Force sensors in cells

2.7 Force sensors in cells

Force sensing proteins can be ion channels or involved in opening ion channels, binding to other proteins and form complexes, undergo conformational changes enhancing protein bindings or stimulate a regulatory pathway. Mechanosensitive (MS) channels change structural conformation when activated. In dorsal root ganglion neurons two types of mechanosensitive ion-channels have been found ([133], [134]), a distinct Ca²⁺ selective and a non-selective cation channel. Even the 31 kDa small bacterial mechanosensitive channel MscS and the larger MscL from Escherichia coli open, if the surrounding membrane is under tension ([135], [136]). Interestingly, MS channels impact other stress sensing structures like SFs. Inhibition of MS ion channels minimizes stress fibre rearrangement, that under substrate stretching conditions reorganize towards the stretching direction ([137]). FAs themselves can be considered force sensors. Stress sensitive proteins at FAs like p130cas ([138], [139]) when activated by vinculin binding, responds to a certain level of sheer stress and detaches from the adhesion site eventually, causing the FAs’

disassembly. The force leads to a local extension of the p130cas protein enhancing phosphorylation by Src kinases([138], [140]). Crk-associated substrate (CAS) binds vinculin, provides phosphorylation sites when stretched and influences focal adhesion size ([140]).

Furthermore, prevention of CAS binding to vinculin causes reduced traction force generation [140]. The probability to maintain a FA decreases exponentially with increasing pulling force [141]. Nonetheless, under constant and tolerable tension FAs are not only growing, but also the SFs gain stability due to reduced binding of severing proteins like cofilin [142].

Non-intuitively, the force a cell receives from a FAs does not depend on the FA size but on their function. In migrating cells, the new built tiny FAs at the cells’ leading edge transmit stronger forces than the larger spots in the mid to back region of the cell [143]. Furthermore, the forces FAs transmit to the cell are not constant but depend on substrate elasticity. Soft substrates provide less stability than stiffer substrates, impeding cell adhesion, migration and varying protein distributions inside a cell. For example, distribution of proliferative proteins like Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) is regulated by substrate stiffness. In MSCs on soft hydrogels YAP/TAZ were predominantly cytoplasmatic, whereas on stiffer substrates they became nuclear. [144] Differentiation also impacts a cells’ traction forces. After one week in osteogenic-differentiation medium, differentiating cells showed continuously more and higher traction forces than non-differentiating or control cells. Whereas in adipogenic medium differentiating and non-differentiating hMSCs showed higher traction forces after the first day, but those decreased thereafter. Also non-differentiating cells were more contractile than differentiating or control cells. [145]

Stem cells are very sensitive to their chemical environment, yet able to sense their physical environment as well [146]. Most tissue cells are adherent cells and need to form contacts with the underlying substrate to survive. Point-like contact regions, so-called focal adhesion sites (FAs), are distributed along the cell-substrate surface. If not being disassembled, FAs

will grow (”mature”) over time. In the beginning, the adhesion sites are tiny spots that connect cell cytoskeleton and substrate via the integrin transmembrane protein family [147], [148]. At the cytoplasmatic side, vinculins are one of the first proteins binding integrins.

Stabilising integrin-substrate bindings [149], it connects FAs to the actin cytoskeleton [149] and recruits talin to FAs [150]. Talin connects integrins to filamentous actin (f-actin).

As soon as talin gets stretched, the folding conformation changes and new binding sites are exposed, so vinculin can bind [151]. A mature FA complex is composed of over 60 different proteins. FAs are interconnected via so-called stress fibres (SFs) that consist of actin filaments, myosin II motor proteins and cross-linking proteins. Walking along two parallel actin filaments or filament bundles at the same time but in opposite directions, myosin motor proteins cause sheer stress throughout the cell. Actin cross-linking proteins like Arp2/3 bind to vinculin at the FAs [152]. Once under tension and bound to SFs, stress-sensitive proteins like zyxin are enabled to bind to FAs [153]. Accumulating more proteins, the FAs develop three diverse layers:

1. integrin signalling layer close to the membrane (integrin cytoplasmatic tails, focal adhesion kinase and paxillin),

2. an intermediate force-transduction layer (talin, vinculin) and

3. on top an actin-regulatory layer (zyxin, vasodilator-stimulated phosphoprotein [VASP] and alpha-actinin) [154].

FAs are able to transduce shear stress inside the cell and sense forces as well. To detect environmental details, different types of force sensors are used by the cell.

2.7.1 Cells reshaping their environment

Cells are not only able to sense forces but capable of applying forces to their surrounding, as well. To our knowledge the first experiments with seeded cells (chicken heart fibroblasts) on silicon substrates were done by Harris et al. in 1980. The adherent cells exerted forces to the substrate via FAs, causing the substrate to wrinkle [155]. The length and height of the wrinkles are related to the applied forces by the cell. But wrinkles of these gels were sometimes larger then the cells themselves and by stretching two different positions, chaotic wrinkling effects showed in the intermediate area. Following up, a more quantitative method to measure substrate deformations is traction force microscopy ([156], [157], [158], [159], [160]). Here fluorescent beads are embedded in a silicon or polyacrylamide substrate.

The position of the beads is measured with and without cells, so a bead displacement map is created showing direction and bead shifting in µm scale. For this method, a higher surface tension is required than in the gels Harris et. al. used to avoid wrinkling effects that add noise to the bead displacement data. To measure the force a cell can possibly apply to a certain position, optical traps or tweezers can be used. Here beads are

2.7. Force sensors in cells 21

coated with binding proteins such as fibronectin or the Arg-Gly-Asp (RGD) fibronectin binding sequence and introduced to the cell. It will form focal contacts to the coating, then the bead can be displaced by using the optical trap. Since the applied forces are controlled, the exerted forces by the cell can be measured in pN range. The cell response is cell-type dependent [161], but can be affirmed by using other methods like the atomic force microscope (AFM) at the same samples [162]. Instead of using optical traps or tweezers, Wang et al. [163] used a magnetometry system. They allowed adherent endothelial cells to bind spherical ferromagnetic microbeads, coated with RGD. Then they applied a strong magnetic field and a weaker one shifted by 90^◦ to be able to twist the beads and apply a distinct shear stress inside the cells. Cells bound to the beads became stiffer and increased their resistance to the applied stress, so that a bead rotation could only be done up to 25^◦. [163] Even MS proteins can be used to measure forces inside a cell. Grashoff et al. [164]

introduced a fusion protein, which can be related to as a tension sensor molecule with a force measuring sensitivity in the pN range. Here two fluorophores mFTP1 and venus are connected via a short flexible amino acid domain, where the fluorophores engage in efficient fluorescence energy transfer. Exposing the construct to a mFTP1 exciting wavelength, the venus signal will increase if the fluorophores are close and decrease otherwise. Comparing the signals, the stretching status of the protein can be followed and the force needed calculated [164].