Cells exposed to mechanical or topological cues

Cells are able to translate mechanical cues into biochemical or electrical signals, a process called mechanotransduction. Not only sensory cells like the hair cells in the inner ear show this capability, but rather all cell-types. Mechanotansduction is crucial for many cellular functions like migration, proliferation or apoptosis.(Lo et al., 2000;

Trichet et al., 2012; Wang et al., 2000) Interestingly, it has also been shown that mechanotransduction can also direct stem cell differentiation. (Engler et al., 2006) However, despite knowledge about multiple effects of mechanical cues on cell behavior, only little is known about the molecular background, by which cells sense the physical properties of their surrounding. Different mediators thought to be involved in mechanotransduction have been reviewed by Donald Ingber.(Ingber, 2006) It is indisputable that stretch-activated ion channels play a role in signal transduction in some cell types like the hair cells in the inner ear. Other mechanical sensors include nuclear structures, cytoskeletal filaments and crosslinkers or cell as well as cell-substrate contacts. To sense changes in cell-substrate rigidity, composition or topography, signaling via integrin-madiated focal adhesion plays a pivotal role.(Geiger et al., 2009)Integrins are transmembrane receptors formed by a α- and a β-integrin subunit.

On the extracellular side, the receptors bind to extracellular matrix proteins like fibronectin or collagen. At the intracellular domain, they are associated to the actin

cytoskeleton via adaptor proteins on the one hand, but on the other hand to a huge number of different proteins, e.g. the protein kinase C (PKC), the tyrosin kinase Src or the focal adhesion kinase (FAK), involved in many cellular processes like migration or proliferation (see Figure 2.6). (Geiger et al., 2009) A detailed picture of proteins and cofactors involved in focal adhesion signaling can be found at http://www.adhesome.org (06.01.2014). The maturation of initial focal complexes, which are formed at the cell edges as initial cell substrate contacts, into focal adhesions has been shown to be dependent on tension generated by the actin network in combination with myosin II.(Geiger and Bershadsky, 2002; Wolfenson et al., 2011)

Figure 2.6: Schematic drawing of focal adhesion complex and mechanotransduction.

Heterodimeric intergrin receptor binds to specific binding motifs on extracellular matrix proteins. Several proteins involved in actin binding and signaling like talin, vinculin, paxillin, FAK or Src bind to the intracellular domain of the receptor. Some of these associated proteins have already been shown to be force-sensitive enabling them to transduce mechanical signals.

Force generation by actin and myosin is a key feature of focal contacts. Inhibition of myosin for example leads to a degeneration of mature focal contacts. (Scheme modified from (Mitra et al.,

2005) and (Geiger et al., 2009))

Following this observation, integrin-mediated mechanotransduction may be facilitated by a mediated exposure of the active or binding site of proteins via stretch-induced conformational changes. An example is the binding of vinculin to talin in nascent focal complexes. As long as no force is applied to talin, some of its vinculin-bindings sites are buried within a five-helix bundle.(Papagrigoriou et al., 2004) Conformational changes, induced by an externally applied force as demonstrated by Sheetz and coworkers, lead to an exposure of additional binding sites and facilitate binding of one or two vinculin molecules, which could lead to force-dependent changes

in signaling. (del Rio et al., 2009) Vinculin, for instance, has been discussed as a mechanosensitive molecule itself, presenting a binding site for MAPK1 (mitogen-activated protein kinase 1) under force-induced conformational changes due to acto-myosin-mediated tensile force.(Holle et al., 2013) MAPK1 plays a role in many cellular processes including proliferation or differentiation. Another player in the mechanotransduction machinery of focal adhesions is the actin-crosslinking protein filamin A. Recently it has been demonstrated, that mechanical load applied to filamin A-crosslinked actin networks reveals a cryptic β-integrin binding site in filamin A. At the same time the filamin A-binding GTPase FilGAP, which influences the activity of the small GTPase Rac, dissociates from filamin A. (Ehrlicher et al., 2011)

Arnold and coworkers found another interesting feature of focal adhesion-based mechanotransduction. (Arnold et al., 2004) In their experiments, they used small gold-nanoparticles coated with Arg-Gly-Asp-peptides (RGD), a well-known ligand of integrins, which is present on the surface of ECM proteins, to vary the spacing between single integrin receptors in cells. Interestingly, mature focal adhesions could only be found in cells seeded on substrates with ligand spacing fewer than 58 nm. Additionally, larger distances between adhesive ligands also resulted in a smaller cell number on the substrate and impaired cell spreading. This behavior has been observed for several cell-types leading to the conclusion that this might be a universal response. This experiment shows that mechanotransduction needs to be amended by a topographic component and, indeed, topographical cues on different dimensions ranging from nm to µm-scale have been found to induce cellular responses as divers as observed for mechanical stimuli. The effects of topographical cues include alignment to topographical features, changes in protein expression and activation and changes in migration. (Curtis and Wilkinson, 1997; Yamamoto et al., 2007) Recently it also been demonstrated that nano-grated polydimethylsiloxane (PDMS) surfaces are able to induce human stem cell differentiation through focal adhesion signaling. (Teo et al., 2013) Further influences of topographical features of the substrate on cell behavior will be discussed in detail in chapter 4.4.

These few examples demonstrate the complexity of integrin-mediated mechanotransduction, which takes place at several levels of focal adhesion organization. Furthermore, mechanotransduction is not an on/off-switch as demonstrated on molecular level by the force-dependent exposure of additional vinculin binding sites on talin rod.(Papagrigoriou et al., 2004) On whole-cell level, the cell reacts to subtle changes in the cell’s mechanical and topographical environment by an adapted response, which includes differentiation into one cell-type or another,

durotaxis, a phenomenon, in which cells migrate along stiffness gradients in substrates towards regions with higher stiffness, or also changes in cellular mechanics.(Engler et al., 2006; Janshoff et al., 2010; Lo et al., 2000; Tee et al., 2011; Teo et al., 2013;

Trichet et al., 2012)

Regarding the variety of proteins involved in mechanotransduction and the multiple processes that are governed by this process, it is not surprising that multiple diseases are associated to deregulations of the mechanosensing machinery. In their review

“Mechanotransduction gone awry”, Jaalouk and Lammerding address different diseases, which can, at least partly, be retraced to misregulation or mutations of proteins involved in mechanotransduction. (Jaalouk and Lammerding, 2009) Besides deafness and diseases related to the eye, the authors also discussed the role of mechanical cues in the context of cancer development, which will be subject of the next subchapter.

2.1.2.1 Role of mechanotransduction in cancer

In 2000 Hanahan and Weinberg published their famous review about the “Hallmarks of cancer”.(Hanahan and Weinberg, 2000) In their review they state six essential alterations, which go along with malignant cell transformation, which are: self sufficiency of growth signals, insensitivity to anti-growth signals, limitless replication potential, angiogenesis, evading apoptosis as well as invasion and metastasis. In recent years it has become clear, that some of these hallmarks can also be related to a malfunction in mechanotransduction.(Jaalouk and Lammerding, 2009) Tumors are generally stiffer than the surrounding normal tissue and this phenomenon is still used for tumor detection by palpation. Recently, it has been found that stiffening of the tumor is governed by oxidation and crosslinking of the ECM, i.e. collagen.(Levental et al., 2009) It has been proposed that this stiffening of the ECM is able to promote tumor growth and progression. Paszek et al. were able to demonstrate that increased substrate rigidity is able to drive malignant transformation by integrin-mediated mechanotransduction (see Figure 2.7). (Paszek et al., 2005) Non-malignant cells grown on stiff matrices exhibited a disruption of normal cell polarity, delocalization of proteins associated to cell-cell adhesions, increased growth and also showed a higher tension.

Figure 2.7: Mechanotransduction in tumor cells and formation of metastasis. A Mechanotransduction, cytoskeletal arrangement and tension are strongly connected to each other. In cancer cells, a stiffened extracellular matric (ECM) leads to more pronounced focal adhesions and activation of focal adhesion kinase (FAK). Downstream mediators of FAK are

among other extracellular signal regulated kinase pathways leading to proliferation and stimulation of Rho-ROCK pathways, increasing the tension of the cytoskeletal network. This increase of tension in turn, enhances the maturation and formation of focal adhesion generating a positive feedback loop. (modified from (Jaalouk and Lammerding, 2009)) B In the formation

of metastasis, transformed cells that detach from the primary tumor are subject to many mechanical cues and therefore need to adapt to different environmental situations (see also (Kumar and Weaver, 2009)). During intravasation into blood or lymphatic vessels cells need to be very deformable. Via the vascular system cancer cells are able to spread and form metastasis.

The authors found these effects to be dependent on integrin-mediated signaling via a extracellular signal regulated kinases (ERK) and Rho-ROCK-dependent tension generation. However, although whole tumors are usually stiffer than their surrounding and single tumor cells are supposed to be under high tension, a critical step in formation of metastasis is the detachment of single cells from the primary tumor as well as intra- and extravasation, a process demanding high deformability of the cell (see Figure 2.7). Thus, it might not be surprising, that cancer cells have often found to be softer than normal cells of the same tissue.(Agus et al., 2013; Cross et al., 2007; Guck et al., 2005; Xu et al., 2012) This effect is thought to be the result of massive cytoskeletal rearrangements in cancer. (Kumar and Weaver, 2009) Additionally, cancer cells have been shown to lose the requirement to adhere to a substrate and show less sensitivity to substrate rigidity, which possibly facilitates their survival while circulating in the vascular system. (Agus et al., 2013; Wang et al., 2000; Wittelsberger et al., 1981) However, malignant transformation is not entirely dependent on mechanotransduction and it needs mutations, i.e. misregulations, in other proteins, for

example involved in cell cycle or DNA repair, to develop cancer. But, the mentioned examples show, that malignant transformation is at least partially governed by mechanical cues leading also to massive structural and mechanical changes of the cell itself. Therefore, I examine cellular mechanics of cell lines with different metastatic potential in chapter 4.3.