Pushing, pulling, and squeezing our way to understanding mechanotransduction

Abstract

Mechanotransduction is often described in the context of force-induced changes in molecular conformation, but molecular-scale mechanical stimuli arise in vivo in the context of complex, multicellular tissue structures. For this reason, we highlight and review experimental methods for investigating mechanotransduction across multiple length scales. We begin by discussing techniques that probe the response of individual molecules to applied force. We then move up in length scale to highlight techniques aimed at uncovering how cells transduce mechanical stimuli into biochemical activity. Finally, we discuss approaches for determining how these stimuli arise in multicellular structures. We expect that future work will combine techniques across these length scales to provide a more comprehensive understanding of mechanotransduction.

Keywords: Mechanical stress; Morphogenesis.

Figure 1. Mechanotransduction converts mechanical stimuli into instructions for biochemical signaling and gene expression

Proteins involved in mediating the cellular response to mechanical cues include stretch-activated ion channels, the transcription factors YAP/TAZ and MRTF-A, and FAK, a key component of cell-matrix adhesions.

Figure 2. Probing mechanotransduction on the molecular scale

(A) Optical tweezers use radiation pressure to exert force on a bead or particle. The schematic on the right depicts an experimental setup for investigating force-dependent formation of the cadherin-catenin complex. From [28]. Reprinted with permission from AAAS. (B) Magnetic tweezers apply force to superparamagnetic beads proportional to the gradient of a magnetic field. The schematic depicts an experimental setup for investigating force-dependent binding of vinculin to talin. From [22]. Reprinted with permission of AAAS. (C) FRET-based techniques provide a way to measure molecular scale forces and changes in conformation. Force can be calculated from FRET measurements by using a tension-sensing module inserted into a protein of interest (e.g. vinculin). Proper calibration provides a link between FRET efficiencies and forces. Adapted with permission from Macmillan Publishers Ltd: Nature [36], copyright 2010. Additionally, changes in fibronectin conformation have been observed by labeling the protein with a green donor and red acceptor fluorophore. Measurements of high FRET (red signal) near the cell interior suggest fibronectin exists in a compact state, whereas low FRET (green signal) near the cell periphery suggests the protein in that region is in an extended state. Adapted with permission [42]. Copyright by the National Academy of Sciences.

Figure 3. Cellular scale techniques to investigate mechanotransduction

(A) Cell-level forces can be calculated by measuring displacements of embedded beads [46] or elastic micropillars [53]. These forces are a function of the displacements, elastic modulus E, and Poisson’s ratio ν. Displacements are exaggerated for ease of viewing. The scanning electron micrograph at right demonstrates the use of elastic micropillars to measure forces exerted by contracting smooth muscle cells. Adapted with permission [50]. Copyright by the National Academy of Sciences. (B) Stresses exerted by cells in vivo can be inferred by monitoring the deformations of fluorescent oil droplets conjugated with adhesion molecules. Adapted with permission from Macmillan Publishers Ltd: Nature Methods [58], copyright 2013. (C) Infrared radiation-mediated release of calcium or calcium chelators from liposomes provides spatiotemporal control of the moduli of alginate-based hydrogels [66]. (D) Micropipette aspiration was combined with microfluidics to apply suction pressure to an array of trapped cells. This technique was used to estimate the threshold cortical tension required to activate mechanosensitive ion channels, as seen by the uptake of a nuclear-localized, membrane-impermeable dye in the bottom image. Adapted from [68] with permission of The Royal Society of Chemistry. (E) Optogenetics provides a new way to precisely apply loads on the cellular scale. Shown here is a photoactivatable form of RhoA-mediated cellular contractility in which stress fiber formation is controlled by exposure to the photo-stimulus. Reprinted with permission from Macmillan Publishers Ltd: Nature Methods [73], copyright 2013.

Figure 4. Techniques to investigate mechanotransduction on the scale of tissues

(A) Laser microdissection used to probe the state of mechanical stress in the yolk syncytial layer (YSL) during early zebrafish development. (EVL; enveloping layer) Reprinted with permission from Springer Science and Business Media [85]. (B) Monolayer stress microscopy uses the traction forces obtained with TFM and incorporates them into a force balance for the epithelium to compute the 2D mechanical stress field within an epithelial layer. Reprinted with permission from Macmillan Publishers Ltd: Nature Materials [87], copyright 2011. (C) Extending TFM to 3D. The tracked motion of fluorescent microspheres embedded within the surrounding gel can be used to estimate the traction stresses induced by 3D micropatterned tissues in culture. With permission from Springer Science and Business Media [90]. (D) Microfluidic channels can be used to induce defined spatial patterns of contractility within developing epithelia. Reprinted with permission [39].