Innovative Tools for Mechanobiology: Unraveling Outside-In and Inside-Out Mechanotransduction

Abstract

Cells and tissues can sense and react to the modifications of the physico-chemical properties of the extracellular environment (ECM) through integrin-based adhesion sites and adapt their physiological response in a process called mechanotransduction. Due to their critical localization at the cell-ECM interface, transmembrane integrins are mediators of bidirectional signaling, playing a key role in "outside-in" and "inside-out" signal transduction. After presenting the basic conceptual fundamentals related to cell mechanobiology, we review the current state-of-the-art technologies that facilitate the understanding of mechanotransduction signaling pathways. Finally, we highlight innovative technological developments that can help to advance our understanding of the mechanisms underlying nuclear mechanotransduction.

Keywords: cytoskeleon; force; integrins; mechanobiology; mechanotransduction; microsystem and macrosystem approaches; signaling/signaling pathways.

Figure 1

Representation of “outside-in” (in gray) and “inside-out” (in brown) mechanotransduction signals in a cell growing in a three-dimensional (3D) fibrous matrix. External forces applied to eukaryotic cells modulate their migration, shape, stiffness, spreading, alignment or adhesion behaviors, whereas the ECM provides multiple cues to cells such as confinement, topography, rigidity and biochemistry. Focal adhesion (FAs, in red) serve as crucial sites for both outside-in and inside-out mechanotransduction through the recruitment of transmembrane integrins. Mechanical signals (curved blue arrows) are converted in biological responses by the nucleus, which is depicted in blue.

Figure 2

Representation of the fundamental quantities involved in the three main modes of deformation used in material characterization: (A) linear elongation, (B) shear deformation and (C) isotropic compression. (D) Viscoelastic models, such as the standard linear model in green, can be obtained from the association of dashpots, that represent viscous Newtonian fluids (in red), and springs that represent ideal elastic solids (in blue). (E) The power law type of relaxation in log–log scale is a line with a slope β, which is the power law exponent. The power law exponents are β = 0 and β = 1 for purely elastic and viscous materials, respectively. Elastic and viscous mechanisms contribute to a complex relaxation response when 0 < β < 1 (Grevesse et al., 2015). (F) The composition and spatial organization of cytoskeletal components significantly influence the cell rheological properties. Epifluorescence image of a C2C12 myoblast immunostained for F-actin (in green), microtubules (in red) and DNA (in blue). The scale bar is 15 μm.

Figure 3

(A) The elasticity of living tissues spans a wide range of rigidities which are organized in three domains: soft (0.1 ≤ E ≤ 1kPa), intermediate (1 ≤ E ≤ 10 kPa) and stiff (10 ≤ E ≤ 100 kPa). (B) Acrylamide (AAm, in black) and bisacrylamide (bis-AAm, in blue) and N-hydroxyethylacrylamide (HEA, in red) monomers were co-polymerized to form a hydrophilic network of polyacrylamide containing hydroxyl groups (hydroxy-PAAm) by random radical polymerization (Grevesse et al., 2013, 2014). (C) The amount of bis-AAm cross-linker allows to modulate the stiffness of hydroxy-PAAm hydrogels. (D) Images of three hydroxy-PAAm hydrogels of various rigidities (from left to right: soft in yellow, intermediate in orange and stiff in red) deformed by a static steel ball that exerts a constant load. The resistance of the hydroxy-PAAm hydrogels against the deformation imposed by the steel ball is proportional to the elastic modulus of the hydrogels. (E) Hydroxy-PAAm hydrogels have superior optical properties, such as high transparency, that do not depend on their mechanical properties.

Figure 4

(A) Microcontact printing allows to from 2D micropatterns of proteins on flat culture substrates from a PDMS structured microstamp (Grevesse et al., ; Versaevel et al., 2014b). Example of fluorescent images of different cell types grown at the single cell and tissue levels on adhesive micropatterns of various geometries and protein coatings. Human Umbilical Vein Endothelial Cell (HUVEC) were grown on (B) a circular micropattern of fibronectin (FN) and stained for tubulin (in red) and DNA (in blue), (C) a rectangular micropattern of FN and stained for vinculin (in red), actin (in green) and DNA (in blue). (D) A cortical neuron was grown on a stripe of laminin (LM) and stained for MAP2 (in green), Tau (in red) and DNA (in blue). (E) A doublet of cortical neurons was grown on a rectangular micropattern of LM and stained for tubulin (in red), actin (in green), and DNA (in blue). (F) A 2D tissue of C2C12 myoblasts was grown on a star-shaped micropattern of FN and stained for actin (in green), troponin T (in red) and DNA (in blue) after differentiation in myotubes. Scale bars are (B) 10 μm, (C) 20 μm, (D) 10 μm, (E) 20 μm, and (F) 50 μm.

Figure 5

Examples of force-application techniques used to probe the rheological properties of cells or to apply well-defined external loads for studying mechanotransduction pathways. From left to right: atomic force microscopy (AFM), micropipette aspiration, optical tweezers, magnetic tweezers and uniaxial stretcher.

Figure 6

(A) Protrusive forces exerted by the lamellipodia of migrating cells can be quantified by measuring the deflection of an AFM cantilever over time. A cantilever of calibrated spring constant is placed vertically perpendicular to the surface in front of the leading edge and its vertical position is measured optically over time. The polymerization of actomyosin filaments exerts a load force that pushes on the cell membrane that deflects the cantilever. (B) Traction force microscopy (TFM) measures the contractile forces exerted by adherent cells on an elastic hydrogel that contains fluorescent embedded beads of ~200 nm in diameter. Cell spreads on the hydrogel, leading to its contraction (stressed state in green). After trypsin detachment, the contractile stress is released, and the fluorescent beads relax (in red). The differences of bead position between stressed and relaxed states serve as markers to visualize the hydrogel deformation in a 2D plane. (C) Gel deformations are estimated using a Fourier-based difference-with-interpolation image analysis. To characterize the contractile forces of each cell, the elastic strain energy stored in the polyacrylamide gel due to cell tractions is calculated as the product of local tractions and deformations, integrated over the spreading area of the cells. The scale bar is 20 μm.

Figure 7

(A) LINC complexes are made of the inner nuclear membrane (INM), SUN protein trimers, the outer nuclear membrane (ONM) and KASH proteins. KASH protein mediates interactions with actin cables through Nesprin 2. (B) A-type and B-type lamins (orange and blue, respectively) form individual polymer networks which are juxtaposed on the inside of the nuclear envelope. Both lamin types are found at the interface between chromatin and the cytoskeleton (Swift and Discher, 2014). The mechanical properties of the lamina can be described by viscoelastic models based on the combination of elastic (spring-like) springs and viscous (flowing) dahspots. The characteristic deformation time of the lamina is therefore related to the ratio of the viscous part (i.e., lamin A) on the elastic one (i.e., lamin B) (Swift et al., 2013). Typical images of nuclei of Madin-Darby Canine Kidney (MDCK) cells stained for lamin A (in orange) and lamin B (in blue). The scale bar is 10 μm. (C) Differential mechanical forces are applied on the nucleus, such as actin contractile forces or microtubules compressive forces, through LINC complexes. The three-dimensional organization of chromosomes is modulated by these mechanical signals, leading to new gene expression programs (Uhler and Shivashankar, 2017).

Figure 8

(A) The optogenetic principle is based on light-sensitive proteins from algae called opsins. (B) Opsins can be expressed in single cells, cell clusters or tissues by using transfection with a gene construct inserted in viruses. By using different wavelengths, optogenetics allows to activate or inhibit for instance neurotransmission, or specific mechanotransduction signaling pathways in living cells, such as neurons.