Controlling Cell Geometry Affects the Spatial Distribution of Load Across Vinculin

Abstract

The shape of adherent cells is known to be a key determinant of cellular processes such as proliferation, apoptosis, and differentiation. Manipulation of cell shape affects stem cell differentiation, gene expression, and the response of cells to mechanical stimulation. Shape sensing is at least partially due to mechanically-sensitive signaling within focal adhesions (FAs). Therefore, we evaluate the dependence of cellular force generation on cellular geometry by measuring loads across the FA protein vinculin using an engineered Fo¨rster Resonance Energy Transfer-based biosensor. To control cellular geometry, vinculin deficient mouse embryonic fibroblasts stably expressing the vinculin sensor were confined to specific shapes of the same area using photopatterning techniques. It was observed that the tension across vinculin increases at the edges of the cells. However, vinculin supporting compressive loads was found in the center of cells, with an increase in compression observed with increasing aspect ratio. This phenomena is consistent with observations of compressive forces directly under the nucleus and supported by our observation of increased nuclear deformation and enhanced apical actin organization, though this is the first time compressive loads across vinculin have been shown to maintain FA assembly. This suggests a new paradigm for mechanosensitivity in adhesion mechanobiology, where the compressive and tensile loads across particular proteins must be considered.

Improving Quality, Reproducibility, and Usability of FRET‐Based Tension Sensors

“…See Materials and Methods for additional details on this analysis. Comparison of over 400 cells individually characterized using the mean‐, median‐, and mode, both with and without bootstrapping, demonstrates that using the bootstrapped mode results in a population average that most closely matches the estimate from previous work but without the use of user‐defined exclusion criteria (Fig. ).…”

“…D). Comparison of these calculations to our previously published cell‐averaged method (mean = 2.25, se = 0.155) demonstrates that the mode‐based estimation provides similar results to our previous approach without the need for user‐defined exclusion criteria used in the previous work.…”

“…( C ) Comparison of the four methods used to estimate k . methods include a previously described cell‐averaged approach and our pixel‐based approach using the mean, median, or mode. Data and error bars are the mean and standard error, respectively, from both constructs across four independent experiments.…”

“…A similar mode‐based approach was incorporated into the analysis of FRET‐based tension sensors to increase the reliability and consistency of characterizing the molecular loads experienced by vinculin. While the intention of this article was to improve FRET measurements of VinTS, our previous work has already validated the function of this sensor . Notably, recent modeling efforts have described the effects of the rotational mobility of the fluorophores and the flexibility of the linker .…”

Using Nesprin Tension Sensors to Measure Force on the LINC Complex

“…The FRET-force relationship of the sensor was previously calibrated and found to have a dynamic range of 1–5 pN [13]. This sensor module was recently shown to respond to compressive loading, with higher FRET than in the unloaded state [14]. We and others have inserted the sensor into a number of proteins at cell-cell [15–18] and cell-matrix [13, 19] adhesions, indicating that the dynamic range of the sensor is well suited to study forces on proteins.…”