Cell Motility and the Cytoskeleton 61:161–171 (2005)
The Last but not the Least: The Origin and Significance of Trailing Adhesions in Fibroblastic Cells Raphaela Rid,1 Natalia Schiefermeier,1 Ilya Grigoriev,1 J. Victor Small,1 and Irina Kaverina1,2* 1
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg, Austria 2 Vanderbilt University Medical Center, Nashville, Tennessee Mature adhesions in a motile fibroblast can be classified as stationary ‘‘towing’’ adhesions in the front and sliding trailing adhesions that resist the traction force. Adhesions formed at the front of motile fibroblasts rarely reach the trailing zone, due to disassembly promoted by intensive microtubule targeting. Here, we show that the majority of adhesions found at the trailing edge originate within small short-lived protrusions that extend laterally and backwards from the cell edge. These adhesions enlarge by sliding and by fusion with neighboring adhesions. A further subset of trailing adhesions is initiated at a novel site proximal to trailing stress fibre termini. Following tail retraction, trailing adhesions are actively regenerated and the stress fibre system is remodeled accordingly; the tensile forces elaborated by the contractile actin system are consequently redirected according to trailing adhesion location. We conclude that persistent and dynamic anchorage of the cell rear is needed for the maintenance of continuous unidirectional movement of fibroblasts. Cell Motil. Cytoskeleton 61:161–171, 2005. ' 2005 Wiley-Liss, Inc.
Key words: cell motility; focal contacts; traction force; mechanosensor
INTRODUCTION
Cell migration is a complex process requiring the continuous formation and disassembly of matrix adhesions. Adhesion sites are initiated via signaling through the Rac or Cdc42 pathways, as small focal complexes
associated with protrusive activity at the cell margin. Focal complexes are short-lived; they either turn over at the active edge of the cell, or mature to form large focal adhesion sites [for reviews, see Small et al., 1999; Katsumi et al., 2004; Webb et al., 2002] in response to
The supplemental materials described in this article can be found at http://www.interscience.wiley.com/jpages/0886-1544/suppmat
J. Victor Small’s present address is IMBA, Austrian Academy of Sciences, Vienna, Austria.
Contract grant sponsor: Austrian Science Fund; Contract grant number: P16066.
*Correspondence to: Irina Kavernia, Dept. of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4213 Learned Lab, MRB III, 465 21st Ave. South, Nashville, TN 37232-8240. E-mail:
[email protected]
Raphaela Rid’s present address is University of Salzburg, Salzburg, Austria.
Received 10 November 2004; Accepted 24 March 2005 Natalia Schiefermeier’s present address is Medical University of Innsbruck, Innsbruck, Austria. Ilya Grigoriev’s present address is Erasmus University, Rotterdam, The Netherlands. ' 2005 Wiley-Liss, Inc.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cm.20076
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actomyosin contraction signaled by the RhoA pathway [for review, see Bershadsky et al., 2003]. Focal adhesions remain stationary or they can slide [for review, see Wehrle-Haller and Imhof, 2002]. Stationary adhesions are found at the front of a motile cell and a subset of these serve as the major ‘‘towing’’ sites supporting the traction forces that drive cell translocation [Beningo et al., 2002]. Trailing ‘‘sliding’’ adhesions at the cell rear resist this towing force. Despite their analogous protein composition, differences have been reported in the dynamic properties and macro-molecular organization of stationary and sliding focal adhesions. Thus, the spacing between integrin molecules in sliding adhesions is more compact than in stationary ones [Ballestrem et al., 2001] and whereas proteins are replaced slowly in stationary adhesions, they undergo fast turnover in sliding adhesions. This fast molecule exchange is thought to be polarized from the distal to the proximal end of sliding adhesions (‘‘adhesion treadmilling’’); it was proposed to be a mechanism that underlies their apparent ‘‘sliding’’ [Wehrle-Haller and Imhof, 2002]. Further, as compared to stationary adhesions, the connection between integrins and cytoskeletal components in the plaque of sliding adhesions is apparently unstable [Laukaitis et al., 2001], such that trailing adhesions can be left behind as integrin-containing ‘‘footprints’’ on the substrate [for review, see Webb et al., 2002]. In earlier studies [Izzard and Lochner, 1980; Abercrombie et al., 1977], trailing adhesions in fibroblasts were seen to arise from the spontaneous redirection of cell migration, such that initially anterior adhesions became localized to the rear. However, these findings have not dispelled a common assumption that trailing adhesions are survivors of adhesions created at the leading edge, which persist after the cell body translocates over them. In either case, trailing adhesions have been attributed with a negative ‘‘resisting’’ role in motility. In the present study, we monitored directional movement of zyxin-GFP-expressing fibroblasts over extended periods, allowing us for the first time to trace assembly and morphogenesis of trailing focal adhesions in persistently moving cells. We show that trailing adhesions arise by two distinct mechanisms. One major subset of trailing adhesions is formed within small short-living protrusions directed away from the main vector of cell relocation. The second one is not associated with protrusive activity but formed underneath pre-existing stress fibres proximal to their trailing ends. Moreover, our observations suggest that dynamic anchorage of the rear edge is necessary for the development of the actin network and thereby contributes to cell guidance by defining the net direction of tensile force. These findings support and extend those of Lo et al. [2004], showing that the proper regulation of stress fibre
contractility is critical for the directional force development during cell movement. MATERIALS AND METHODS Cells
Goldfish fin fibroblasts (line CAR, ATCC) were maintained in basal Eagle medium with Hepes and non-essential amino acids and with 12% FBS at 278C. They were transfected transiently as described previously [Kaverina et al., 1999]. In brief, the transfection mixture containing 2 to 4 lg of DNA and 14ml of Superfect lipofection agent (Qiagen, Chatsworth, CA) in 400 ll of serum-free medium was incubated for 30 min at room temperature. Then, 1.2 ml of medium containing 5% serum was added to the mixture, which was then applied to subconfluent monolayer cultures on 30-mm Petri dishes for 4 h at 278C. Then, the medium was replaced by full culture medium containing 12% serum. After 2 to 12 h, cells were replated onto fibronectin-coated coverslips and used for microscopy after at least 48 h. Only cells with a very moderate level of expression were used for experiments; their morphology and motile behavior did not differ from those of non-transfected cells. Fibronectin (Sigma, St. Louis, MO) was coated onto polylysine-treated coverslips by incubation on a drop of 50 mg/ml fibronectin in PBS for 1 h at room temperature (RT); after rinsing in PBS, these coverslips were used without drying. Fibronectin was stored as a stock solution in 2 M urea at 48C. For polylysine coating, coverslips were incubated in aqueous 100 mg/ml polylysine for 30 min at RT, rinsed with water, dried, and UV sterilized. Transfection Constructs
Human zyxin in a pEGFP-N1 (Clontech, Palo Alto, CA) vector was kindly provided by Prof. Jurgen Wehland and coworkers (GBF, Braunschweig, Germany). Murine EB1 in a pEGFP-N1 (Clontech) vector was donated by Dr. Anna Akhamanova (Erasmus University, Rotterdam, The Netherlands). Murine H1 calponin in pEGFP-C1 vector was kindly provided by Dr. Mario Gimona (Institute of Molecular Biology, Salzburg, Austria). Microinjection
Injections were performed with sterile Femtotips (Eppendorf) held in a Leitz Micromanipulator with a FemtoJet pressure supply (Eppendorf). Cells were injected with a continuous outflow mode from the needle under a constant pressure of between 20 and 40 hPa. Tetramethyl Rhodamine (5-TAMRA; Molecular Probes, Eugene, OR) conjugated vinculin from turkey
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Fig. 1. Adhesions that are formed at the leading edge of a directionally moving fibroblast are disassembled in front of the perinuclear region: A. Selected video frames illustrate adhesion (arrow) formation at the leading edge (time point 00 ), growth behind the protruding front (time point 490 ) and disassembly in the peri-nuclear region (time point 770 ) in a moving fish fibroblast. Zyxin-GFP. A representative example from total of 10 recorded directionally moving cells. See also supplementary video Fig1.mov. B. Time changes of the area of individual
adhesion sites at the front of a moving cell. The area of 34 measured adhesions randomly chosen from different cells reached it’s maximum at different time points within 70 minutes; then, all adhesions diminished at different time points within 120 minutes. 5 examples are shown. C. Life history of an adhesion highlighted in (A) (76 minute sequence). Zyxin-GFP. Adhesion is shown for every 10 30@. Note subsequent growth and disassembly of the adhesion.
gizzard was kindly provided by Drs. K. Rottner (GBF, Braunschweig, Germany) and M. Gimona (Institute of Molecular Biology, Salzburg, Austria). Small aliquots in 2 mg sucrose/mg protein were stored at 708C. Before use, the fluorescent vinculin was dialyzed against 2 mM Tris-Acetate, pH 7.0, 50 mM KCl, 0.1 mM DTE, and used at a concentration of 1 mg/ml.
RESULTS
Video Microscopy and Image Analysis
CAR cells were observed in an open chamber at room temperature on an inverted microscope (Axiovert 135TV; Zeiss, Austria) equipped for epifluorescence and phase contrast microscopy. Video microscopy was performed with a 100/NA 1.4 Plan-Apochromat with or without 1.6 intermediate magnification. Tungsten lamps (100 W) were used for both transmitted and epiillumination. Data were acquired with a back illuminated, cooled CCD camera from Princeton Research Instruments driven by IPLabs software (both from Visitron Systems, Germany) and stored as 16-bit digital images. The microscope was additionally equipped with shutters (Optilas GmbH, Germany) to allow separate recordings of video sequences in phase contrast and fluorescence channels and with a filter wheel for two fluorescent channels. Times between frames were 5 to 60 sec.
Anterior Adhesions in Motile Fibroblasts Do Not Reach the Rear of the Cell
Focal adhesion dynamics was followed in zyxinGFP expressing fish fibroblasts uni-directionally moving on a fibronectin substrate (Fig. 1, video Fig1.mov). Only cells that translocated in one direction more than one cell length were taken into consideration. Normally, dot-like adhesions that formed at the protruding leading edge grew for 20–80 min, and then subsequently diminished until complete disassembly after 40–120 minutes (Fig. 1B,C). These adhesions were immobile relative to the substratum during their lifetime (see also Fig. 3A). For the majority of these adhesions, complete disassembly was observed before their position was reached by the central part of the cell body, including nucleus (Fig. 1A). Adhesions Disassembly Is Associated With Intensive Microtubule Targeting in the Advancing Lamella
The frequency of microtubule targeting for adhesions in the front half of the cell was analyzed in cells expressing GFP-EB1 and microinjected with rhodaminlabeled vinculin. Facts of co-localization of growing microtubule tips carrying GFP-EB1 with focal adhesions
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were considered as targeting events. As shown in Figure 2E, the peak of targeting frequency was observed for adhesions that were positioned 20 to 40 lm behind the leading edge. Notably, the most mature (largest) adhesions were found in the same zone (Fig. 2D). Hence, a reduction of adhesions followed a period of intensive targeting by microtubules (Fig. 2A–C; videos Fig2B.mov and Fig2C.mov). The extent of adhesion disassembly correlated with the number of preceding targeting events. Small Protrusions at the Cell Rear Provide the Most Common Source for Trailing Adhesions
Consistent with the observations above, analysis of the life histories of trailing adhesions showed that only 8% originated at the leading edge of the cell and then 16% originated from lateral regions (Fig. 3B). The most common sites of initiation of trailing adhesions (53%) were protrusions at the rear and flanks of the cell. Commonly, short-lived lamellipodia extended backwards at an otherwise stable cell edge (Fig. 4, video Fid4AB.mov). Adhesions formed in these protrusions matured rapidly in response to tensile force applied from the moving cell body and developed into sliding trailing adhesions. Figure 3A depicts the tracks of trailing adhesions during their life cycle and shows that they commonly arise from the rear and, less frequently, from the flanks of the cell. At later stages, sliding trailing adhesions either fused with newly formed ones or detached from the substrate as newer ones took over their function (e.g., complementary video 4). Novel Site of Adhesion Initiation in Front of Trailing Adhesions
Additional focal adhesions were found to form at the rear of cell body in front of sliding trailing adhesions (Fig. 5, video Fig5.mov). In contrast to the known adhesion types, the origination of these adhesions did not require protrusive activity. The sites of their initiation were located under the proximal parts of stress fibres attached to sliding trailing adhesions. In the process of maturation, additional adhesions fused with the approaching sliding adhesion at the end of the same stress fibre (Fig. 5B). In sum, 23% of nascent adhesion sites that later developed into trailing adhesions were associated with stress fibres (Fig. 3B). Persistent Cell Movement Requires the Regeneration of Trailing Adhesions After Complete Detachment of Cell Rear
The locomotory cycle of fibroblastic cells often involves a stage of complete detachment of cell rear.
Detachment is followed by enhanced protrusion at the leading edge of the cell [Chen, 1979; Dunn, 1980]. We observed that the boost of protrusion was restricted to the front of the cell only for 1–2 minutes after detachment (Fig 6A, time 140 –160 ). This could be explained by a fast redistribution of cell mass to the front due to contraction of stress fibres upon the release of their rear ends [Dunn, 1980], as well as by increase of towing at the front adhesions upon loss of resistance [Munevar et al., 2001]. Subsequent to this event, when there was no more directional force, we observed formation of protrusions in a non-polarized manner around the cell perimeter. In the example shown, the leading edge of the cell did not advance significantly for 8–15 minutes (Fig6A, time 160 –200 ). Protrusions at the former rear of the cell served for initiation of new adhesion sites that provided attachment for the trailing edge (Fig. 6B, 160 –170 ). As soon as new trailing adhesions were formed, leading edge advance was restored and polarized movement continued (Fig. 6C, 300 –400 ). See also video Fig6.mov.
Fig. 2. Adhesion release in the front of the perinuclear region is promoted by microtubule targeting activity: A. Video frames from a sequence showing disassembly of adhesions behind the protruding edge (scattered line). TAMRA-vinculin marked adhesion pattern is shown at time points 10@ and 80 30@. Inset area from frame 10@ is shown at the right. On this image, EB1 localizations from three subsequent frames (time points 00 in red, 10@ in green and 20@ in blue) are overlaid; margins of focal adhesions at time point 10@ are shown in white. Growing microtubule tips target large adhesions (arrows) that disassembly at frame 80 30@. A representative example from total of 6 recorded directionally moving cells is shown. B. Video frames from a sequence showing growing plus-ends of microtubules (green) approaching adhesions (red). Localization of EB1 patches according to adhesions suggests that each of them will target an adhesion in time period from 0@ to 25@ after the current frame (compare with EB1 localization in A). Majority of plusends are concentrated close to the most mature adhesions behind the leading edge. Note that adhesions in an oval frame undergo more intensive targeting than those in rectangular frame. GFP-EB1 and TAMRAvinculin. See also supplementary video Fig2B.mov. C. Long-term observations of large adhesions in the zone of maximal maturation following microtubule targeting shown in A. Note effective disassembly of adhesions. Adhesions in oval frame are destroyed faster than those in rectangular frame (compare with A). TAMRA-vinculin. See also supplementary video Fig2C.mov. In B and C a representative example from total of 8 (other than in A) recorded directionally moving cells is shown. First, GFP-EB1 and TAMRA-vinculin in each cell were recorded for 3 minutes at 5 seconds/frame rate and then, TAMRA-vinculin in each cell was recorded for 20 minutes at 30 seconds/frame rate. D. Correlation between the distance from the advancing cell edge and the area of individual adhesion sites. The area of adhesions located farther than 40 lm from the leading edge goes down. E. In the same cells as D, correlation between the distance from the advancing cell edge and the number of microtubule tips (targeting events). Most intensive targeting occurs between 20 and 40 lm from leading edge. Analysis of 4 directional moving cells (D and E), other than in (A, B and C), recorded at 5 seconds/ frame rate for 8 minutes. Each 5th frame was quantified.
Trailing Contacts: Origin and Importance
Figure 2.
Trailing Adhesions Influence the Direction of Motility Via Stress Fibre Organization
The direction of cell relocation was parallel to an axis bisecting the main protrusion (towing) and main trailing adhesion (resisting) zones (Fig. 7A, C). When the trailing adhesions were released, the rear anchorage sites were often shifted, which resulted in the reorientation of this axis and, in consequence, in the direction of motility (Fig. 7A, video Fig7A.mov). Release of trailing adhesion entailed that associated stress fibers lost their anchorage sites (Fig. 7B). Upon adhesion release, stress fibres were remodeled and established connection with the new trailing adhesions (Fig. 7B, video Fig7B.mov). Notably, stress fibre remod-
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Fig. 3. Trailing adhesions are commonly originated not at the leading edge but elsewhere: A. Tracks of adhesion relocation in a motile fibroblast. Location of all adhesion site found in the first frame (blue cell outline) until the last of these adhesions is detached (red cell outline) is shown (black). Note that sliding adhesion tracks start most frequently at the rear, sometimes at the flanks and very rarely at the front. B. Respective input of alternative mechanisms to trailing adhesion initiation. First pie shows input of adhesions formed beneath stress fibre termini (grey) compared to those associated with protrusions (orange). Second pie presents in detail respective input of adhesions formed at front (faint yellow), lateral (dark yellow) and rear (red) protrusions. Results of analysis of 110 trailing adhesions from 5 directionally moving cells are shown. Number of adhesions in each group and percentage from the total is presented at pie slices.
eling was accompanied by correction of cell movement to the new direction of the cell axis (Fig 7C, compare with Fig. 7A). Since stress fibres respond to tension and serve as a connection between the towing and resisting adhesions, their organization reflects the direction of the traction force. Thus, it is possible to map the net direction of movement from the stress fibre pattern. We estimated the force direction from the geometric vector sum of stress fibres (Fig 7D). Directions deducted in this way showed good correlation with the direction of locomotion for the both initial and resulting stress fibre configuration before and after the release of trailing adhesion.
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We conclude that while protrusion produces the traction force, trailing adhesion can tune the direction of cell translocation by influencing stress fibre organization. DISCUSSION The Disslolution of Adhesions in Front of the Cell Body is Promoted by Microtubule Targeting
According to the observed dynamics of the EB1, which allows observation of microtubule dynamics in thicker regions of the cell, the dissolution of anterior adhesions under the cell body is potentiated by microtubule targeting events. Consistent with this observation is the finding that focal adhesions failed to disassemble under the body of microtubule-depleted cells that were forced to move by the asymmmetrical application of myosin inhibitors [Kaverina et al., 2000]. Contrary to the claims of [Ballestrem et al., 2000], destabilisation by microtubules is not restricted to trailing adhesion sites. Rather, the present findings support the idea [for review, see Small et al., 2002] that microtubules transduce signals to release tension at focal adhesion sites both in the front and rear of a cell. Trailing Adhesions are Originated by Specific Mechanisms and not by Chance
The initiation of primary focal complexes is typically restricted to lamellipodia and filopodia, sites of intensive actin polymerization. Further maturation of focal adhesions entails the development of tensile stress against the substrate, provided by myosin-dependent contraction [for reviews, see Vasiliev 1985; Chrzanowska-Wodnicka and Burridge, 1996; Bershadsky et al., 2003; Kaverina et al., 2002]. This transition has been observed in several studies using fluorescent probes tagged to adhesion components [Ballestrem et al., 2001; Laukaitis et al., 2001; Zaidel-Bar et al., 2003; Anderson and Cross, 2000]. For migrating fish fibroblasts, around 75% of trailing adhesions originated from posterior and lateral protrusion events and grew through steps of retraction, sliding and fusion. In this context, the protrusive activity on the cell margin at the rear illustrated a Fig. 4. Most frequently trailing adhesions are formed at small protrusions at the back of moving cell: A, C. Video frames from a sequence illustrating adhesions within small short-term protrusions (thin arrows) at the rear of directionally moving cells (note star for a reference point). Overview for B and D, respectively. B, D. Examples of formation and maturation of a trailing adhesion within a small protrusion (arrow). Note absence of cell extension at first frame (310 in B and 00 in D), initiation of adhesion within protrusion (670 in B and 30 in D) and typical trailing adhesion upon maturation (1060 in B and 130 in D). Zyxin-GFP. Representative examples from total of 33 adhesions present in 6 of 6 analyzed directionally moving cells. See also supplementary video Fig4AB.mov.
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deliberate intention on the part of the cell to create trailing attachments; in other words trailing adhesions are active rather than passive elements of cell migration. A second, novel site of adhesion formation was identified beneath and in advance of existing trailing adhesions. These sites are likely analogous to the ‘‘hot spots’’ of actin polymerization identified in the stress fibres of smooth muscle cells, proximal to adhesion foci [Kaverina et al., 2003; Burgstaller and Gimona, 2004]. In cultured smooth muscle cells, such sites show a different composition of associated proteins [Burgstaller and Gimona, 2004] and seed Arp2/3 dependent actin polymerisation that can be enhanced in response to phorbol esters [Kaverina et al., 2003]. Arp2/3 dependent actin polymerisation is closely linked to adhesion formation in lamellipodia [for review, see DeMali and Burridge, 2003]. Arp2/3 has recently been found to bind vinculin in a Rac-dependent manner [DeMali et al., 2002] and thus can be implicated in the Rac-dependent recruitment of the adhesion molecules to protrusions. We conclude that the regions in stress fibres proximal to focal adhesions constitute specific subdomains that provide regulatory environment similar to protrusions and combining the potential for adhesion initiation, with inherent tensile stress for adhesion development. Analyzing recovery after photobleaching of sliding adhesions, [Ballestrem et al., 2001] proposed that integrins cycle though the length of sliding adhesions in a treadmilling-like mode. Accordingly, molecules are incorporated in the sliding adhesion at their proximal ends. This treadmilling activity may be a manifestation of the same ‘‘hot spot’’ of adhesion formation described here. Trailing Adhesions are Required to Maintain the Stress Fibres to Support Cell Body translocation
The migration of fibroblasts is thought to be driven by strong propulsive traction forces applied by nascent focal adhesions in the front of the cell [Beningo et al., 2002]. The translocation of the cell body is effected by the transduction of these forces through the actin cytoskeleton framework. Stress fibres play a critical role in
Fig. 5. Long-life trailing adhesion can partially arise from additional adhesions that are formed in front of it: A. Video frames from a sequence showing typical long-life adhesion (thin arrow) that slides without detachment at the rear of motile fibroblast (note star for a reference point). Overview for B. B. Additional adhesions formed in the front of sliding trailing adhesion (arrows) in the sites where stress fibres are connected to adhesions. Later, they fuse with the main adhesion (960 -1000 ). Zyxin-GFP. A representative example from total of 14 adhesions present in 5 of 6 analyzed directionally moving cells. See also supplementary video Fig5.mov.
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Fig. 6. Polarized advance of the leading edge of fibroblastic cell requires trailing adhesion: A. Cell outline upon complete release of trailing adhesions. Protrusion is enforced during the release between time 140 (red outline) and 160 (green outline). No visible forward protrusion is detected in two next 2-minute intervals (180 and 200 , blue outlines). B. Complete detachment of trailing adhesion (150 ) is rapidly followed by the formation of new adhesion (arrow) accompanied with protrusion backwards (160 , 170 ). The rear part of the cell is shown.
Zyxin-GFP. C. Leading edge advance (red to green line) before (left) and after (center, right) detachment of trailing adhesion. Three 10-minute periods are shown. Directly after detachment (frames 15– 25) the leading edge does not extend until new trailing adhesions are formed (thin arrows, see also B). Zyxin-GFP. A representative example from total of 5 rear release events in 4 out of 6 analyzed recorded cells. See also supplementary video Fig6.mov.
this translocation [Munevar et al., 2001] and could not form along the axis of movement without anchorage at trailing adhesions. Using local application of adhesion disrupting peptides, Munevar et al., [2001] were able to distinguish distinct functional differences between leading and trailing adhesions. The disruption of anterior adhesions caused a drastic decrease in traction forces. Disruption of trailing adhesions had no such effect, and traction force was continuously produced by the ‘‘towing’’ anterior adhesions. According to our observations, however, ‘‘towing’’ at the front is not sufficient for continuous movement: upon trailing edge retraction, a short forward thrust was followed by a phase of re-formation of adhesions at the rear (Fig. 6). Thus, trailing adhesions appear to have a feedback effect on protrusion at the front.
Position of Trailing Adhesion Influences Direction of Cell Movement
Trailing adhesions appear to exhibit the property of rudders in cell movement in that the selective dissociation of a subset of trailing adhesions can lead to a reorientation of the cell axis. Rapidly locomoting keratocytes exhibit sliding adhesions on their flanks [Anderson and Cross, 2000] and mechanical disruption of adhesions on one flank causes a reorientation of cell movement [Anderson et al., 1996] in a similar, rudder-like mode. We earlier showed that trailing adhesions in fibroblasts are subject to high frequencies of targeting by microtubules [Kaverina et al., 1999]. We conclude that this targeting constitutes an active tuning of adhesions at the tail and that a cooperation of adhesion modulation and
Fig. 7. Trailing adhesions have a strong influence on the direction of motility: A. The cell changes the direction of movement (double chevron) according to the main tensile force axis (long hollow arrow); the axis, in turn, is arranged between the leading protrusion and the main trailing adhesion (white arrows). Zyxin-GFP. A representative example from total of 3 zyxin-GFP-expressing cells that changed direction during recording (out of 6 recorded cells). See also supplementary video Fig7A.mov. B. Organization and direction of stress fibres depends on trailing adhesions. Trailing adhesion release results in stress fibre remodeling and reorientation (arrows). GFP-H1-calponin. A representative example from total of 4 GFP-H1-calponin-expressing cells that changed direction during recording (B, C and D). See also supplementary video Fig7B.mov. C. Phase contrast image of the cell shown in (B). Black arrows indicate
main trailing anchorage zones. The direction of cell movement (double chevron) approximately corresponds to the direction from the middle of anchorage zone to the leading protrusion (long hollow arrow). Compare with (A). D. Schematic presentation of inputs of forces produced by individual stress fibres in the direction of main tensile force axis for the time frame 00 from (B). Directions of individual stress fibres (middle) were estimated from processes image. All stress fibers were presented as vectors directed to the leading edge, and geometrically summarized. Vector sum of all stress fibres (shown at lower magnification, left) corresponds to the direction of cell movement (long hollow arrow, compare with (C)). E. Schematic presentation of stress fibre forces for time frame 770 from (B). Vector sum of all stress fibers (left) corresponds to the direction of cell movement (long hollow arrow, compare with (C)).
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protrusive activity determines the net orientation of movement. This cooperation is likely coordinated via mechanical signals transduced through the stress fibre network. The absence of such a coordination mechanism may explain the random movement of fibroblasts lacking myosin IIb, which is restricted in its localization to stress fibre bundles [Lo et al., 2004]. These cells exert disorganized forces on the substrate leading to random multiple protrusive activity and are therefore unable to directional movement. In the same context, stable protrusion can be mechanically induced by directional application of tensile stress to a cell plated on a flexible substrate [Lo et al., 2000] by substrate deformation. A number of recent studies reveal possible mechanisms for mechanosensory facilitating of protrusion. For instance, organization of adhesion pattern in the front of the cell can be directly regulated by tensile force [for review, see Geiger and Bershadsky, 2002; Katsumi et al., 2004]. Direction of force decides on location, where integrins make strong connection with extracellular matrix. Artificial focal complex formation can be induced by force application [Galbraith et al., 2002] that stabilizes integrin-cytoskeleton linkage [von Wichert et al., 2003]. New adhesions that come under local stress mature earlier than others [Riveline et al., 2001] and provide a stable base for advancing leading edge. Also, mechanical force can determine cell motility reactions by means of stretch-activated ion channels. Ca2þ channels are involved in the modulation of cell movement [Lee et al., 1999] and local concentrations of intracellular Ca2þ can enhance traction at the front of fibroblasts [Munevar et al., 2004]. Accordingly, spatial activation of ion channels has been implicated to play a role in tension-induced fibroblastic motility. These mechanisms, among others, could contribute to the fine tuning of protrusion localization by trailing adhesions. In this study, we show that trailing adhesions, which support stress fibre directionality are necessary for persistent forward movement of fibroblasts, and that they are actively generated at the rear of the cell. We suggest the existence of a feedback, mechanosensing mechanism that maintains trailing adhesion renewal in moving cells. The nature of this mechanism remains to be investigated.
ACKNOWLEDGMENTS
We thank Drs. Anna Akhmanova, Mario Gimona, and Juergen Wehland for providing expression constructs.
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