Combining mechanical and optical approaches to dissect cellular mechanobiology

Abstract

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra-and intermolecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.

Mechanical Forces in Cutaneous Wound Healing: Emerging Therapies to Minimize Scar Formation

“…Advancements in the past decade pertaining to the exploration of mechanical forces on the individual cell and system levels can be attributed to developments in nanotechnology, fluorescence energy transfer-based mechanosensors, atomic force microscopy, traction force microscopy, and magnetic twist cytometry. [63][64][65][66][67] Fibroblasts have been extensively studied in biomechanical wound models, and physical forces are known to influence the expression of ECM genes and inflammatory genes involved in scar formation. [68][69][70] Fibroblasts grown in mechanically loaded three-dimensional collagen lattices resembling connective tissue develop dendritic extensions that enable them to migrate and remodel their matrices.…”

Optical measurement of arterial mechanical properties: from atherosclerotic plaque initiation to rupture

“…3). 83,84 Traction forces are then calculated from the bead displacement vector field by solving the inverse problem using a variety of approaches, which include the generation of a quadrilateral mesh to evaluate the traction field 85 or a more computationally efficient fast Fourier transform-based algorithm that recasts the relationship between bead displacements and traction into Fourier space to recover the traction field. 83 TFM has been used to measure traction forces in a variety of arterial cell types to evaluate EC cellcell contacts, 86 to understand the modulation of EC behavior and function in response to ECM stiffening, 82 and to evaluate the tensile properties of stress fibers in VSMCs.…”

Building gyms for cells : development of combinatorial screening platforms for mechanical stimulation

“…Another effect commonly observed was the higher mechanical strength of strained cell hydrogel constructs 34,39 . When non-physiological strains were applied, such as equibiaxial strains applied to smooth muscle cells that reside in an anisotropically strained environment in vivo, the loss of the healthy phenotype was observed 32 .…”

“…For simplicity, the studies where cells were cultured inside hydrogels and a strain was applied are considered in this section. For strain-stimulating cells within hydrogels, increasing the throughput of the experimental platform has also been reported [37][38][39] .…”

“…Calcium levels within the cell are important regulators of functions such as cell contractility and cell motility 38 . In response to mechanical stimuli, the modulation of stretch activated calcium channels leading to variations in intracellular calcium is often an early response and hence of interest for our test platforms 39 . Some biological processes of interest whose mechanical regulation can be investigated using the developed platforms include vascularization, stem cell differentiation and tissue injury by large forces.…”