The frequency of catastrophes is higher at focal adhesion sites

In order to visualize microtubule dynamics clearly in relation to focal adhesions, we applied total internal reflectance fluorescence (TIRF) microscopy to illuminate only the ventral layer of cells attached to a glass substrate (Axelrod, 1989). Fish fibroblasts were transfected with mCherry-tubulin or 3xGFP-EMTB to visualize microtubules and GFP-paxillin or mCherry-paxillin to mark adhesion sites (Fig. 1A-C). Under TIRF conditions, we assumed that colocalization of a microtubule with an adhesion site indicates their close physical interaction. In live-cell time-lapse sequences, we observed that, when growing microtubules reached an adhesion site, they frequently underwent catastrophe and shrank. Individual microtubules often underwent multiple catastrophe events at a focal adhesion followed by subsequent rescues (Fig. 1A), whereas only few catastrophes occurred in adhesion-free areas (Fig. 1B). Analysis of overall adhesion numbers showed that approximately 40% of catastrophes occurred at adhesion sites, 12% at the cell edge and 48% elsewhere (Fig. 1D). As the overall adhesion area is considerably smaller than the adhesion-free ventral cell surface, these numbers indicate that adhesions serve as preferential sites for catastrophes.

A simple quantification of catastrophe events within a large area is insufficient for the analysis of the induction of microtubule catastrophes at local sites. The reason is that, because catastrophe is an event in which a microtubule switches from growth to shrinkage, it can occur only to a growing but not to a shortening plus-end. Thus, only those sites where growing microtubule ends were present were taken into consideration. Accordingly, we measured the length of elongation for microtubules while growing through adhesions or through an adhesion-free area. To obtain average persistent microtubule elongation, we normalized the average microtubule elongation by the average number of catastrophe events for each group of data. Strikingly, persistent microtubule elongation without catastrophes was seven times lower at adhesion sites (Fig. 1E) than elsewhere. These data suggest a specific mechanism that triggers microtubule catastrophes at adhesion sites.

Adhesions trigger catastrophes by combined mechanical and biochemical factors

A crucial question is whether catastrophes at adhesions are triggered by a specific biochemical mechanism or as a consequence of the increased density of actin filaments at the adhesion plaque. Such nonspecific mechanisms could include mechanical restraint or spatial restriction for free tubulin penetration. In order to answer this question, we analyzed the dynamics of mCherry-EB3-marked plus-tips of microtubules in the vicinity of GFP-paxillin-containing focal adhesion sites (Fig. 2).

Kymographic analysis of the dynamics of microtubule tips revealed two scenarios: either EB3 was released from a microtubule at an adhesion site, indicative of a catastrophe (Fig. 2A), or a growing tip proceeded through an adhesion (Fig. 2B). In the second case, the velocity of microtubule tips was notably reduced while moving through the adhesion site (Fig. 2B). In adhesion-free cytoplasm, ∼70% of microtubules grew at a rate of 0.1 μm/second or more (`fast microtubules') and ∼30% of microtubules grew more slowly (`slow microtubules'). At focal adhesions, the proportion of slow microtubules increased by up to 50% within the same microtubule population (Fig. 2D). At the same time, the catastrophe ratio dramatically increased at adhesion sites. For microtubules approaching adhesions, the probability of catastrophe increased from less than 5% on the approach route to ∼25% at the adhesion (Fig. 2F).

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Fig. 2.

Microtubule tip dynamics at focal adhesions. (A) Kymograph of a microtubule catastrophe event at a focal adhesion (FA). The microtubule is marked with mCherry-EB3 (red), and the focal adhesion is marked with GFP-paxillin (green). (B) Kymograph of a microtubule tip (mCherry-EB3, red) that does not undergo catastrophe at the adhesion (GFP-paxillin, green). The microtubule tip changes its growth dynamics at the focal adhesion. (C) An example of assigned area zones for microtubule growth paths near focal adhesions (GFP-paxillin, green). Cytoplasmic zones close to the cell center are encoded as zones `–2' and `–1'. The zone adjacent to a focal adhesion is encoded as zone `0'. A focal adhesion contains zones `1-3', according to its length. Cytoplasmic zones towards the cell periphery are encoded as zones `1out' and `2out'. Each zone is 1 μm in size. (D) The distribution between fast (growth rate >0.1 μm/second) and slow (growth rate <0.1 μm/second) microtubules for each zone. In the cytoplasm (yellow background), ∼70% of microtubules grow at a speed of 0.1 μm/second or faster. At focal adhesions (green background), the percentage of fast microtubules reduces down to ∼50%. (E) Dependence of the microtubule catastrophe ratio (percentage of approaching microtubules undergoing catastrophe) on the time a microtubule spends in the cytoplasm zone (zone –1, green), at the adhesion base (zone 0, blue) and in the adhesion (zone 1, red). (F) Total microtubule catastrophe ratio for each zone. The percentage of approaching microtubules undergoing catastrophe increases from 2% in the cytoplasm to 25% at focal adhesions. A total of 139 microtubules were quantified in three 3xGFP-EMTB- and mCherry-paxillin-co-transfected cells within 15 minutes. Bar, 1 μm.

Combined, these data reveal a correlation between microtubule tip speed and catastrophe ratio. The percentage of `slow MTs' increases (Fig. 2D, red line) at the same location at inner adhesions (zones 0-1) where catastrophes are induced (Fig. 2F, blue line). These data suggest that the dense focal adhesion structure suppresses microtubule growth and thus increases the probability of catastrophe. This mechanism, however, is not specific for adhesions. We found that the catastrophe ratio of individual microtubules correlated inversely with their plus-tip velocity, both for microtubules growing through adhesions and through adhesion-free areas (Fig. 2E), suggesting that the microtubule catastrophe rate can be increased by cytoplasmic rigidity or spatially restricted concentration of tubulin dimers equally at adhesion plaques and at other locations. Strikingly, for microtubules with the same growth rate, the catastrophe ratio was higher at adhesion sites than elsewhere (Fig. 2E).

We have further tested whether softening of the adhesion structure decreases the ability of adhesions to induce catastrophes. For this purpose, we plated cells on a soft gelatin gel cushion (Seals et al., 2005), where strong tensile forces cannot be developed (Discher et al., 2005). Adhesions formed under these conditions were smaller than those on glass substrates (<2 μm versus <4 μm in length), but the catastrophe ratio at such adhesions was similar (23% on glass and 32% on gelatin gel). Together, these data indicate that a specific trigger of biochemical catastrophe acts at adhesion sites.

In line with this finding, some microtubules continued to polymerize when their plus-ends were apparently prevented from advancing by an obstacle. The polymerizing plus-ends of such microtubules remained immobile while the length of the microtubule increased, resulting in loop formation (supplementary material Fig. S1). Thus, mechanical obstacles do not necessarily cause microtubule catastrophe. Combined, our results strongly suggest that a biochemical mechanism, rather than a mechanical mechanism or spatially restricted concentration of tubulin dimers, drives induction of microtubule catastrophe at focal adhesion sites.

Induction of catastrophe does not depend on the maturation stage of adhesions

Having determined that a specific biochemical mechanism at the adhesion sites enhances microtubule catastrophe activity, we aimed to identify focal adhesion proteins potentially involved in the process. As focal adhesion composition and signalling changes during their life course, we first investigated whether induction of catastrophe depends of the adhesion maturation stage. To distinguish initial (early) focal adhesions from mature (late) adhesion sites, we used two adhesion markers: paxillin, which is present in the majority of adhesion sites, and zyxin, which has been shown to incorporate into adhesions at a later maturation stage (Zaidel-Bar et al., 2003). Simultaneous transfection of the microtubule marker GFP-EMTB, mCherry-paxillin and Cerulean-zyxin allowed us to compare microtubule dynamics at early (containing only paxillin) versus late (containing paxillin and zyxin) adhesions (Fig. 3A,B).

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Fig. 3.

Microtubule catastrophes do not depend on the maturation stage of focal adhesions. (A,B) A frame from a video sequence of a cell co-transfected with mCherry-paxillin (A, red) and Cerulean-zyxin (B, cyan). Paxillin marks both early (hollow arrows) and late (filled arrows) focal adhesions. Zyxin is a marker for late focal adhesions only (filled arrows). Bar, 5 μm. (C) Microtubule catastrophe ratio at early versus late adhesions. Approximately 90% of approaching microtubules undergo catastrophe both at early and late adhesions. A total of 117 catastrophe events in four cells were quantified.

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Fig. 4.

Catastrophes can occur in the zyxin-free zone of focal adhesions. (A) A frame from a video sequence of a cell co-transfected with mCherry-paxillin (red) and GFP-zyxin (green). Bar 10 μm. (B,C) Enlargement of areas at the leading edge (B) and trailing edge (C) of the cell (paxillin, red; zyxin, green). The intensity profiles of paxillin (red lines) and zyxin (green lines) along a line (white) of width one pixel are shown. Paxillin extends more distantly towards the cell edge. The image is a representative example from 15 cells. (D) A frame from a video sequence of a cell co-transfected with GFP-EMTB (blue) to visualize microtubules, Cerulean-zyxin (green) to mark late adhesions and mCherry-paxillin (red) to mark both early and late adhesions. Bar, 10 μm. The boxed area is enlarged to the right (E,F). (E) Enlarged frame sequence of microtubules (green) and paxillin (pink). (F) Enlarged frame sequence of microtubules (green) and zyxin (red). A catastrophe occurred at the 20-second time-point at the distal zyxin-free end of a focal adhesion. Time, seconds. Arrows show the direction of microtubule movement. (G) Enlarged frame sequence of the focal adhesion from E and F, showing zyxin (green) and paxillin (red). The arrows point at the zyxin-free distal end of the focal adhesion.

Overall quantification based on time-lapse live-image sequences revealed that ∼90% of adhesion-associated catastrophes occur at late adhesions, and only ∼10% at early adhesions (Fig. 3C). Nevertheless, taking into account that late adhesions are approached by a significantly higher number of microtubules, the final analysis showed that both types of adhesions were equally effective in catastrophe induction: 90% of approaching microtubules underwent catastrophe at adhesions (Fig. 3D) regardless of their maturation stage.

Paxillin-associated adhesion structures are sufficient for induction of microtubule catastrophe

Although both zyxin and paxillin are present in late adhesions, we found that these adhesion proteins are distributed unevenly within single elongated mature adhesion sites: paxillin extends more distantly towards the cell edge than zyxin (Fig. 4). Such uneven distribution was found both at adhesions behind the leading edge (Fig. 4B) and sliding adhesions at the trailing edge (Fig. 4C) of the cell. We observed that a considerable number of microtubules underwent catastrophe at the distal ends of late adhesions (Fig. 1B). Detailed analysis of these catastrophes showed that they occurred at paxillin-rich adhesion domains devoid of zyxin (Fig. 4D-G). These data suggest that paxillin-associated adhesion structures are sufficient for induction of microtubule catastrophe.

Displacement of paxillin from focal adhesions by microinjection of LIM domains 2 and 3 decreases the number of catastrophes specifically at adhesion sites

We further examined the involvement of paxillin in the regulation of catastrophe by displacement of paxillin from adhesion sites. For this purpose, individual LIM domains of paxillin, including LIM2, LIM3 and tandem LIM2-LIM3 domains were produced as GST-fusion proteins (see Materials and Methods) and microinjected into cells. One hour after injection, cells were fixed and processed for immunostaining. Single GST-LIM2 (Fig. 5A,B) and GST-LIM3 (data not shown) showed mostly diffuse staining in the cytoplasm, with only occasional weak focal adhesion localization, whereas GST-LIM2-LIM3 was localized robustly to focal adhesions (Fig. 5C,D). Moreover, live-cell imaging of GFP-paxillin-expressing cells showed that injected LIM2-LIM3 domains caused displacement of GFP-paxillin from adhesion sites within 45 minutes (Fig. 5E,F). Immunostaining of injected cells confirmed paxillin displacement (data not shown) while another core adhesion component, vinculin, remained in the adhesions (Fig. 5D).

Notably, although LIM2-LIM3 domains have been shown to bind to tubulin (Brown and Turner, 2002; Herreros et al., 2000), no detectable localization to microtubules has been observed for the injected protein (Fig. 5C). Thus, if the LIM2-LIM3 domains of paxillin interact with microtubules in vivo, it can only occur in the vicinity of focal adhesions.

In order to determine whether substitution of paxillin by the LIM2-LIM3 domain at adhesion sites modulates microtubules at this location, we microinjected LIM2-LIM3 into fish fibroblasts co-transfected with GFP–β-tubulin and dsRed-zyxin. Time-lapse live-image sequences were recorded before the injection as well as 5 minutes after the injection. We found first that microtubules more often exhibited persistent growth resulting in an increase of microtubule length and density at the cell periphery (Fig. 6A,B). Analysis of microtubule dynamics revealed that, for each cell, the percentage of catastrophe events in the adhesion sites was 40% lower than in the same cell before injection (Fig. 6G,H). Typically, microtubules grew processively through three to four focal adhesions during a single growth phase in comparison with one to two adhesions in the same cell before injection (Fig. 6C-F). Along with a statistically significant decrease of catastrophe frequency at adhesions, no changes were observed in microtubule catastrophes in adhesion-free cytoplasm (Fig. 6G,H). This effect appeared to be specific for LIM2-LIM3 domains as microinjections of the paxillin LIM1 domain alone and either LIM1-LIM2 or LIM3-LIM4 domains had no influence on microtubule dynamics (data not shown).

Furthermore, we found that injection of LIM2-LIM3 paxillin domains in GFP-EB1-expressing cells resulted in an increased number of growing microtubules but had no effect on the velocity of microtubule growth. In particular, the number of EB1 comets indicating polymerizing microtubule plus-tips was increased by 30% 5 minutes after LIM2-LIM3 microinjection (supplementary material Fig. S2). However, the rate of microtubule growth did not change significantly (supplementary material Fig. S2), indicating that, other than decreasing the rate of catastrophe at the adhesion site, injection of LIM2-LIM3 protein had no detectable effect on microtubule polymerization.

Cells

Goldfish fin fibroblasts (line CAR, no. CCL71; American Type Culture Collection) were maintained in DMEM with nonessential amino acids, 25 mM HEPES and with 10% FBS at 27°C. For experiments, cells were plated onto coverslips or glass-bottomed dishes (MatTek) and coated with human serum fibronectin (BD Biosciences) for at least 48 hours. Fibronectin was coated onto coverslips or glass-bottomed dishes (50 μg/ml in PBS for 30 minutes at room temperature). Gelatin gel cushion was prepared essentially as described previously (Seals et al., 2005). In brief, 2.5% gelatin in PBS containing 2.5% sucrose was allowed to polymerize for 30 minutes on glass-bottomed dishes (300 μl per dish). Next, the gels were crosslinked by 0.5% glutaraldehyde for 45 minutes. Free aldehyde groups were blocked by 1 mg/ml NaBH₄. Finally, the gels were coated with fibronectin, as described above.

DNA constructs and transfection

mCherry-EB3 and Cerulean-EB3, made by replacing GFP in a pEGFP-EB3 construct (kind gift of A. Akhmanova, Rotterdam, The Netherlands) with Cerulean (kind gift of D. W. Piston, Nashville, TN) or mCherry (kind gift of R. Tsien, San Diego, CA), EGFP-EB1 (kind gift of A. Akhmanova, Rotterdam), pEGFP-tubulin (kind gift of J. Wehland, Germany), mCherry-tubulin (kind gift of R. Tsien) and 3xGFP-EMTB [microtubule-binding domain of E-MAP-115 (ensconsin) fused to three copies of EGFP (Bulinski et al., 1999), a kind gift of J. C. Bulinski, New York, NY] was used for microtubule plus tips and microtubule visualization. Neither tubulin fusion proteins (Rusan et al., 2001) nor EMTB (Faire et al., 1999) change microtubule dynamics and are routinely used for in vivo studies. GFP-paxillin (West et al., 2001), mCherry-paxillin (kind gift of S. Hanks, Nashville, TN), mCherry-zyxin, made by replacing GFP in a pEGFP-zyxin construct (kind gift of J. Wehland) with mCherry, and Cerulean-zyxin, constructed by cloning zyxin from mCherry-Zyxin into a mCerulean-C1 vector, were used for visualization of adhesion sites. Subconfluent monolayer cultures on 30 mm Petri dishes were used for transfection. For each dish, the transfection mixture was prepared as follows: 1.5-2 μg total DNA and 12 μl of Superfect lipofection agent (Qiagen) were mixed in 200 μl of serum-free medium. After 30 minutes of incubation at room temperature, a further 1.3 ml of medium containing 5% serum was added. Cells were incubated in this mixture for 4 hours at 27°C and the medium then replaced by normal medium containing 10% serum. After 24 hours, cells were replated at a desired dilution onto coverslips or glass-bottomed dishes for microscopy.

Microinjections

Injections were performed with sterile Femtotips (Eppendorf) held in a Leitz Micromanipulator with a pressure supply from an Eppendorf Microinjector 5242. Cells were injected with a continuous outflow mode from the needle under a constant pressure of between 20 and 40 hPa.

Proteins for microinjection

Cy3-tubulin was kindly provided by F. Severin (Max Planck Institute, Dresden, Germany). It was stored at a concentration of 20 mg/ml in aliquots at –70°C. For microinjections, Cy3-tubulin aliquots were diluted 1:3 with Tris-acetate injection buffer (2 mM Tris-acetate, pH 7.0, 50 mM KCl and 0.1 mM DTE) and used on the same day. GST only, GST-LIM1, GST-LIM2, GST-LIM3 and GST-LIM2-LIM3 fusion recombinant proteins were expressed in Escherichia coli and purified as described previously (Brown et al., 1998). GST, GST-LIM1, GST-LIM2, GST-LIM3 and GST-LIM2-LIM3 were stored at a concentration of 1.6 mg/ml at –70°C.

Video microscopy of transfected cells

TIRF live-cell videos were acquired on a Nikon TE2000E microscope equipped with a Perfect Focus System and Nikon TIRF2 System for TE2000 using a TIRF 100× 1.49 NA oil-immersion lens and back-illuminated EM-CCD camera Cascade 512B (Photometrics) driven by IPLab software (Scanalytics). A 40 mW argon laser (Melles Griot) and 10 mW DPSS laser 85YCA010 (Melles Griot) were used for excitation. A custom-made double-dichroic TIRF mirror and emission filters (Chroma) in a filter wheel (Ludl) were used. Three-channel movies were made with the TIRF setting as described above for two channels and wide-field fluorescence using a BrightLine-CFP 2432A filter cube (Semrock) for the third channel.

Video microscopy of injected cells

Cells were injected and observed in an open chamber at room temperature on an inverted microscope (Axiovert 135TV; Zeiss) equipped for epifluorescence and phase-contrast microscopy. Injections were performed at an objective magnification of 40× (1.3 NA Plan Neofluar) and video microscopy with a 100× 1.4 NA Plan-Apochroma objective. Double fluorescence was achieved by a GFP-RFP Pinkel filter set (Chroma) in a custom-made filter wheel. Tungsten lamps (100 W) were used for both transmitted and epi-illumination. Data were acquired with a back-illuminated, cooled CCD camera from Princeton Research Instruments driven by IPLabs software. Paxillin displacement from focal adhesions was observed by live TIRF imaging of GFP-paxillin-expressing cells, as described above.

Photobleaching

Cells coexpressing mCherry-alpha-tubulin and GFP-paxillin where photobleached for 10 seconds with a 10 mW DPSS laser 85YCA010 (Melles Griot) by focusing laser light in the focal plane with a custom-made lens (Nikon) placed in the position of the filter cube. Two-channel movies were recorded using TIRF microscopy, as described above, for 2.5 minutes after bleaching.

Immunofluorescence microscopy

For immunostaining, cells were extracted for 1 minute in 0.25% Triton X-100 in cytoskeleton buffer (10 mM MES, 150 mM NaCL, 5 mM EGTA, 5 mM glucose and 5 mM MgCl₂, pH 6.1) and fixed for 20 minutes in 3% paraformaldehyde in cytoskeleton buffer. Immunostaining was performed using polyclonal goat antibodies against glutathione S-transferase (GST) and mouse antibodies against vinculin.

Quantitative analysis

Microtubule catastrophe: time-lapse movies of cells expressing 3xGFP-EMTB to label microtubules and mCherry-paxillin to mark adhesion sites (5 seconds/frame) were processed by background subtraction and intensity adjustment using ImageJ software. The coordinates of each catastrophe event were recorded and then checked to determine whether there was an adhesion site with the same coordinates.

Average microtubule elongation: time-lapse movies of cells expressing mCherry-tubulin and GFP-zyxin (5 seconds/frame) were processed by background subtraction and intensity adjustment using ImageJ software. The trajectories of microtubule plus-end movement were followed using a plug-in of ImageJ (`manual tracking'). Microtubule elongation in the cytoplasm or at adhesion sites was normalized by the number of catastrophes in each location for each microtubule. Then the average elongation distance was calculated for all microtubules. Statistical analysis was performed in Microsoft Excel.

Tip dynamics at adhesion sites: time-lapse movies of cells expressing mCherry-EB3 and GFP-paxillin (5 seconds/frame) were processed for intensity adjustment using ImageJ. For uniform analysis, only the most common cases when microtubule tips approached adhesions from their proximal ends were considered. Narrow areas around adhesion sites were delineated and combined into kymographs. The area was divided into 1-μm-long zones (Fig. 2C). For each microtubule tip, the time it spends in each zone and place of catastrophe was calculated manually. Microtubules that pass through the zone in 1-2 frames (10 seconds or less) were considered as fast microtubules and in three frames or more (>15 seconds) as slow microtubules. For each zone, the number of approaching microtubule tips and disappearing tips was calculated. For zones –1, 0 and 1, the probability of a microtubule undergoing catastrophe versus the time the microtubule tip spent in the zone was calculated. Statistical analysis was performed in Microsoft Excel.

Catastrophes at early and late adhesions: time-lapse movies of cells expressing 3xGFP-EMTB, mCherry-paxillin and Cerulean-zyxin (5 seconds/frame) were used. Trajectories of microtubule plus-end movement were followed using a plug-in (`manual tracking') of ImageJ software. The coordinates of each catastrophe were recorded and then checked against the localization of paxillin and zyxin by using ImageJ software. Statistical analysis was performed in Microsoft Excel.

Analysis of microtubule dynamics after microinjection: calculations of microtubule growth velocity and catastrophe events were made using MetaMorph software, and statistical analysis was performed in Microsoft Excel.