Force generated by actomyosin contraction builds bridges between adhesive contacts
2010; Springer Nature; Volume: 29; Issue: 6 Linguagem: Inglês
10.1038/emboj.2010.2
ISSN1460-2075
AutoresOlivier Rossier, Nils C. Gauthier, Nicolas Biais, Wynn Vonnegut, Marc-Antoine Fardin, Philip Avigan, Evan Heller, Anurag Mathur, Saba Ghassemi, Michael S. Koeckert, James Hone, Michael P. Sheetz,
Tópico(s)Neuroscience and Neural Engineering
ResumoArticle11 February 2010free access Force generated by actomyosin contraction builds bridges between adhesive contacts Olivier M Rossier Olivier M Rossier Department of Biological Sciences, Columbia University, New York, NY, USAPresent address: CNRS, UMR 5091, Université Bordeaux 2, Bordeaux 33077, France Search for more papers by this author Nils Gauthier Nils Gauthier Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Nicolas Biais Nicolas Biais Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Wynn Vonnegut Wynn Vonnegut Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Marc-Antoine Fardin Marc-Antoine Fardin Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Philip Avigan Philip Avigan Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Evan R Heller Evan R Heller Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Anurag Mathur Anurag Mathur Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author Saba Ghassemi Saba Ghassemi Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author Michael S Koeckert Michael S Koeckert Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author James C Hone James C Hone Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author Michael P Sheetz Corresponding Author Michael P Sheetz Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Olivier M Rossier Olivier M Rossier Department of Biological Sciences, Columbia University, New York, NY, USAPresent address: CNRS, UMR 5091, Université Bordeaux 2, Bordeaux 33077, France Search for more papers by this author Nils Gauthier Nils Gauthier Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Nicolas Biais Nicolas Biais Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Wynn Vonnegut Wynn Vonnegut Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Marc-Antoine Fardin Marc-Antoine Fardin Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Philip Avigan Philip Avigan Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Evan R Heller Evan R Heller Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Anurag Mathur Anurag Mathur Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author Saba Ghassemi Saba Ghassemi Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author Michael S Koeckert Michael S Koeckert Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author James C Hone James C Hone Department of Mechanical Engineering, Columbia University, New York, NY, USA Search for more papers by this author Michael P Sheetz Corresponding Author Michael P Sheetz Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Author Information Olivier M Rossier1, Nils Gauthier1, Nicolas Biais1, Wynn Vonnegut1, Marc-Antoine Fardin1, Philip Avigan1, Evan R Heller1, Anurag Mathur2, Saba Ghassemi2, Michael S Koeckert2, James C Hone2 and Michael P Sheetz 1 1Department of Biological Sciences, Columbia University, New York, NY, USA 2Department of Mechanical Engineering, Columbia University, New York, NY, USA *Corresponding author. Department of Biological Sciences, Columbia University, Fairchild Center, Room 713, MC 2408, 1212 Amsterdam Avenue, New York, NY 10027, USA. Tel.: +1 212 854 4857; Fax: +1 212 854 6399; E-mail: [email protected] The EMBO Journal (2010)29:1055-1068https://doi.org/10.1038/emboj.2010.2 There is an Article (March 2010) associated with this Have you seen ...?. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another. Introduction Distributed throughout the body, connective tissues hold organs in place and are composed of an extensive extracellular fibrous network in which cells are embedded. The extracellular matrix (ECM) is a complex heterogeneous mixture of proteoglycans and protein fibres (collagen, fibronectin (FN) and laminin) that are assembled by cells to structure the tissues. ECM architectures are often discontinuous, comprised of adhesive fibres separated by micron-sized gaps as in the cornea (Nishida et al, 1988) or the intestinal mucosa (Toyoda et al, 1997). In vivo, the ability of cells to sense and respond to the structural heterogeneity of their environment is fundamental for tissue homeostasis. In particular, cell responses to gaps in ECM are of physiological importance during wound closure (Jester et al, 1999), but also for regulation of micron-sized fenestra, allowing migration of immune cells through the ECM as in the intestinal mucosa or skin dermis (Desaki and Shimizu, 2000; Stoitzner et al, 2002). In connective tissues, fibroblasts adhere to collagen fibres and develop bridges suspended above the interstices of the fibrous network (Nishida et al, 1988; Toyoda et al, 1997). Bridging in response to gaps is general as many cell types also exhibit bridges between adhesive fibres in 3D cell-derived or engineered matrices (Rovensky et al, 1999; Friedl and Brocker, 2000; Thibault and Buschmann, 2006; Schnell et al, 2007; Doyle et al, 2009) and above the non-adhesive regions on 2D surfaces (Lehnert et al, 2004; Zimerman et al, 2004; Thery et al, 2006b). Advances in surface patterning (Chen et al, 1997) enabled deposition of ECM with a precise geometry on 2D substrates that are ideal for optical microscopy and recapitulate ECM heterogeneity in tissues. Cell spreading on patterned surfaces shows that bridges can form over distances up to 20 μm (Lehnert et al, 2004; Zimerman et al, 2004). In all situations, bridges are actin-based structures characterized by inwards curved edges (Zand and Albrecht-Buehler, 1989; Jester et al, 1994; Thery et al, 2006a; Bischofs et al, 2008). Bridges are anchored along the edges of the adhesive regions by focal adhesions (FA) (Chen et al, 2003; Goffin et al, 2006; Thery et al, 2006a), which are known to assemble under cytoskeletal tension (Balaban et al, 2001; Riveline et al, 2001). This suggests that bridges are exerting mechanical tension on their anchoring contacts. However, no direct measurement of forces exerted by bridges has been performed to date. Traction forces produce cytoskeletal tension and stabilize cell shape. They are powered by non-muscle myosin-II (MII) (Sims et al, 1992; Cai et al, 2006). Traction forces are stable in time and space despite the rapid dynamics of actin filaments (Galbraith and Sheetz, 1997; Cai et al, 2006). Owing to the large distances between adhesions, bridges are structures whose stability could not be explained only by cell-adhesion structures. Bridges contain actomyosin networks that can produce cytoskeletal tension thought to be important for the maintenance of a structured cytoplasm between contacts (Thery et al, 2006a). Contractile filaments that support cytoskeletal tension are remodelled with a rapid turnover (Hotulainen and Lappalainen, 2006). How cells orchestrate turnover with continuous force generation at the molecular level is still unclear? A flowing web of actin and MII could provide cohesion of the cytoplasm as in the cortex of amoeboid cells (Yumura et al, 1984). In mammalian fibroblasts, however, no similar structure has been reported (Ponti et al, 2004; Giannone et al, 2007). MII is crucial for actin treadmilling in the lamellipodia in which actin filaments polymerize at the leading edge, flow backwards and depolymerize (Watanabe and Mitchison, 2002; Medeiros et al, 2006; Giannone et al, 2007). By alignment and organization of actin filaments through contraction, MII is essential for actin dynamics, bundle assembly and the stability of cytoskeletal shape (Sims et al, 1992; Wang and Ingber, 1994). However, traction forces are also known to control elongation of FA, which have been shown to be sites of actin polymerization in stress fibres (Hotulainen and Lappalainen, 2006). Such a mode of actin assembly was postulated to depend on MII-based forces, but the level of forces needed was not known (Endlich et al, 2007). A corollary of this hypothesis is that MII-generated forces can also regulate either the level or the localization of actin assembly within an existing actin network such as in the bridges? In mammalian fibroblasts, two isoforms of MII coexist (MIIA and MIIB) and are involved in distinct cellular processes with different localizations (Conti and Adelstein, 2008). For example, MIIA produces the majority of traction forces of mouse embryonic fibroblast (MEF) cells on continuous adhesive substrates (Cai et al, 2006) and is responsible for developing a cohesive actomyosin network within the cytoplasm (Cai et al, 2010), whereas MIIB is responsible for collagen fibre movement (Meshel et al, 2005). In a similar manner, MII isoforms could be used at distinct tasks or in different regions in bridges. In an effort to unravel the function of contractility in the maintenance of bridges, we used an original force measurement device and found that bridges generated large forces on their anchoring adhesions. When we analysed the dynamics of actomyosin components within bridges on patterned substrates, they showed a treadmilling flow that enabled the replenishment of filaments for continuous force generation. Thus, MII-based contractility was needed for self-sustained cytoskeleton dynamics and maintenance of the cellular bridges, but the two MII isoforms had different functions in the stability of bridges. Bridges were much more dynamic than earlier imagined for a cytoskeleton network of stable filament bundles. Bridges provided a framework to study how cytoskeletal tension, MII contraction, actin polymerization and FA assembly cooperate in an integrated manner to generate a self-sustained stable structure. Results Bridges are actin-enriched structures characterized by 'bow-tie'-shaped cytoskeleton with concave edges To better understand the basis of bridge formation and maintenance, we used patterned substrates with alternating large non-adhesive stripes (polyethylene glycol (PEG) regions of 3.5–20 μm in width) and small adhesive stripes (FN-coated lines of 1.5 to 6 μm in width) (Supplementary data). When MEFs were plated on these surfaces, they spread over adhesive as well as non-adhesive areas (Figure 1A), but extended predominantly along the FN stripes where they developed anchoring FAs (Figure 1B). Cells spanning non-adhesive gaps formed non-adherent bridges that maintained cytoplasmic integrity between adhesions (Figure 1A, box i). Bridges were easily recognized by the concave cell edges over the non-adhesive regions. After 1–2 h of spreading and extension, MEFs reached a fully spread area that was independent of the width of non-adhesive gaps (Supplementary Figure S1) or FN stripes (ATOTAL=3219±98 μm2, n=193 for all the patterned substrates tested) with a significant fraction of the cell covering non-adhesive surfaces. We defined a bridging index Bri as the fraction of total cell area in the bridges: Bri=ABRIDGE/ATOTAL (e.g. Bri=ABRIDGE/ATOTAL=68.8±0.8%, n=14 for FN line width 4.75 μm, gap 18 μm, with ATOTAL=3301±324 μm2). Interestingly, the Bri of cells on a given pattern was related to the fraction of non-adhesive area on the pattern [fNA=gap width/(gap width+FN stripe width)] and was ranging from 80 to 97% of fNA value. Figure 1.Cellular bridges are actin-rich structures characterized by 'bow-tie'-shaped cytoskeleton and concave edges. FA and strongest traction forces are generated at the sides of concave edges. (A) Typical morphology of MEFs spread for 2 h on patterned FN lines. (i) Close-up of cell (i) in the outlined box (Bri=66%). Top panel: bridges highlighted in red (ABRIDGE) developed above the non-adhesive regions with arch-shaped ends. The cell regions in contact with FN lines are in green (AFN); bottom panel: actin cytoskeleton in the same cells (EPI and TIRF images). (B) Adhesion proteins: paxillin immunostaining by EPI and with GFP-integrin β3 by TIRF. (C) Top left: original geometry of PDMS force-sensing pillar device; bottom left: scanning electron micrograph of MEFs onto pillars; top right: differential interference contrast micrograph of MEF exerting onto pillars, traction forces represented with red arrows (force scale bar=12 nN); bottom right: plot of the amplitude (red circles) of forces exerted on each pillars of the bottom row in the differential interference contrast micrograph, as well as separated force components: Fx along the axis of the rows (green circles) and Fy directed perpendicularly (blue circles). FN lines are outlined with yellow dashed lines. Scale bars: (A) 50 μm, A (i) 30 μm, (B, C) 15 μm. Download figure Download PowerPoint The actin cytoskeleton was more concentrated in the bridges compared with the adhesive FN stripes (Figure 1A) and assembled into a 'bow-tie' shape in each bridge. The wide parts of the 'bow-tie' were near the concave edges designated type 1 regions where bundles of actin fibres appeared anchored to the edges of FN lines and formed arrowheads. Interestingly, comparing epifluorescence (EPI) and total internal reflection fluorescence (TIRF) microscope images of cytoskeletons in bridges (Figure 1A), we observed that concave edges were significantly above the glass because the actin fibres associated with concave edges appeared only in EPI, but not in TIRF. In the body of bridges defined as type 2 regions, bundles of actin fibres, oriented parallel to the FN stripes, connected the actin arrowheads in type 1 regions on both ends of bridges. The actin meshwork in type 2 regions was more condensed at the centre of the non-adhesive region. There were fewer apparent connections to the FN stripes than in type 1 regions. FA and strongest traction forces are generated at the sides of concave edges Organization of FA in cells plated on patterned substrates also confirmed the existence of two types of regions within bridges. FA structures are found only along adhesive stripes (Figure 1B) and are mostly localized at the edges of FN stripes as seen in earlier studies (Chen et al, 2003; Thery et al, 2006a). Interestingly, the adhesion proteins, paxillin and integrin β3 accumulated with the greatest density along the edges of FN stripes adjacent to concave type 1 regions (Figure 1B). Moreover, FA had different morphologies and orientations in the two regions. In type 1 regions, FA were long and oriented towards the non-adhesive areas, whereas in type 2 regions, FA were dot-like aggregates aligned along the matrix edges, as seen with green fluorescent protein (GFP)-α-actinin (Figure 3A). Type 1 adhesion-rich patches increased in size with the width of the non-adhesive gaps (Supplementary Figure S1). Thus, type 1 regions had the greatest concentration of actin bundles and the largest FA. As FA contacts grow with force (Balaban et al, 2001; Riveline et al, 2001; Galbraith et al, 2002), we postulated that the greatest forces might be concentrated in type 1 regions. Figure 2.Formation and extension of bridges correlate with actomyosin assembly and contractions. (A) Differential interference contrast: formation of arch-shaped edges (arrowheads) during late spreading on a patterned substrate. Arrows indicate coexisting transient protrusions. GFP-β-actin (TIRF): when bridges formed (2.8 min), actin cables bent towards the middle (box ii, arrowheads, 7.0 min). GFP-MRLC (TIRF): when bridges formed, MRLC is enriched above non-adhesive regions and condensed into bow-tie-shaped fibres at longer times. (B) Bridge extension during a cycle of extension of actin-rich protrusions (arrow) followed by contraction into MII bundles (arrowhead). Plot of positions of FN-attached regions (blue and red dots) and bridge edge (green dot) versus time during one cycle. Scale bars: (A) 15 μm (except for differential interference contrast panel: 18 μm) and (B) 13 μm. Download figure Download PowerPoint Figure 3.Actomyosin dynamics within bridges exhibit two distinct patterns, (1) 'contractile' treadmilling at the concave edges of bridges and (2) 'cross-linking' in the body of bridges. (A) GFP-α-actinin (TIRF): speckles decorated the 'bow-tie'-shaped actin cytoskeleton as well as FAs; velocity field: particle image velocimetry analysis of speckles' dynamics permitted to define a 'contractile' treadmilling pattern (region 1-red arrows) at concave edges of mature bridges and in newly formed bridges, and a 'cross-linking' pattern (region 2-blue arrows) in the body of bridges. Velocity scale bar=25 nm/s. (B) GFP-MRLC (TIRF): similar dynamical patterns were observed with MII ongoing (i) 'contractile' treadmilling in region 1 as seen with MRLC speckles in snapshot (arrowhead) and in kymograph taken along the red line (arrowhead: corresponding speckle trajectory) and (ii) 'cross-linking' dynamics in region 2 as seen in kymograph of MRLC speckles along actin fibre (blue line ii). Images were filtered with the ImageJ plugin SpotTracker. Scale bars: (A, B) 15 μm. Download figure Download PowerPoint To measure the forces on FA contacts, we fabricated a PDMS pillar device (Tan et al, 2003; Cai et al, 2006) with rows of force-sensing pillars (2 μm diameter, 5 μm height, spaced by 1 μm with a spring constant: k=37.7 nN/μm) separated with gaps (5–15 μm) similar in size to non-adhesive gaps on patterned substrates (Figure 1C) (Supplementary data). To ensure that cells only adhered to the top of the pillars, we stamped the tops of the pillars with FN and blocked adhesion to their sides with pluronic F-127 (Supplementary Figure S2). Cellular bridges formed with the same morphology as on the 2D-micropatterned substrates (Figure 1C). We measured the magnitude of the deflection of pillars from their equilibrium position to determine the cell traction forces. The largest traction forces produced by bridges were in the vicinity of the concave edges of bridges (width: 1–4 pillars, i.e. 2–12 μm) with a magnitude of 5–20 nN/pillar (Figure 1C and Supplementary Video 1). Forces were directed parallel to the concave edges along the anchored actin bundles in region 1. In type 2 regions, forces were oriented perpendicular to the rows of pillars and were smaller. When pillars were pulled by two adjacent bridges, they often had no deflection or a deflection parallel to the rows of pillars, resulting from the sum of the forces from both bridges. We conclude that FA size and orientation correlated with the traction forces and further established the differences between regions 1 and 2. In particular, the concave region 1 had the highest forces and seemed to contribute to bridge development. Formation of bridges correlates with actomyosin assembly and contractions To understand the steps that led to extension of bridges, adhesion localization and force generation in bridges, we studied how cells spread and expand their area on patterned substrates. At early stages, MEFs rapidly spread regardless of the substrate coating and then abruptly started MII-dependent contractions as described earlier on continuous adhesive substrates (Giannone et al, 2004). Contractions caused inwards bowing of cell edges over the non-adhesive regions, while outwards extension continued over the adhesive regions (Figure 2A and Supplementary Video 2). Live-cell imaging of β-actin (GFP-β-actin) and MII regulatory light chain (GFP-MRLC that binds to all the isoforms of the MII heavy chain) revealed that actin and MII were preferentially recruited above non-adhesive areas as contraction started (Figure 2A and Supplementary Videos 3 and 4). As a consequence, there were stronger forces in between FN stripes than along them. After MII recruitment, actin filaments bent inwards, while staying anchored at their ends to the sides of concave edges (Figure 2A, box ii), and over time, MII fibres contracted to the middle of the non-adhesive regions (Figure 2A, box iii). Thus, the 'bow-tie' shape of bridges over non-adhesive regions appeared to be caused by actomyosin contraction. After the initial spreading, cells expanded their projected area through extension of existing bridges. They extended through cycles of protrusions and contractions along the FN lines (Figure 2B and Supplementary Video 5) (Giannone et al, 2004). Contractions formed adhesions at sites further along the FN stripe that grew as the concave edge extended. Originating from FN stripes, protrusions above non-adhesive areas were rich in actin, but contained very diffuse MII (Figure 2B, arrow). They ruffled back along the curved edge of the bridge except near new FA sites where they provided links to the actomyosin network in the concave region. Actin filaments then condensed into thick cables that colocalized with GFP-MRLC in the concave edges (Figure 2B, arrowheads). Such thickening of actin filaments correlated with an enhanced concentration of MII (Supplementary Video 5). Bridge extension relied on extension of FN-attached regions and followed them with the same velocity (Figure 2B). After 1–2 h of spreading and extension, MEFs reached a fully spread area. Interestingly, during the whole process of cell spreading and extension to the final spread area, the bridging index Bri stayed constant ±7% (Figure 4C). Figure 4.Generation of forces induced by MII activity is essential for the formation and maintenance of bridges. (A) Reversible collapse of bridges induced by MII inhibition with MEF initial spreading (t=0 to 40 min), perfusion of BBI (t=50 to 100 min), and drug washout (t=100 min). (B) Plot of the variation versus time of the total projected area, ATOTAL (black circles), the bridge area, ABRIDGE (red circles) and the cell area above the adhesive region, AFN (green circles), for the cell depicted in (A). (C) Plot of the variation versus time of the bridging index, Bri (black circles), for the cell depicted in (A). Bri was constant during cell spreading and decreased from 69 to 32% during BBI perfusion. (D) During BBI treatment, force generation by bridges represented with red arrows (force scale bar=5 nN) stopped after 5–10 min (red circles indicated pillars where force generation was stopped); then bridge tearing (left panel: arrowheads) and/or bridge retraction (middle panel) led to bridges' collapse. Plot of the variation during BBI perfusion versus time of the traction force exerted on pillars adjacent to the concave edges (marked by asterisks: yellow for pillar in left panel and blue in middle panel). Scale bars: (A) 30 μm and (D) 15 μm. Download figure Download PowerPoint Actomyosin dynamics within bridges exhibit two distinct patterns: (1) 'contractile' treadmilling at the concave edges and (2) 'cross-linking' in the body of bridges To study actomyosin dynamics in bridges, we followed α-actinin, an actin binding protein and MRLC. Actomyosin dynamics in bridges showed two different behaviours in the two distinct regions of the 'bow-tie'-shaped cytoskeleton. Both proteins were found to label intensely the 'bow-tie' cytoskeleton (Figure 3A and B and Supplementary Videos 6 and 7). Speckles of GFP-α-actinin were tracked as fiduciary markers and analysed by particle image velocimetry to obtain the velocity fields (Figure 3A). In the vicinity of the concave edges (width 5–30 μm), GFP-α-actinin speckles originated from the adhesion-rich patches located at the FN borders and flowed with velocities between 4 and 10 nm/s towards the middle of the non-adhesive region, indicating treadmilling of actin from the edges of FN stripes to the centre of the non-adhesive region. GFP-MRLC speckles also formed at the border of FN stripes by FA sites and flowed with similar velocities as GFP-α-actinin speckles to the centre of non-adhesive regions, indicating that concave regions 1 were continuously contracting (Figures 3B, box i and 7A, box ii and Supplementary Videos 7 and 14). We suggest that this dynamic pattern common to both GFP-α-actinin and MRLC speckles is indicative of actomyosin 'contractile' treadmilling. In the middle section of bridges (region 2), within the actin bundles, GFP-α-actinin and GFP-MRLC speckles oscillated in synchrony without net movement (α-actinin: Figures 3A and 5B and Supplementary Videos 6 and 9; MRLC: Figure 3B, box ii and Supplementary Video 7). Owing to their function in maintaining connections across the cell, actomyosin bundles in region 2 appear to be supporting high tensions and oscillations in the contractions gave rise to the fluctuations in position as in a tug of war. Occasionally, fibres appeared to rupture causing the aggregates near the 'break' to separate as previously seen for surgically broken stress fibres anchored to mature FA (Rajfur et al, 2002; Kumar et al, 2006) (Figure 7A and Supplementary Video 14). However, contrary to the latter case, there was rapid healing of the ruptures. After an initial rapid separation, movement of GFP-MRLC aggregates slowed and new aggregates formed in the gap that restored normal density (Figure 7A, red arrowheads, box i). Oscillation of the new aggregates around their initial positions indicated that mechanical continuity of the network was restored (Supplementary Video 14). In newly formed bridges, actin dynamics presented only 'contractile' treadmilling flow patterns as found in region 1 (Figure 3A, bottom bridge and Supplementary Video 6). This indicated that the actin dynamics in region 2 only appeared when the concave edges moved sufficiently far from each other. Contraction and force generation by MII is essential for the formation and maintenance of bridges Actomyosin dynamics could have been governed by two major factors: contraction mediated by MII and/or actin polymerization. To test whether MII contractility was involved in maintenance of bridges, we added blebbistatin (BBI), an inhibitor of MII ATPase activity (Straight et al, 2003), to spread cells with established bridges. After 2 min of BBI application, bridges started to collapse into thin tubular processes that were complete in <30 min (Figure 4A and B and Supplementary Video 8). No retraction fibres (Mitchison, 1992) were left, reinforcing the suggestion that there were no adhesions in non-adhesive areas. In addition, the perinuclear region rounded as the cytoplasm retracted. However, the frequency and duration of extensions from or along the FN stripes seemed unchanged. Further, cellular extension along FN stripes increased (Figure 4B). In addition, in separate experiments, MII inhibition did not block initial spreading along the FN stripes or across non-adhesive areas. However, after initial spreading, bridges did not form even though processes extended along the adhesive stripes and across non-adhesive regions (data not shown). Seven to ten minutes after BBI was removed, the bridges started to reform slowly as evidenced by a Bri increase (Figure 4C), and perinuclear cytoplasm extension with nuclear flattening. Inhibition of the Rho-associated kinase (ROCK), an upstream activator of MII, with the drug Y-27632 caused similar disruption of bridges. To determine the relationship between MII and force generation by bridges, we measured forces generated by cells on pillars during MII inhibition with BBI. MII inhibition caused gradual loss of force over 5–10 min after the drug application, similar to the time course of the initial loss of bridges (Figure 4D). The collapse of the bridges occurred as on patterned substrates (Supplementa
Referência(s)