The Nanoscale Architecture of Force-Bearing Focal Adhesions

Abstract

The combination of micropillar array technology to measure cellular traction forces with super-resolution imaging allowed us to obtain cellular traction force maps and simultaneously zoom in on individual focal adhesions with single-molecule accuracy. We achieved a force detection precision of 500 pN simultaneously with a mean single-molecule localization precision of 30 nm. Key to the achievement was a two-step etching process that provided an integrated spacer next to the micropillar array that permitted stable and reproducible observation of cells on micropillars within the short working distance of a high-magnification, high numerical aperture objective. In turn, we used the technology to characterize the super-resolved structure of focal adhesions during force exertion. Live-cell imaging on MCF-7 cells demonstrated the applicability of the inverted configuration of the micropillar arrays to dynamics measurements. Forces emanated from a molecular base that was localized on top of the micropillars. What appeared as a single adhesion in conventional microscopy were in fact multiple elongated adhesions emanating from only a small fraction of the adhesion on the micropillar surface. Focal adhesions were elongated in the direction of local cellular force exertion with structural features of 100-280 nm in 3T3 Fibroblasts and MCF-7 cells. The combined measure of nanoscale architecture and force exerted shows a high level of stress accumulation at a single site of adhesion.

Bone‐forming cells with pronounced spread into the third dimension in polymer scaffolds fabricated by two‐photon polymerization

“…The mechanical forces exerted by the NBDS fibroblast cells on the walls may be similar to those resulting in the bending of polydimethyl siloxane (PDMS) micropillar arrays described in the literature 21. This mechanical action may also explain the observation in Figure 8, where it seems that the cells exert forces on the scaffold walls, which leads to partial destruction of the polymer structure.…”

The integrin expression profile modulates orientation and dynamics of force transmission at cell–matrix adhesions

“…Micropillars were used for cellular traction force measurements according to methodology described previously (Trichet et al, 2012;van Hoorn et al, 2014). A negative silicon wafer master was made using a two-step deep reactive ion etching (DRIE) process.…”

“…A mask with 10610 mm arrays with circles of 2 mm diameter and 4 mm center-to-center distance in a hexagonal grid was used as a negative for the micropillar arrays and a mask with two rectangular spacers of 1062 mm was aligned on the sides of the arrays. The etching depth was varied for the micropillar arrays to make short and long pillars, calculated to have a bending stiffness of 66 nN/mm and 16 nN/mm, respectively, using finite element modeling (van Hoorn et al, 2014). Using a published elastic model (Ghibaudo et al, 2008), we calculated that these bending stiffnesses corresponded to a Young's modulus in continuous (e.g.…”

“…Pillar deflections were determined with ,30 nm precision using a specifically designed Matlab script (van Hoorn et al, 2014). Briefly, the exact pillar locations were determined from the labeled fibronectin fluorescence image using a fit to the cross-correlation function between a perfect binary circle and the local fluorescence of one pillar.…”

Elucidating the molecular mechanisms underlying cellular response to biophysical cues using synthetic biology approaches

“…More recently, the experimental combination of live cells on micropillars with super-resolution microscopy has been demonstrated and allows for the relation between cell traction forces and the number of molecules (fluorescently tagged paxillin in this case) in a focal adhesion complex to be determined. 29 However, nanotopographic surfaces do not take into account an out-of-plane component which is relevant for certain cell types e.g. epithelial cells, which form curved assemblies e.g., tubular vessels.…”