Single-Molecule Force Imaging Reveals That Podosome Formation Requires No Extracellular Integrin-Ligand Tensions or Interactions.

Podosomes are integrin-mediated cell adhesion units involved in many cellular and physiological processes. Integrins likely transmit tensions critical for podosome functions, but such force remains poorly characterized. DNA-based tension sensors are powerful in visualizing integrin tensions but subject to degradation by podosomes which ubiquitously recruit DNase. Here, using a DNase-resistant tension sensor based on a DNA/PNA (peptide nucleic acid) duplex, we imaged podosomal integrin tensions (PIT) in the adhesion rings of podosomes on solid substrates with single molecular tension sensitivity. PIT was shown to be generated by both actomyosin contractility and actin polymerization in podosomes. Importantly, by monitoring PIT and podosome structure in parallel, we showed that extracellular integrin-ligand tensions, despite being critical for the formation of focal adhesions, are dispensable for podosome formation, as PIT reduction or elimination has an insignificant impact on structure formation and FAK (focal adhesion kinase) phosphorylation in podosomes. We further verified that even integrin-ligand interaction is dispensable for podosome formation, as macrophages form podosomes normally on passivated surfaces that block integrin-ligand interaction but support macrophage adhesion through electrostatic adsorption or Fc receptor-immunoglobin G interaction. In contrast, focal adhesions are unable to form on these passivated surfaces.