MACRONANO

Macrophages infiltrate all body tissues which are heterogeneous in composition, stiffness and architecture. This process is critical for immune protection. However, it is also reported to accelerate the progression of several chronic diseases. Control of macrophage migration thus appears as a fundamental issue in biology but also as a pharmacological challenge. We have shown that macrophage mesenchymal migration in 3D environments requires adhesion structures called podosomes. As such, these structures constitute potential targets for new therapeutic approaches to control macrophage migration. Podosomes are dynamic adhesion structures organized as a sub-micron size cone-shaped F-actin surrounded at its base by a ring of adhesion proteins, integrins, and proteins linking integrins to the actin cytoskeleton. Podosomes are interconnected through a radial F-actin network. Podosomes are thought to play a role in cell mechanosensing through the generation of a protrusive force which has never been measured due to the lack of an adequate method to measure forces perpendicular to the substratum. Thus, understanding of the mechanisms involved in the mechanosensing process at the cell and podosome scales requires the emergence of new technologies of high spatial resolution at the interface between biology and physics. We have recently developed a method dedicated to the evaluation of the forces that podosomes generate to protrude in the extracellular matrix. It consists in measuring by Atomic Force Microscopy the deformations produced by macrophages on a compliant film of Formvar and has been called Protrusion Force Microscopy (PFM). With PFM, we have been able to show that each podosome exert oscillatory forces of ~90nN (Labernadie et al. in revision). Now, our objective is to develop new methodologies based on nanotechnologies to further investigate macrophage mechanosensing properties. The project comprises four tasks: - the identification of proteins located at the podosome ring involved in force generation at podosomes using PFM; - the assessment of podosome mechano-sensing properties, by creating local patterns in the Formvar film of tunable stiffness by Focused Ion Beam milling and measuring the podosome force on these surfaces by PFM; - the determination of the role of the organisation of podosomes as a network, that will be controlled by plating macrophages on designed adhesive landscapes obtained by surface micro-patterning; - the investigation of the mechanics of macrophage migration in a designed 3D structure, obtained by 3D 2-photon lithography. Finally, we will complete a theoretical model that we designed to explain the force generation measured by PFM, so as to include the elements in the podosome ring, lateral cables and podosome interactions/positioning which may be important to control the oscillatory force generation. The results of our project need to be analysed along a biological perspective and a technological one. Indeed, we will generate on the one hand, new non-existing quantitative data on the mechanical and mechano-sensing properties of human macrophages and on the other hand, a validated technology, generic to all cell types, for the dynamical (real time), multi-modal and quantitative investigation of single cell behaviour. To achieve these objectives we will integrate several methods from nanoscience to macrophage biology: atomic force microscopy (AFM) in liquid medium, soft lithography, local etching using Focused Ion Beam technology (FIB), 3D two-photon lithography, Total Internal Reflection Fluorescence Microscopy (TIRF) and RNA interference-based protein depletion (siRNA).