Actin polymerization is vital for maintaining cell shape and integrity, establishing cell polarization, internalizing membrane vesicles and sensing environmental forces. During cell migration, lamellipodial and filopodial protrusions powered by actin polymerization push the cell membrane forward, whereas podosomes and invadopodia couple actin polymerization to the delivery of matrix metalloproteinases to create paths for cells to migrate through dense extracellular matrices. Remarkably little is known at the structural level about how the molecular players of the actin machinery cooperate inside cells to generate forces through network assembly and contractility.
The actin cytoskeleton is linked to the plasma membrane through the integrin adhesome, a complex network of hundreds of interacting proteins. How adhesion molecules are organized to regulate the structural functions of cell-matrix adhesions remains elusive, partly because classical structural techniques either alter morphological details or rely on protein extraction and purification.
Among cellular actin structures, we chose to investigate the nanoscale architecture of human macrophage podosomes. Branched actin networks and adhesion proteins are closely linked in these mechanosensitive adhesion structures, therefore representing ideal tools to investigate the coupling of both machineries. Podosomes present a central core of actin filaments surrounded by an adhesion ring comprising integrins and transducer proteins linking integrins to the actin cytoskeleton. How podosomes enable cells to sense and respond to their environment remains obscure.
The proposed research aims at employing technological advances in cryo-electron tomography (cryo-ET) to reveal the structural basis of key aspects of podosome function and mechanics. Building on our complementary expertise in cryo-ET and podosome mechanobiology, we propose to picture in unperturbed cellular environments (“in situ”):
1- The molecular architecture of actin filaments and their regulators, including actin branches and crosslinkers, and elucidate the molecular mechanism by which protrusion forces are generated and transmitted.
2- The composition and structure of a megadalton protein adhesion complex and its role in podosome mechanosensing.
To sum up, we will combine cryo-ET, nanoscale force measurements, quantitative 3D nanoscopy and computational modelling to reveal the nanoscale organization of the actin polymerization and adhesion machineries in podosomes, and elucidate in situ the structural mechanisms of cellular mechanosensing. These results will shed light on the structural determinants of force generation in podosomes, and provide high-resolution structures of key actin- and adhesion-related proteins not attainable by any other structural approach.
Monsieur Renaud Poincloux (INSTITUT de PHARMACOLOGIE et de BIOLOGIE STRUCTURALE - CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IPBS - CNRS INSTITUT de PHARMACOLOGIE et de BIOLOGIE STRUCTURALE - CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
MPI Max Planck Institute of Biochemistry
Help of the ANR 200,000 euros
Beginning and duration of the scientific project: September 2021 - 24 Months