Regulation of podosome dynamics and function by the podosome cap and base

• Applicant: Professor Dr. Stefan Linder
• Subject Area: Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 502808895

Podosomes are prominent adhesion and invasion structures which are especially abundant in monocytic cells such as macrophages. They fulfill a plethora of critical functions such as cell-matrix adhesion, extracellular matrix degradation, and mechanosensing, and are thus crucial for immune cell migration and invasion. This multitude of functions requires exquisite spatiotemporal control. Podosomes thus contain more than 300 different components, and their internal architecture is accordingly complex. The current project is focused on two poorly understood podosome substructures, the cap on top of the actin core, and the base at the cell-matrix interface. We have identified the actin binder and endocytosis regulator swiprosin-1, the formin INF2, and the actin regulator drebrin as novel components of the podosome cap, as well as the unconventional myosins Myo1e and Myo1f as components of the base. We could already show that each of these proteins regulates specific aspects of podosome dynamics, architecture and function, including dynamic contact with microtubule plus tips, a prerequisite for regular podosome turnover. However, the respective molecular mechanisms are unclear. It is also unknown how both substructures integrate with the core and ring substructures to form a fully functional podosome. We thus aim to unravel the molecular mechanisms of these novel cap and base components, their contributions to podosome dynamics and function, and to identify their interactors. The work programme is structured in 4 work packages, with WP1-3 focusing on distinct proteins at the cap or base, while WP4 represents an integrative approach to identify both specific interactions between components of the cap, base and other substructures, and also to unveil more global interactomes, as well as substructure architecture.To investigate this, we will apply a combination of techniques, including i) molecular biological techniques such as siRNA-mediated knockdown, expression of fusion, mutant and rescue constructs, coimmunoprecipitation, GST/MBP pulldown, actin cosedimentation, and phosphoinositide binding assays, ii) microscopical techniques such as confocal live cell imaging and various superresolution techniques including 3D STED or SoRA, iii) software-based analysis of podosome parameters, architecture, and contact with microtubule plus tips, and iv) advanced cell-based assays such as matrix degradation assays, proximity ligation assays, as well as BioID labeling, coupled with mass spectrometry. For this, we have teamed up with national (Dirk Mielenz; swiprosin-1) and international (Mira Krendel: Myo1e, Myo1f) experts.Results gained from this study will reveal the roles and molecular mechanisms of podosome cap and base in the regulation of podosome architecture, turnover and function. Collectively, these data will also be crucial for a better understanding of environmental sensing and invasive migration of primary human immune cells in health and disease.

• DFG Programme: Research Grants