Abstract
Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces.
Highlights
Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance
Whereas focal adhesions (FAs) assemble into fibrillar microscale structures that apply contractile forces to the substrate[14,21,22,23,24,25], podosomes assemble into a columnar architecture consisting of an actin core surrounded by a ring complex containing adhesome proteins including integrin receptors[26]
With tension-gauge tethers (TGTs), which limit the maximum magnitude of integrin tension, we demonstrate that podosomes apply integrin forces in their rings, but that these forces are required to stabilize podosomes
Summary
Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Whereas FAs assemble into fibrillar microscale structures that apply contractile forces to the substrate[14,21,22,23,24,25], podosomes assemble into a columnar architecture consisting of an actin core surrounded by a ring complex containing adhesome proteins including integrin receptors[26]. To the best of our knowledge, no quantitative force maps have been reported validating the role of adhesion receptor mechanics in opposing actin protrusion and mechanically linking the substrate and the cytoskeleton within podosomes. We first quantify podosome-mediated depletion as a marker of actin core protrusion on SLBs. we introduce molecular tensionfluorescence lifetime imaging microscopy (MT-FLIM), producing pN maps of integrin tension during receptor–ligand clustering at the cell membrane. Treatment with pharmacological inhibitors showed that podosome tensile forces are a direct consequence of actin polymerization
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