Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring

Abstract

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Modular actin nano-architecture enables podosome protrusion and mechanosensing

“…It is generally accepted that podosome-mediated protrusive force generation is regulated by an interplay between actin polymerization and myosin IIA activity (21,22,42). Furthermore, recent in silico modelling of podosome force distribution strongly suggested that protrusive force generation in the core is balanced by local pulling force in the ring at the level of single podosomes (20). An explanation, however, for how protrusive and pulling forces are transmitted within the podosome structure remained elusive since no clear structural connection between the core and the ring has been described so far.…”

“…Neighboring podosomes are interconnected by a network of actin filaments that radiate from the podosome core and facilitate a mesoscale (1.5-10 µm) connectivity (17)(18)(19). While individual podosomes are thought to function as micronsized protrusive machineries (20)(21)(22), their mesoscale connectivity presumably facilitates long-range basement membrane exploration for protrusion-permissive spots (18,23). An adequate structural framework, however, that explains podosome protrusion and mechanosensing is still lacking.…”

Super-resolved live-cell imaging using Random Illumination Microscopy

“…In Figure 1C, we compare the reconstructions of the same sample of tagged F-actin network in podosomes 123 obtained by second-order statistics dSTORM (NanoJ software, Super Resolution Radial Fluctuation) 124 (Gustafsson et al, 2016) and RIM. Podosomes are actin-rich, cell adhesion structures applying protrusive 125 forces on the extra cellular environment, recently observed by 3D dSTORM (Bouissou et al, 2017).…”

Superresolution architecture of pluripotency guarding adhesions

“…Talin can adopt multiple conformations in the cell, ranging from a head to tail auto-inhibited conformation to a fully extended, integrin and actin bound mechanically stretched conformation (Yao et al, 2016). To evaluate the 3D FA distribution of talin as well as its vertical extension, we imaged both N-terminally (talin-N) and C-terminally (talin-C) tagged talin-1 (Liu et al, 2015;Bouissou et al, 2017). Overall, the Z-position values for talin-N ( Z centre =72.8± 12.3 nm ) and for talin-C ( Z centre =103.5± 20.5 nm ) are higher than those reported for U2OS cells (Liu et al, 2015).…”