating lines of investigation will not only lead to novel data on the function of podosome substructures and their respective role in regulation of actomyosin contractility, but also reveal the molecular mechanisms that allow mechanotransduction at podosomes. Our investigations will thus help to uncover new regulatory principles that link key functions of adhesion structures such as substrate adhesion, mechanosensing and extracellular matrix degradation, which are crucial not only for macrophage biology, but also for cell migration and invasion in genera

• DFG Programme: Research Grants