Mechanotechnology N4 Pdf

[Fito Coulter]

Thankyou for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Akin to the widely studied focal adhesions (FAs), podosomes have been shown to exert mechanical forces and to respond to ECM stiffness14,15,16,17,18,19,20. Whereas FAs assemble into fibrillar microscale structures that apply contractile forces to the substrate14,21,22,23,24,25, podosomes assemble into a columnar architecture consisting of an actin core surrounded by a ring complex containing adhesome proteins including integrin receptors26. The actin core protrudes from the cell body, applying nN compressive forces onto the underlying substrate16,27,28. Given that a static cell cannot experience a force imbalance29, it is widely recognized that podosomes apply opposing tensile forces, with some disagreement as to the requirement for integrin adhesion forces26,27,28,30,31,32,33. Mathematical modeling suggests that these tensile forces are localized to the podosome ring27, and there are two lines of experiments that support this model. The first comes from biophysical measurements of talin extension18 and vinculin tension28 within podosomes. These measurements are indirect as they fail to map the molecular forces applied by the podosome itself. The second class of measurements reports bulk substrate deformations using traction force microscopy (TFM). While TFM provides a direct measure of cellular stresses, the spatial and force resolution of the method precludes mapping the forces at the podosome ring complex. A more sensitive variant of TFM that is interferometry based offers improved force sensitivity but still averages deformations of the substrate34 and thus cannot quantify receptor forces. 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.

Further confounding podosome mechanical models, recent results demonstrated the formation of podosome-like adhesions on supported lipid bilayers (SLBs)35,36. SLBs are phospholipid membranes that self-assemble onto a glass slide. Lipids are confined in the z-direction but are laterally fluid37. Thus, on SLBs, podosomes are reported to form even in the absence of traction forces35,38, which is confounding since podosomes apply compressive forces on the SLB.

Finally, we model pN integrin forces in podosomes and demonstrate that podosomes exert nN vertical forces on an SLB, in agreement with previously measured protrusion forces16. Our work offers receptor-level quantitative maps of integrin tension on fluid substrates and provides direct experimental evidence that podosomes are mechanosensors with local pN sensitivity.

We hypothesized that FLIM could be used to map receptor forces on fluid membranes, because fluorescence lifetime is sensitive to energy transfer but is independent of dye concentration54. We functionalized SLBs with FRET-based DNA tension probes containing binary DNA hairpins with a tunable F1/2 threshold and two linker arms; the F1/2 threshold is defined as the force equilibrium at which 50% probes open (Fig. 2)23. The bottom arm was hybridized to a biotinylated quencher strand containing an internal deoxythymidine BHQ1 modification. We selected this site for the quencher to ensure that the probe was FRET quenched, as absorbance spectroscopy demonstrated that conventional MTFM probes are static quenched and thus poorly suited for FLIM (Supplementary Fig. 6). The upper arm of the DNA hairpin was hybridized to a Cy3B ligand strand containing cRGD. At rest, closed probes are FRET quenched, with a low fluorescence intensity and a short fluorescence lifetime. When integrin receptors bind and transport DNA tension probes into nascent integrin adhesions, the probes are clustered, causing an increase in fluorescence intensity. If the applied force equals or exceeds F1/2, then the DNA hairpin unfolds, causing an increase in both fluorescence intensity and fluorescence lifetime (Fig. 2a).

To support our MFM result, we performed emission-resolved polarization measurements49 (Supplementary Fig. 17). Here, any global organization in the lateral component of podosome forces within the ring will lead to bright nodes in fluorescence anisotropy. In agreement with a model of vertical force generation, we found no organized pattern in podosome fluorescence anisotropy. This confirms MFM measurements, indicating that podosomes on SLBs lack an organized traction force component in the plane of the SLB.

A key question in the podosome literature is whether podosome tensile forces are generated directly from core polymerization or whether actomyosin contractility is required to generate tension in the ring27,28,31,32. Our work validates a model (Fig. 7c) of polymerization-induced tension on the ring, as integrin tension was abolished when actin polymerization was perturbed. Few works have reported podosome formation on substrates lacking ligand67,68, leading to a hypothesis that the podosome architecture can sustain tension without receptor forces19. Our TGT data disagree with this model, demonstrating that firm integrin adhesion is required to form stable and protrusive podosomes. In contrast to works performed on rigid substrates28,69,70,71, our data support a model of local podosome force balance that is independent of myosin IIa contractility. Van den Dries et al. reported that myosin IIa inhibition led to increased actin core intensity by shifting the feedback between contractility and protrusion28. We observed a similar increase in core depletion area and tension signal with Rho kinase inhibitor. It will be important to identify which myosins plays a role in this vertical force balance feedback on an SLB. An interesting candidate is myosin 1, which has been shown to localize to podosome cores and to apply forces when anchored to a fluid membrane72,73,74.

Our results should be considered in the context of a few important caveats. While fibroblasts provide a robust model to study podosomes in vitro, to our knowledge, fibroblasts have not been shown to form podosomes in vivo. Another corollary point is that the mechanical properties and the stiffness of the substrate will likely influence podosome dynamics. While MT-FLIM quantitatively maps integrin tension on fluid substrates, this signal is subject to some spatiotemporal convolution, and fast changes in tension will be obscured during the 1-min acquisition. Furthermore, regions of low photon counts such as podosome depletion regions may be disproportionately affected by the diffraction limit. These issues will be addressed as superresolved and faster FLIM electronics become more widely available.

Nevertheless, our work provides valuable mechanobiology tools and insight. SLBs offer a unique landscape to study the minimal mechanical machinery required for podosome formation, and DNA probes provide a powerful method to map and manipulate molecular forces. While RGD ligands on an SLB are more mobile than in physiological ECM, their mobility can recapitulate degraded ECM75, which is relevant to podosome and invadosome biology1,13. During cancer cell invasion, cells alternate between periods of integrin and invadosome-mediated matrix degradation and migration30,76. Thus, podosome retraction following adhesion photocleavage may provide a model to understand how changes in tension can regulate function. While MT-FLIM has some current limitations in its temporal resolution, this method offers a unique solution to mapping forces at fluid interfaces and is a departure from past methods in its incorporation of a parameter that uniquely reports forces and is not subject to intramolecular fluorescence crosstalk. PC probes provide a method to perturb individual adhesion structures with minimal disruption to the cell body and without changing the extracellular environment77. Beyond podosome biology, MT-FLIM and PC probes will be useful in studying immune cell interactions, Notch-Delta signaling, and adherens junctions42,78,79. Fluorescence lifetime is an improved indicator of density versus tension, and because hairpin probes unfold specifically under receptor tension, all measured forces can be attributed to integrins rather than to the vertical force balance vector that arises at the contact line between a liquid droplet applying pressure on a solid substrate80. In conclusion, we introduce and apply powerful DNA mechanotechnology tools to demonstrate the role and regulation of receptor forces on fluid substrates.

3a8082e126