Muscle cells are the best-known example of a cell in the human body that can contract. These cells contain bundles of filaments made of proteins called actin and myosin, which can generate pulling forces. However, many other cells in the human body also rely on similar “contractile actomyosin bundles” to help them stick to each other, to maintain the correct shape or to migrate from one location to another. These bundles in the non-muscle cells are often called “ventral stress fibers”.

Ventral stress fibers develop from structures commonly referred to as “arcs”. Previous work has clearly established that ventral stress fibers are sensitive to mechanical forces. However, the underlying mechanism behind this process was not known, and it remained unclear how external forces could promote these actomyosin bundles to assemble, align and mature.

Tojkander et al. documented the formation of ventral stress fibers in migrating human cells grown in the laboratory. This revealed that pre-existing arcs fuse with each other to form thicker and more contractile actomyosin bundles. The formation of these bundles then pulls on the two ends of the stress fibers that are attached to sites on the edges of the cell.

Tojkander et al. also showed that this tension inactivates a protein called VASP, which is also found at these sites. Inactivating VASP inhibits the construction of actin filaments, which in turn stops the stress fibers from elongating and allows them to contract. Further experiments then revealed that ventral stress fibers are maintained and can even become thicker under a sustained pulling force. Conversely, stress fibers that were not under tension were decorated by proteins that promote the disassembly of actin filaments. This subsequently led to the disappearance of these fibers.

Future studies could now examine whether the newly identified pathway, which allows mechanical forces to control the assembly and alignment of stress fibers, is conserved in other cell-types. Furthermore, and because the assembly of such mechanosensitive actomyosin bundles is often defective in cancer cells, it will also be important to study this pathway’s significance in the context of cancer progression.

Cell migration is essential for embryonic development, wound healing, immunological processes and cancer metastasis. Cell migration is driven by assembly and disassembly of protrusive and contractile actin filament structures. The force in protrusive actin filament structures, including lamellipodium and filopodia at the leading edge of cell, is generated through actin polymerization against the plasma membrane. In contractile actin filament bundles, such as stress fibers, the force is generated by sliding of bipolar myosin II bundles along actin filaments. Notably, whereas the assembly-mechanisms of protrusive actin filament structures are relatively well understood, general principles underlying the assembly of contractile actomyosin bundles have remained elusive (Pollard and Cooper, 2009; Bugyi and Carlier, 2010; Michelot and Drubin, 2011; Burridge and Wittchen, 2013).

The most prominent contractile actomyosin structures in most cultured non-muscle cells are stress fibers. Beyond cell migration, stress fibers guide adhesion, mechanotransduction, endothelial barrier integrity, myofibril assembly, and receptor clustering in T-lymphocytes (Burridge and Wittchen, 2013; Wong et al., 1983; Sanger et al., 2005; Tojkander et al., 2012; Yi et al., 2012). Due to their intrinsic properties, stress fibers have become an important model system for studying the general principles by which contractile actomyosin bundles are assembled in cells. Stress fibers can be divided into three main categories based on their protein compositions and interactions with focal adhesions (Small et al., 1998). Dorsal (radial) stress fibers are connected to focal adhesions at their distal ends and rise towards the dorsal surface of the cell at their proximal region (Hotulainen and Lappalainen, 2006). They elongate through vectorial actin polymerization at focal adhesions (i.e. coordinated polymerization of actin filaments, whose rapidly elongating barbed ends are facing the focal adhesion, is responsible for growth of dorsal stress fibers). These actin filament bundles do not contain myosin II, and dorsal stress fibers are thus unable to contract (Hotulainen and Lappalainen, 2006; Cramer et al., 1997; Tojkander et al., 2011; Oakes et al., 2012; Tee et al., 2015). However, dorsal stress fibers interact with contractile transverse arcs and link them to focal adhesions. Transverse arcs are curved actin bundles, which display periodic α-actinin – myosin II pattern and undergo retrograde flow towards the cell center in migrating cells. They are derived from α-actinin- and tropomyosin/myosin II- decorated actin filament populations nucleated at the lamellipodium of motile cells (Hotulainen and Lappalainen, 2006; Tojkander et al., 2011; Burnette et al., 2011; 2014). In fibroblasts and melanoma cells, filopodial actin bundles can be recycled for formation of transverse arc –like contractile actomyosin bundles (Nemethova et al., 2008; Anderson et al., 2008). Ventral stress fibers are defined as contractile actomyosin bundles, which are anchored to focal adhesions at their both ends. Despite their nomenclature, the central regions of ventral stress fibers can bend towards the dorsal surface of the lamellum (Hotulainen and Lappalainen, 2006; Schulze et al., 2014). Migrating cells display thick ventral stress fibers that are typically oriented perpendicularly to the direction of migration, and thinner ventral stress fibers that are often located at the cell rear or below the nucleus. At least the thick ventral stress fibers, which constitute the major force-generating actomyosin bundles in migrating cells, are derived from the pre-existing network of dorsal stress fibers and transverse arcs. However, the underlying mechanism has remained poorly understood (Burridge et al., 2013; Hotulainen and Lappalainen, 2006).

Stress fibers and focal adhesions are mechanosensitive structures. Stress fibers are typically present only in cells grown on rigid substrata and they disassemble upon cell detachment from the matrix (Mochitate et al., 1991; Discher et al., 2005). Furthermore, after applying fluid shear stress, stress fibers align along the orientation of flow direction in endothelial cells (Sato and Ohashi, 2005). Also focal adhesions develop only on rigid surfaces, and applying external tensile force promotes their enlargement (Chrzanowska-Wodnicka and Burridge, 1996; Pelham et al., 1999; Riveline et al., 2001). Focal adhesions contain several mechano-sensitive proteins, including talin, filamin and p130Cas, whose activities and interactions with other focal adhesion components can be modulated by forces of ~∼10–50 pN range (Sawada et al., 2006; del Rio et al., 2009; Ehrlicher et al., 2011). Furthermore, the protein compositions of focal adhesions are regulated by tension supplied by myosin II activity and external forces applied to the cell (Zaidel-Bar et al., 2007; Kuo et al., 2011; Schiller et al., 2011). Importantly, despite wealth of information concerning mechanosensitive focal adhesion proteins, possible effects of tensile forces on actin filament assembly at focal adhesions have remained elusive. Furthermore, the mechanisms by which tension contributes to the alignment of stress fibers and actin dynamics within these actomyosin bundles have not been reported.

Here we reveal that formation of mature contractile actin bundles from their precursors is a mechanosensitive process. We show that arc fusion during centripetal flow is accompanied by increased contractility that inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, thus insuring formation of ventral stress fibers. Conversely, activation of AMPK allows generation of contractile ventral stress fibers in cells growing on compliant matrix, where their formation is normally prevented. Furthermore, we provide evidence of mechanosensitive actin filament disassembly by ADF/cofilins during stress fiber assembly. These data provide support to a new mechanobiological model explaining the principles of assembly and alignment of ventral stress fibers in migrating cells.

Ventral stress fibers play an important role in cell adhesion, morphogenesis and migration, but how these and other contractile actomyosin bundles are generated has remained elusive. Here we have revealed several new aspects concerning the mechanisms underlying the assembly of contractile ventral stress fibers. We provide evidence that: (1) Formation of ventral stress fibers from their precursors (arcs and dorsal stress fibers) is a mechanosensitive process. (2) Arcs fuse with each other during centripetal flow to form thicker and more contractile actomyosin bundles, which apply tension to focal adhesions located at their ends. (3) This tension activates AMPK-mediated phosphorylation of VASP that leads to inhibition of vectorial actin polymerization at focal adhesions. (4) AMPK-mediated VASP phosphorylation is necessary for assembly and proper alignment of contractile ventral stress fibers. Conversely, activation of AMPK can bypass the need of stiff matrix for ventral stress fiber assembly. (5) ADF/cofilin–mediated disassembly of non-contractile dorsal stress fibers is important for the proper maturation of the stress fiber network. We propose that similar mechanosensitive actin filament assembly and disassembly may have general role in formation and alignment of diverse contractile actomyosin bundles in different cell-types.

A working model for mechanosensitive assembly of contractile ventral stress fibers is presented in Figure 10. Nascent adhesions appear at the lamellipodium of migrating cell and a fraction of them matures to focal adhesions (Burnette et al., 2011; Choi et al., 2008). Dorsal stress fibers are initiated from focal adhesions located at the leading edge of cells, and elongate through ‘vectorial’ actin polymerization at focal adhesions, mediated at least by VASP and Dia1 formin (Hotulainen and Lappalainen, 2006; Watanabe et al., 1999; Gateva et al., 2014). Similarly to filopodia, where VASP localizes at the tip-complex and promotes assembly of unipolar actin filament bundles, VASP at focal adhesions is expected to catalyse polymerization of an unipolar actin filament bundle towards the cell center. Elongating dorsal stress fibers associate with multiple myosin II containing arcs, which are derived from the lamellipodial actin structures (Figure 10A). During centripetal flow of this spider-net like structure, arcs fuse with each other to generate a thicker, more contractile bundle (Figure 10B and C). As a result, those focal adhesions that are attached via dorsal stress fibers to the ends of the contractile arc sense strong myosin II-generated tension, leading to their enlargement and turning along the direction of the arc. In support to this, ventral stress fibers apply strong traction forces to the substrate through their terminally located focal adhesions (Figure 2 and Möhl et al., 2012). Moreover, tension-mediated maturation of terminal focal adhesions leads to inhibition of vectorial actin polymerization, which is at least partially mediated by phosphorylation of VASP (Figure 10C). However, because VASP-depletion did not result in a compete inhibition of vectorial actin polymerization after releasing the tension in ventral stress fibers (Figure 7), other proteins are also likely to contribute to vectorial actin polymerization at focal adhesions. It is also important to note that, although the actin polymerization activity of VASP is inhibited at focal adhesions under high tension, VASP protein is still present in that location. Thus, VASP may contribute to integrity of the adhesions at the tips of ventral stress fibers through its other activities, including actin filament bundling (Bear and Gertler, 2009). Furthermore, VASP phosphorylation is not expected to inhibit all actin dynamics in focal adhesions, because VASP appears to specifically contribute to vectorial actin polymerization at focal adhesions, whereas other proteins such as formins may promote the turnover of other focal adhesion-associated actin filament populations.

Importantly, inhibition of vectorial actin polymerization is essential for proper alignment and contractility of the ventral stress fiber, because continuous elongation of the actin filament bundle from focal adhesions would counteract myosin II-driven shortening of the actomyosin bundle. Finally, we propose that mechanosensitive binding of cofilin-1 to those dorsal stress fibers and dorsal stress fiber regions, which are not under myosin II-applied tension, leads to disassembly of these ‘non-productive’ regions of the stress fiber network. Consequently, the focal adhesions located at the distal ends of ‘central’ dorsal stress fibers, which are oriented perpendicularly to the contractile arcs and hence do not sense strong myosin II–derived tension, diminish in size and disappear. On the other hand, contractile arcs and mature ventral stress fibers are protected from ADF/cofilin-mediated actin filament disassembly (Figure 10D and E). Consistent with this model, dorsal stress fibers and arcs form in cells grown on soft substrata, whereas contractile ventral stress fibers fail to assemble in compliant matrix (Figure 1A).

Focal adhesions are mechanosensitive structures (e.g. Iskratsch et al., 2014). Their maturation from nascent adhesions and maintenance require relatively small forces that can be generated by retrograde actin flow without myosin II-driven contractility (Oakes et al., 2012; Stricker et al., 2013). However, focal adhesions mature into larger, elongated adhesions under stronger, myosin II-derived tension (Geiger et al., 2009). Myosin II -generated force also affects the protein composition and dynamics in focal adhesions (Kuo et al., 2011; Schiller et al., 2011; Wolfenson et al., 2011). Here, we provide evidence that vectorial actin polymerization, which drives elongation of stress fibers, is strictly controlled in focal adhesions. Thus, different force regimes seem to have distinct effects on actin dynamics and molecular composition of focal adhesions. While weak forces exerted by retrograde actin flow appear to be required for VASP recruitment and to promote vectorial actin polymerization at focal adhesions, stronger force applied by contractility of the myosin II-containing ventral stress fiber efficiently inhibits actin polymerization at focal adhesions. Importantly, during this process VASP is phosphorylated by AMPK, whose activity at least in muscle cells can be controlled by tension through a currently uncharacterized mechanism (Blair et al., 2009). It is likely that activities or localizations of additional actin-polymerization associated proteins in focal adhesions are regulated by contractility. Indeed, an interaction partner of VASP, palladin (Gateva et al., 2014), as well as tropomyosins Tm1 and Tm5NM1 (Figure 10—figure supplement 1) are enriched in focal adhesions located at the tips of dorsal stress fibers, but absent from adhesions at the tips of ventral stress fibers. Furthermore, formins contribute to actin filament nucleation and/or processive polymerization in focal adhesions, and it is likely that also their activities are regulated during this process. Recent studies demonstrated that low-regime forces (<3 pN) increase the actin polymerization activity of formins (Courtemanche, et al., 2013; Jégou et al., 2013). Furthermore, the activity of formins can be controlled by a local increase in G-actin levels (Higashida et al., 2013). In the future, it will be interesting to examine the effects of imposed external forces, comparable in magnitude to tension applied by a myosin II-containing ventral stress fiber, on formins. However, similar to shown here for VASP, we propose that possible regulation of formin activity in focal adhesions is more likely controlled through biochemical signalling cascades than direct mechanical regulation of the formin molecule. This is because culturing cells on hyaluronic acid containing soft gels can produce a similar formation of ventral stress fibers that is otherwise observed only in cells cultured on rigid substrates (Chopra et al., 2014). Furthermore, several tyrosine kinases play an important role in focal adhesion mechanosensing, and their inactivation can shift the stiffness regime for assembly of large focal adhesions and ventral stress fibers (Prager-Khoutorsky et al., 2011).

Figure 10 with 1 supplement see all

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In addition to vectorial actin polymerization at focal adhesions, actin filaments within the stress fiber network undergo turnover with a half-life of an approximately one minute (Hotulainen and Lappalainen, 2006). We propose that maintenance or disappearance of individual stress fibers depends on a balance between assembly and tension-sensitive disassembly of actin filaments. In addition to its role in actin polymerization in focal adhesions, VASP contributes to actin filament assembly within the stress fiber network (Gateva et al., 2014; Smith et al., 2010; Hoffman et al., 2012). Our data propose that stress fibers are maintained or become thicker under tension, whereas mechanosensitive binding of ADF/cofilins to stress fibers that are not under tensions shifts the balance from steady state (or net assembly) to net disassembly. Eventually, this leads to disappearance of the stress fiber and the focal adhesion associated with its end.

What are the functions of dorsal stress fibers? Our data demonstrate that arc fusion during centripetal flow occurs preferentially at the intersections with dorsal stress fibers. This suggests that dorsal stress fibers may functions as ‘rails’ to facilitate coalescence of adjacent arcs in the 3D-environment inside lamellum. However, arc fusion and formation of focal adhesion–attached ventral stress fibers can occur also in VASP-depleted cells, which either do not contain dorsal stress fibers or where these actin filament bundles are very thin and fragile (Figure 4—figure supplement 1A and B; Video 6). Furthermore, many cell-types including epithelial cells can assemble peripheral actomyosin bundles resembling ventral stress fibers in the apparent absence of dorsal stress fibers. Thus, focal adhesion–attached contractile actomyosin bundles can be generated at least in non-motile cells without prominent dorsal stress fibers. It is, however, important to note that the stress fiber network is typically poorly organized in VASP-depleted U2OS cells (Figure 4—figure supplement 1B), suggesting that dorsal stress fibers are required for proper alignment of ventral stress fibers in migrating cells. Furthermore, dorsal stress fibers play an important role in directional cell migration (Kovac et al., 2013).

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Collectively, our findings reveal that mechanosensitive actin filament assembly and disassembly are essential for generation of contractile ventral stress fibers, and function as selection processes to ensure proper alignment of ventral stress fibers perpendicularly to the direction of cell migration. In the future, it will be important to identify the signalling pathway regulating mechanosensitive phosphorylation of VASP in focal adhesions. Here it will be especially interesting to examine the possible contribution of mechanosensitive Ca²⁺ channels and Ca²⁺ -activated CaMKK family kinases, because the latter can activate AMPK kinase (Carling et al., 2008). Furthermore, it will be important to reveal how the activities of other proteins contributing to actin polymerization at focal adhesions are regulated by myosin II -dependent tension. Finally, it will be interesting to examine how mechanosensitive actin filament assembly and disassembly contribute to generation and proper alignment of stress fibers and other contractile actomyosin bundles in the tissue environment.

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Author details

1. Sari Tojkander

   Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   ST, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Gergana Gateva

   Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   GG, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Amjad Husain

   Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States

   Contribution

   AH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Ramaswamy Krishnan

   Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States

   Contribution

   RK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Pekka Lappalainen

   Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   PL, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   pekka.lappalainen@helsinki.fi

   Competing interests

   The authors declare that no competing interests exist.

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