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Originally published In Press as doi:10.1074/jbc.M404054200 on April 21, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28715-28723, July 2, 2004
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Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions*

Grégory Giannone{ddagger}§, Philippe Rondé{ddagger}, Mireille Gaire{ddagger}, Joël Beaudouin||, Jacques Haiech{ddagger}, Jan Ellenberg||, and Kenneth Takeda{ddagger}**

From the {ddagger}Laboratoire de Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, Unité Mixte de Recherche CNRS 7034, Université Louis Pasteur de Strasbourg, 67401 Illkirch, France and ||Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Received for publication, April 12, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Focal adhesion kinase (FAK) activity and Ca2+ signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca2+ sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca2+ variations at FA sites and FA dynamics. Discrete subcellular Ca2+ oscillators initiate both propagating and abortive Ca2+ waves in migrating U87 astrocytoma cells. Ca2+-dependent FA disassembly occurs when the Ca2+ wave reaches individual FAs, indicating that local but not global Ca2+ increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca2+ elevation. FAK-related non-kinase domain-Ycam had a faster, Ca2+-insensitive recovery half-time (11 s), which is consistent with the effect of Ca2+ on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca2+ and FAK autophosphorylation was correlated to intracellular Ca2+ levels, we propose that local Ca2+ elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular adhesion and migration involve remodeling and reorganization of focal adhesion (FA)1 sites. In FAs, aggregated integrins span the plasma membrane and mediate interactions between extracellular matrix components and cytoskeletal proteins. In migrating fibroblasts, FAs are mainly immobile, serving as traction points, except in restricted retractile cell edges, where they display centripetal movements (1). This local regulation of FA movement involves asymmetric signal transduction and probably directs cell polarization and migration. Despite the observation of an increasing Ca2+ gradient from front to rear in migrating fibroblasts (2, 3), Ca2+ is not often suggested to be a polarity signal in chemotaxis. This is surprising, given the large number of potential Ca2+-sensitive targets involved in integrin signaling and cell migration (4–8) and the tight spatio-temporal regulation of Ca2+ elevations (9). In support of a spatially restricted action of Ca2+ during cell migration, Ca2+ oscillations induce cell rear retraction in lymphocytes and keratinocytes (10, 11). Using fluorescent protein fusion constructs targeted to FAs, we showed recently (12) that Ca2+ elevations trigger local disassembly of FAs in human U87 astrocytoma cells. Here, we again made chimeric proteins using focal adhesion kinase (FAK) as an FA-targeting module and yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca2+ sensor (13) to measure both local Ca2+ variations at FAs by fluorescence resonance energy transfer (FRET) between enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), and FA dynamics. Here, we describe temporal and spatial correlations linking local Ca2+ elevations to disassembly of individual FAs.

In the hierarchical organization of FA-associated proteins, FAK appears as an early component in FA formation (14). The integrin/adhesion-dependent increase in FAK Tyr397 autophosphorylation relies on intermolecular FAK transphosphorylation (15) mediated by the formation of integrin clusters. The central role of FAK in the formation of a functional FA is also emphasized by its scaffolding function, allowing direct interaction and translocation of signaling proteins such as Src, growth factor receptor binding protein-2, paxillin, phospholipase C-{gamma}, phosphatidylinositol 3-kinase, and p130cas (16) toward FAs. Furthermore, FAK interacts with FA structural proteins, including integrins, talin, {alpha}-actinin and tensin, and via paxillin with both vinculin and F-actin (17). In FAK-deficient cells, reduced motility is accompanied by an increased number of FAs (18), suggesting that FAK tyrosine kinase activity is involved in the regulation of FA turnover (19). Expression of the dominant negative FAK-related non-kinase domain (FRNK) inhibits integrin-stimulated migration and phosphorylation of endogenous FAK and other FA components such as paxillin and tensin (20–22). Thus, FAK activity likely governs FA dynamics and, hence, motility. However, no studies have yet addressed the possible regulation of FAK signaling by modification of its partitioning between FAs and the cytosol.

These considerations led us to examine FAK-Ycam and FRNK-Ycam molecular dynamics using fluorescence recovery after photobleaching (FRAP). We report a rapid exchange of both FAK-Ycam and FRNK-Ycam between cytosolic and FA compartments. FRNK-Ycam, which induced a decrease in tyrosine phosphorylation content at FA, had a shorter recovery half-time compared with that of FAK-Ycam. The local Ca2+ increases triggering FA disassembly prompted us to investigate the effect of Ca2+ on the dynamics of FAK-Ycam and FRNK-Ycam. Ca2+ elevation slowed the recovery half-time of both FAK-Ycam and FAK-GFP at FAs while having no effect on FRNK-Ycam. Several studies report that FAK autophosphorylation levels are increased by Ca2+ elevation (23–25); in agreement, we found that rises in Ca2+ and Ca2+ buffering, respectively, increased and decreased FAK Tyr397 phosphorylation at FAs. Thus, the extended residency of FAK at FAs when Ca2+ is elevated correlates with increased FAK tyrosine phosphorylation, FA disassembly, and cell-edge retraction. Therefore, it is possible that enhanced residency of FAK at FAs triggered by Ca2+ rises might play a role in Ca2+-dependent FA disassembly. Taken together, our results suggest a tight regulation of FAK association at FAs, which likely directs the activation status of FAK-associated targets involved in FA disassembly.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Cells—Eagle's minimal essential medium, fetal calf serum (FCS), HEPES, L-glutamine, penicillin, streptomycin, gentamycin, and trypsin-EDTA solution were obtained from Invitrogen. Fura Red-AM, BAPTA-AM, and pluronic acid F-127 were obtained from Molecular Probes. Matrigel and the monoclonal antibody (mAb) directed against the kinase domain of FAK were obtained from BD Biosciences. The human U87 astrocytoma cell line was obtained from American Type Cell Culture. Cells were maintained at 37 °C in a humidified incubator gassed with 5% CO2 in air on type I collagen-coated (0.06 mg/ml) plastic dishes in Eagle's minimal essential medium supplemented with 10% heat-inactivated FCS, 0.6 mg/ml glutamine, 200 units/ml penicillin, 200 units/ml streptomycin, and 0.1 mg/ml gentamycin.

Expression Plasmids and Transfection—As described previously (12), fluorescent FA-targeted protein was made by fusion of FAK cDNA from human T lymphocytes (pCDM8-FAK plasmid (26), kindly provided by S. B. Kanner) next to the 3' end of Ycam (pcDNA3-Ycam2 plasmid (13), kindly provided by R. Y. Tsien). FRNK-Ycam was made by following a similar protocol. All constructs were verified by sequencing. A FAK-GFP plasmid (34) was kindly provided by L. H. Romer. The plasmids were isolated (JetStar Plasmid kit, Genomed) before transfection by electroporation. Cells were selected 24 h later using 800 µg/ml G418 (Sigma) and maintained with 400 µg/ml G418. To obtain >80% expressing cells, cells were sorted using a FACStar cell sorter (BD Biosciences) before use.

Measurement of Intracellular Ca2+ and Focal Adhesion Dynamics—Global intracellular Ca2+ levels were measured using Fura Red by confocal microscopy (Kr/Ar laser, Bio-Rad 1024) on an inverted microscope (Nikon Eclipse TE300), with 40x oil-immersion CFI Plan-Fluor objective (numerical objective 1.3) simultaneously with FA dynamics using emitted fluorescence of EYFP (12). Cells (5 x 104 cells/ml) expressing FAK-Ycam or FRNK-Ycam were grown to sub-confluence on Matrigel in Petri dishes in which a 2-cm diameter hole had been cut in the base and replaced by a thin (0.07 mm) coverslip. After 48 or 72 h, cells in the culture medium were loaded with 10 µM Fura Red-AM and 0.03% pluronic acid (45 min) in the incubator and then washed 2x with Ringer's solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2,2mM MgCl2,10 mM HEPES, 11 mM glucose, pH 7.4). Imaging of single U87 cells in Ringer's solution containing 10% FCS was done at 32 °C (488 nm excitation), and emitted fluorescence was collected simultaneously at 585 ± 10 nm for Ca2+ (Fura Red) and at 522 ± 16 nm for FA dynamics (EYFP) at 10-s intervals, usually for 30 min. Images were taken at the bottom of the cell surface. FA dynamics were analyzed using NIH Image, with fluorescence intensities from manually outlined, FA-containing regions of interest of ~3–5 µm2, as described previously (12). FA movement is illustrated in the figures using a time-lapse, three-color overlay representation (1); images are shown after color inversion. Intracellular Ca2+ measurements based on FRET between ECFP and EYFP and restricted to FAs were made using FAK-Ycam on a confocal system as above, except that excitation was at 440 from a He/Cd laser. Simultaneous measurements of local Ca2+ measurements (FRET between ECFP and EYFP) and FA dynamics (EYFP emission) were done at the European Molecular Biology Laboratory on a temperature-controlled stage (32 °C) of a Zeiss LSM 510 confocal system equipped with a 60x oil-immersion objective (1.4 numerical objective); excitation was at 413 nm from a Kr laser. Cells expressing FAK-Ycam were grown to sub-confluence on Matrigel in Petri dishes when using the Bio-Rad 1024 or in Labtech chambers when using the Zeiss LSM 510. Single cells were imaged in Ringer's solution plus 10% FCS, and emitted fluorescence was collected simultaneously at 485 nm for ECFP and at 530 nm for EYFP at 0.5–1 Hz from ~20–25 µm2 regions of interest containing individual FAs. Thus, Ycam FRET-dependent Ca2+ measurements are based upon FAK-Ycam expressed both in FAs (which have a smaller surface, typically 3–5 µm2) and in the cytosol surrounding FAs. Intracellular Ca2+ variations were represented by the ratio of EYFP/ECFP emission (R530/485) and normalized to fluorescence intensity in the regions of interest at the beginning of the recording. Images were analyzed as above.

Fluorescence Recovery after Photobleaching Experiment—As described previously (27), FRAP experiments were done on a custom confocal microscope at the European Molecular Biology Laboratory at 32 °C with simultaneous excitation at 488 nm and 514 nm (100 mW Ar ion laser). For quantitative measurements, the photobleached region corresponded to a rectangle enclosing the selected FA or cytoplasmic region. Fluorescence within the rectangle was measured at low laser power before bleach and then photobleached with full laser power (100% power, 100% transmission) for ~6 s (which effectively reduced the fluorescence to background levels in fixed cells; data not shown). Recovery was followed with low laser power at ~1-s intervals until a steady plateau was reached. Negligible bleaching occurred during imaging of recovery, as verified in control experiments. Time was corrected by setting time zero as equal to the half-time of the bleaching period. The post-bleach intensities were normalized slightly upward to correct for the loss of total fluorescence because of photobleach (typically <2%). Fluorescence recovery time courses were normalized to pre-bleach intensity. We compared relative recovery rates for FAK-Ycam, FAK-GFP, and FRNK-Ycam in various conditions, using the half-time for recovery of fluorescence toward the asymptote. Mobile and immobile fractions were calculated by comparing the intensity ratio in the bleached area just before the bleach and after recovery. FRAP data are given as mean ± S.D. for nFAs.

Immunostaining—FAK-Ycam and FRNK-Ycam cells were rinsed with phosphate-buffered saline (PBS) and fixed 15 min with 3% paraformaldehyde at room temperature. After three washes with PBS, cells were treated for 10 min with 0.2% Triton X-100 in PBS, 0.2% bovine serum albumin (BSA) and incubated 30 min with PBS/3% BSA. Cells were washed three times with PBS, 0.2% BSA and incubated 1 h with either the anti-FAK P-Tyr397 mAb (1/1000) or the anti-P-Tyr Ab (1/1000) in PBS, 0.2% BSA. For FAK Tyr397 or total Tyr staining, cells were washed three times and incubated 1 h with a TRITC-labeled goat anti-mouse Ab (1/100). Fluorescence was observed with a Bio-Rad confocal system and a 40x objective, as above. EYFP and TRITC were excited at 488 and 568 nm, respectively, and fluorescence was collected at 522 and 585 nm, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Local Active Ca2+ Oscillators Trigger Propagating Ca2+ Waves—Recently, we showed that Ca2+ spikes observed in migrating U87 cells represent a Ca2+ wave that spreads throughout the cell (28). Here, Ca2+ measurements restricted to FAs were made using FAK-Ycam. U87 cells having stable expression of FAK-Ycam were grown to sub-confluence on Matrigel and imaged in Ringer's solution containing 10% FCS, conditions necessary to observe Ca2+ spikes (12) and functional integrins. We observed clear kinetic differences in Ca2+ elevations at discrete FAs (Fig. 1A), which is in agreement with a propagated wave. The delays measured for the appearance of Ca2+ spikes at individual FAs located in different parts of a cell (Fig. 1C) allowed calculation of a Ca2+ wave speed of 20 ± 10 µm/s (n = 48 waves, 7 cells). An interesting feature is the presence of active Ca2+ oscillators, which reliably trigger Ca2+ wave propagation. These sites were defined as the nearby FAs displaying the earliest increase in Ca2+ during spiking (Fig. 1, compare red and light-blue FAs) and were characterized by a distinct and relatively constant frequency of oscillation. Between one and three active Ca2+ oscillators were detected in different cells (n = 22). Both regenerative, propagating Ca2+ waves (which couple to remote conveying Ca2+ oscillators; see Fig. 1B, first four spikes) and subcellularly restricted or abortive Ca2+ waves (which do not propagate throughout the cell; see Fig. 1B, spikes 5–7 of red FA) appear to be generated by active oscillators. Both types of waves probably share common underlying mechanisms, because the propagation speed for abortive Ca2+ waves (21 ± 12 µm/s; n = 52 waves, 9 cells) was the same as for regenerative waves. The location of active Ca2+ oscillators seems not to be correlated with the direction of cell migration, as we have observed polarized cells having active Ca2+ oscillators at both poles (not shown). However, it should be noted that our experiments were done without any directed signals (low density plating, homogeneous serum stimulation). Note also that our methods did not allow us to resolve whether FAs are the sites where integrin-dependent Ca2+ signaling is first generated. Nevertheless, we used FAK-Ycam, which concentrates FAs, to measure local Ca2+ rises around individual FAs in an effort to demonstrate the compartmentalized nature of Ca2+ elevations.



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  		FIG. 1.
Abortive and regenerative Ca2+ waves are initiated by local oscillators. A, subcellular changes in internal Ca2+ in U87 cells were measured using FA-targeted Ycam-2 in regions of interest surrounding the five FAs indicated in color. B, local Ca2+ changes were visualized by changes in FRET (R530/485; excitation 440 nm) from each of the five FAs (the colors of the traces correspond to the colors of the FAs in A; the traces are paired to provide comparison of Ca2+ signaling at FAs in adjacent regions of the cell). Note the differences in when peak Ca2+ increases were observed and the varying oscillator frequency at different FAs. Active oscillators having a sustained frequency are localized in the region containing the red and light-blue FAs. C, higher time-resolution representation of the second Ca2+ spike in B, showing that the increases in Ca2+ at the red and light-blue FAs occur earliest, which is in agreement with these regions triggering a Ca2+ wave that propagates throughout the cell.

 
Local Subcellular Ca2+ Increases Trigger Disassembly of Neighboring FAs—We also reported that Ca2+ spikes trigger FA disassembly (12), based upon simultaneous measurements of global Ca2+ variations and dynamics of FAs or actin stress fibers. We tested whether the spatially restricted Ca2+ increases described above were capable of triggering adjacent FA disassembly (Fig. 2). Color overlays (blue, red, green) of three sequential images of EYFP emission (Fig. 2A) distinguish immobile FAs (black) versus motile FAs (rainbow). Disassembly of peripheral FAs (Fig. 2A, boxed areas 1 and 2), which corresponds to dissipation of EYFP fluorescence (Fig. 2, C and D, green traces) and subsequent cell-edge retraction (Fig. 2A, blue and red dominant colors in the zone close to areas 1 and 2), were coincident with global Ca2+ oscillations (calculated as the sum of subcellular Ca2+ increases; see Fig. 2B, red trace, spike 4). As described previously, this disassembly occurs simultaneously with retraction of associated stress fibers and cell-edge movement. The local Ca2+ rise in region 1 (Fig. 2B, green trace) mirrors the global Ca2+ signal, unlike for region 2 (Fig. 2B, blue trace). Disassembly of FA 2 (at 340 s; see Fig. 2D, green trace) is triggered by a local rise in Ca2+ in region 2 (Fig. 2B, blue trace; Fig. 2D, red trace). Although this early Ca2+ increase appears to be weakly propagated to region 1, disassembly of FA 1 occurs later at 370 s, coincident with a strong local Ca2+ increase in region 1 (Fig. 2B, green trace; Fig. 2C, red trace) which hardly propagated to region 2. These data demonstrate that disassembly of individual FAs is associated with local increases in Ca2+. This spatial and temporal correlation was observed in all cells where Ca2+ increases in different parts of the cell were clearly separated in time, with the latency between the onset of local Ca2+ increases and FA disassembly being always shorter (15 ± 2 s, n = 7 FAs, 6 cells) when compared with global Ca2+ increases measured from the entire cell (39 ± 8 s, n = 7 FAs, 6 cells).



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  		FIG. 2.
Spatio-temporal correlation of local Ca2+ increases and focal adhesion disassembly. Simultaneous measurements of subcellular changes in Ca2 and FA dynamics in U87 cells using FAK-Ycam. A, color overlay of three sequential images (blue, red, and green at times i, ii, and iii in C and D) of EYFP emission (522 nm) in a FAK-Ycam cell having Ca2+ oscillations. Immobile FAs are black, and mobile FAs appear as rainbows (blue to green). Boxed areas 1 and 2 enclosing FAs 1 and 2 are shown enlarged (right panels, black arrows represent the direction of FA movement). Cell-edge retraction is visualized by the blue and red dominant colors in the zone close to areas 1 and 2. B, green and blue traces show local Ca2+ variations at FA 1 and FA 2, respectively (evaluated by changes in Y-cam FRET, R530/485); the red trace represents global Ca2+ oscillations measured from the entire cell surface. C and D, local Ca2+ variations (red traces) and FA dynamics (green traces, EYFP emission) at FA 1 (C) and FA 2 (D). Disassembly of FA 2 is associated with a Ca2+ rise in region 2 (dotted line at 340 s in B and D). The later disassembly of FA 1 is triggered by a Ca2+ increase in region 1 (dotted line at 370 s in B and C).

 
We reported previously (28) that integrin-dependent Ca2+ oscillations in U87 cells were reduced by depletion of Ca2+ stores and abolished by non-selective inhibition of Ca2+ channels by Cd2+ and La3+. However, Ga3+, which inhibits stretch-dependent Ca2+ channels, did not affect the generation of Ca2+ oscillations in U87 cells (not shown), unlike in keratinocytes (11).

To show directly that Ca2+ increases because of Ca2+ entry cause FA disassembly, we bathed FAK-Ycam cells in Ca2+-free external medium (with 10% FCS) containing 5 mM EGTA (to empty internal Ca2+ stores) and 5 µM ionomycin (Fig. 3). Upon local and repeated microperfusion of the same solution (but containing, in addition, 2 mM Ca2+), transient increases in Ca2+ were observed (using Fura Red; see Fig. 3D, solid trace)to trigger FA disassembly (Fig. 3D, dotted traces). Note the retraction of the cell edge after Ca2+ application (Fig. 3B). Although we cannot rule out that the experimental protocol used might give rise to Ca2+-sensitive outside-in integrin signaling, on balance, given the present knowledge concerning how external Ca2+ affects integrin function (for review, see Ref. 70), we feel it is quite plausible that the observed elevations in Ca2+ provoked by ionomycin-assisted Ca2+ entry trigger FA disassembly in a qualitatively similar way to integrin-dependent Ca2+ oscillations (Fig. 2).



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  		FIG. 3.
Ionomycin-induced Ca2+ ncreases trigger FA disassembly. A–C, EYFP emission (522 nm) at FAs (boxed area is enlarged in C) in a FAK-Ycam cell before (A) and after (B) local microperfusion of 2 mM Ca2+ solution (bath contained 5 µM ionomycin and 0 µM Ca2+/5 mM EGTA). Note the cell-edge retraction (B, arrow). D, decrease in Fura Red emission (585 nm, continuous trace) corresponds to increase in Ca2+ in the same cell. The onset of FA disassembly (dotted traces corresponding to FA 1 and FA 2 in C) is coincident with the rise in Ca2+.

 
FAK Molecular Dynamics Are Characterized by a Fast Equilibrium between FAs and Cytosol—In motile U87 cells, we also observed predominantly static FAs, as found in fibroblasts (11), together with restricted zones where FAs disassembled and formed, respectively, at the cell rear and at the migration front (12). FAs need to be relatively stable structures in time and space to support traction of the cytoskeleton necessary to propel the cell body (11). Cell-edge retraction triggered by FA disassembly stops when the next stable FA is encountered. We occasionally observed a slow redistribution of FAK-Ycam fluorescence from a disassembling FA to an adjacent FA (Fig. 4). As before, color overlays (blue, red, green) of three sequential images of EYFP emission (Fig. 4A) distinguish immobile FAs (black) from motile FAs (rainbow). This exchange of FAK (Fig. 4A, right panels), which occurs over several minutes, is seen as a loss of FAK-Ycam EYFP fluorescence from a disassembling FA 1 (Fig. 4A, dotted circle in upper right panel; Fig. 4B, green line) and an increase of EYFP fluorescence in an adjacent, (initially) stable FA 2 (Fig. 4A, upper right panel, solid circle; Fig. 4B, blue trace).



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  		FIG. 4.
Redistribution of FAK to adjacent FAs during FA disassembly. A, color overlay of three sequential images (blue, red, and green at times i, ii, and iii in B) of EYFP emission (522 nm) in a FAK-Ycamexpressing cell showing mobile (rainbow) and static (black) FAs. Right panels, enlargements of the boxed area. Upper right panel, disassembly of an isolated FA (area enclosed by the dotted line); the other two encircled regions show FAK redistribution between FA 1 and the adjacent FA 2. Lower right panel, image subtraction at time ii minus time i; in this representation, loss of EYFP signal from the disassembling FA appears black, and gain of EYFP signal in the adjacent FA appears white. B, time course of EYFP signals for the two FAs encircled in A. Disassembly of FA 1(red trace) is accompanied by a simultaneous increase in EYFP signal in FA2(blue trace, arrow in upper right panel in A) that was initially stable but then subsequently disassembled.

 
To test whether the disappearance of FAK-Ycam fluorescence during FA disassembly corresponds to the dissociation of a stable complex containing FAK and FA structural components, as suggested by Fig. 4, the molecular dynamics of FAK-Ycam exchange between the cytosolic and FA compartments were studied by FRAP (Fig. 5). Experiments were made to determine the turnover of FAK-Ycam at immobile FAs. The EYFP moiety of Ycam was bleached at individual FAs, and subsequent time-lapse imaging of bleached regions allowed visualization of FAK-Ycam movement during recovery (Fig. 5B, second column). The recovery of FAK-Ycam at FAs was surprisingly fast, with a half-time of 17 ± 4 s (Fig. 5D, n = 12 FAs), which, together with the 79 ± 4% recovery, indicates the presence of a small immobile fraction. This supports the idea that there is a large and rapid exchange flux of FAK between cytosolic and FA compartments, with FAK molecular dynamics being much faster compared with both FA formation and disassembly (which take >5 min). FRAP experiments done on small areas of the peripheral cytosol revealed a FAK-Ycam recovery half-time of 3 ± 1 s (Fig. 5C, n = 13), which is much faster than at FAs and is most likely diffusion-limited. This result indicates that additional factors, for example binding to various FA components, are involved in regulating FAK exchange between FAs and the cytosol. Therefore, the fast exchange of FAK between cytosolic and FA compartments contrasts with the stability of FAs and suggests that FAK forms transient interactions with FA components.



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  		FIG. 5.
Regulation of the molecular dynamics of FAK-Ycam at FAs by Ca2+ elevation. A, U87 cells expressing FRNK-Ycam (paired images, 1), FAK-Ycam in basal Ca2+ condition (paired images, 2), or after ionomycin-induced Ca2+ elevation (paired images, 3) imaged before and after recovery from bleaching (respectively left and right panels) of an isolated FA (boxed areas). B, time-lapse sequences showing recovery after bleaching of corresponding FAs in A (bp, before photobleaching; ap, after photobleaching; the time after photobleaching is indicated in s; far, fluorescence after recovery). The rows correspond to sequential images of the boxed FAs in the FRNK-Ycam cell (1), the FAK-Ycam cell (2) and the FAK-Ycam cell + ionomycin (3) in A. C–F, kinetics of recovery of FAK-Ycam and FRNK-Ycam fluorescence in FA and cytosolic compartments after bleach. Fluorescence intensity in the bleached region was measured and expressed as relative recovery. Note the shorter recovery half-time at FAs for FRNK-Ycam compared to FAK-Ycam (D) and the longer recovery half-time for FAK-Ycam after ionomycin (F). Data are mean ± S.D. (representative error bar at the end of each trace). The sizes of peripheral FAs in FRAP experiments were always between 2–3 µM diameter.

 
FRNK Molecular Dynamics Are Faster than Those of FAK—FRNK acts as a dominant-negative of FAK, lacking the N-terminal domain, the kinase domain, and the Tyr397 autophosphorylation site, but including the C-terminal FA-targeting sequence (20). Accordingly, both the total amount of tyrosine phosphorylation (Fig. 6A) and FAK Tyr397 phosphorylation (Fig. 6B) were decreased in FRNK-Ycam-transfected cells compared with adjacent non-transfected cells. To test whether FAK and FRNK displayed the same molecular dynamics, FRAP experiments were done using FRNK-transfected cells. The recovery of bleached FRNK-Ycam at FAs (Fig. 5B, first row) was significantly faster than for FAK-Ycam (Fig. 5B, second row), with a recovery half-time of 11 ± 3 s (Fig. 5D, n = 13 FAs), whereas the immobile fraction was similar (16 ± 6%). However, no significant differences were noted between FAK-Ycam and FRNK-Ycam dynamics after cytosolic bleaching, with a FRNK recovery half-time of 2 ± 1 s (Fig. 5C, n = 8). These data indicate that the faster recovery of FRNK at FAs is not due to differences in cytosolic diffusion of FAK compared with FRNK, and that the slower recovery of FAK is due to a longer residency time at FAs.



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  		FIG. 6.
FRNK-Ycam and Ca2+, respectively, decrease and increase FAK Tyr397 phosphorylation at FAs. A and B, FRNK-Ycam-transfected cells plated for 2 days on Matrigel were fixed and permeabilized; total tyrosine phosphorylation (A) or endogenous FAK Tyr397 phosphorylation (B) was compared with adjacent untransfected cells using mAbs. FRNK-Ycam-transfected cells (middle panel) displayed lower total tyrosine (A, left) and FAK Tyr397 phosphorylation (B, left) at FAs compared with untransfected cells. C, FAK-Ycam-transfected cells were loaded (lower panels) or not (upper panels) with BAPTA for 24 h before fixation, permeabilization, and FAK Tyr397 phosphorylation staining. Ca2+ buffering with BAPTA induced a dramatic reduction of FAK Tyr397 phosphorylation at FAs (left, lower panel) compared with control cells (left, upper panel).

 
Calcium Elevation Prolongs FAK Association at FAs—Only a few studies have reported a correlation between Ca2+ elevations and FAK-mediated increases in tyrosine phosphorylation (23–25). In U87 cells, thapsigargin-induced Ca2+ increases produce a rapid (<30 s) enhancement of FAK Tyr397 phosphorylation (12), as also seen for ionomycin-induced Ca2+ elevation (not shown). In agreement, Ca2+ buffering with BAPTA induced a dramatic reduction of FAK Tyr397 phosphorylation at FAs (Fig. 6C). Because enhanced migration associated with sustained FA turnover is dependent upon FAK activity (18, 19, 29), activation of FAK targets, and FA-localization of FAK (22, 30), it is possible that increases in Ca2+, which trigger FA disassembly, might also have regulatory effects on the activation and/or localization of FAK.

Therefore, we used ionomycin to artificially trigger Ca2+ elevation and then examined the molecular dynamics of FAK-Ycam and FRNK-Ycam in FA and cytosolic compartments by FRAP (Fig. 5). Despite extensive disassembly of FAs observed in most cells after ionomycin application (not shown), sufficient remaining stable FAs allowed us to carry out FRAP experiments. The FA residency time of FAK-Ycam was significantly increased (Fig. 5B, third row) after ionomycin treatment, with a recovery half-time of 29 ± 7 s, compared with 17 ± 4 s in control conditions (Fig. 5F, n = 18 FAs). Furthermore, the immobile FAK-Ycam fraction was slightly but significantly increased, to 32 ± 5%, compared with 21 ± 4% in control conditions. By contrast, the exchange of FRNK-Ycam between the cytosol and FAs was not affected by an increase of Ca2+ (recovery half-time = 11 ± 3 s, immobile = fraction 17 ± 9%; Fig. 5E, n = 10 FAs). Likewise, in the presence of ionomycin, the cytosolic recovery half-time of FAK-Ycam (3 ± 2 s; Fig. 5FM, n = 13) was the same as in control (Fig. 5C). Thus, increases in Ca2+ slow FAK-Ycam dynamics at FAs, which are possibly linked to enhanced tyrosine phosphorylation, but not those of FRNK-Ycam, indicating that Ca2+ elevation does not result in a nonspecific modification of the dynamics of components at FAs. Furthermore, the FAK-Ycam recovery half-times (tens of seconds) are in sharp contrast with the much slower kinetics (>5 min) of FA disassembly (and formation), suggesting that a long-lived dissociation (or association) of FAK from (with) an FA complex occurs during these processes.

To verify that the Ca2+-sensitivity of FAK-Ycam dynamics at FAs was unrelated to the conformational changes of Ycam induced by Ca2+ binding (13) after ionomycin treatment, we repeated FRAP experiments on cells transfected with FAK-GFP (34). As illustrated, both FAK-GFP and endogenous FAK were revealed in Western blots, with localization of FAK-GFP in FAs (Fig. 7A). Compared with FAK-Ycam dynamics in untreated cells (recovery half-time = 17 ± 4 s, immobile fraction = 21 ± 4%; see Fig. 5D), FRAP experiments showed that FAK-GFP dynamics at FAs were faster with recovery half-times of 10 ± 3 s (Fig. 7B, n = 34 FAs), with a similar immobile fraction of 24 ± 7%. These faster dynamics may be related to the smaller size of FAK-GFP compared with FAK-Ycam. After treatment with ionomycin, FAK-GFP recovery half-times (Fig. 7B) were significantly increased (22 ± 5 s; n = 26 FAs), with a small increase also in the immobile fraction (34 ± 6%). These slowing effects of increased intracellular Ca2+ on FAK-GFP dynamics are similar to those found for FAK-Ycam after ionomycin treatment (recovery half-time = 29 ± 7 s, immobile fraction = 32 ± 5%; see Fig. 5).



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  		FIG. 7.
Ca2+ elevation slows the molecular dynamics of FAK-GFP at FAs. A, FAK-GFP expression (right panel, Western blot probed with a FAK mAb) and localization to FAs (left panel) in transfected U87 cells. B, kinetics of recovery of FAK-GFP at FAs after photobleaching in control conditions and after ionomycin treatment. Note a similar slowing of FAK-GFP dynamics in the presence of elevated intracellular Ca2+, as found for FAK-Ycam (Fig. 5). Data are mean ± S.D. Bar = 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the relative immobility of most FAs and their life span on the order of at least several minutes, this study shows that the association/dissociation of FAK and FRNK with FAs is a fast dynamic process, with increases in Ca2+ leading to longer association of FAK but not FRNK at FAs. This result is the first indication that FAK signaling might be regulated by modification of its partitioning between FAs and the cytosol. Note that both FA formation and disassembly have much slower kinetics, and that the molecular dynamics of FAK are about 10-fold faster compared with other FA proteins (31–33). Simultaneous measurements of local Ca2+ variations at FAs and FA dynamics reveal that Ca2+-dependent FA disassembly occurred when the Ca2+ wave, which is composed of transient Ca2+ subcellular elevations, reached the vicinity of the FA. Because compartmentalized Ca2+ oscillations trigger local FA disassembly and Ca2+ elevation or buffering induce, respectively, an increase or decrease in FAK autophosphorylation, we propose that Ca2+-induced increases in FAK association with FAs might be involved in FA disassembly.

FAK functions as a molecular adaptor interacting with both signaling and structural proteins (16), which is consistent with FAK being a multifunctional protein playing both signaling and structural roles (17). The continuous fast exchange of FAK-Ycam between cytosolic and FA compartments reported here emphasizes a signaling function for FAK, given the immobility of most FAs in migrating cells (1, 19). In control conditions, after photobleaching FAK-Ycam, we found an unexpectedly fast recovery half-time of 17 s, which is the fastest so far described for an FA protein. This finding is consistent with recent work showing that FAK autophosphorylation is intermolecular, and that FAK cannot form stable homo-oligomers (15); rather, FAK requires external proteins for transient transphosphorylation and subsequent action on its targets. By comparison, recovery half-times after photobleaching of 2–5 min (31) or >10 min (32) have been reported for the integrin {beta}3 subunit; for {alpha}-actinin, the recovery half-time after photobleaching was 2–3 min (32, 33). Even if {beta}3 integrins move slowly inwards at FAs (31), our results indicate that FAK and FRNK rapidly associate and dissociate from a more stable platform at FAs composed of at least integrins. In agreement with a predominant signaling role for FAK, chromophore-assisted laser inactivation of FAK at FAs does not lead to stress fiber detachment (34). Nevertheless, redundant interactions that characterize FA-associated proteins could underlie protein exchange without dissipation of FA architecture and might reconcile fast FAK dynamics with a structural function. Consistently, low affinity interactions between FA components and integrins have been described (35), which may facilitate protein-exchange dynamics. Note that chromophore-assisted laser inactivation of {alpha}-actinin, which exchanges between FAs and the cytosol more slowly than FAK, leads to disruption of integrin-cytoskeleton interactions (34). The fast dynamics of FAK-Ycam suggest that FAK exchange between FA and cytosolic compartments may be constant during the relatively slower process of FA disassembly (>5 min). Thus, FAK-Ycam fluorescence probably reflects the size of the integrin-containing platform with which FAK associates and which is more stable.

The identical ability of FAK and FRNK to localize at FAs, which are unaltered by the Ycam tag, is due to the FAT sequence (36) that contains binding sites for paxillin and talin (37, 38), both of which seem to be involved in FA targeting. The shorter FA association time for FRNK compared with FAK may result from FRNK being unable to mimic FAK signaling or from a difference in affinity for FA components. Indeed, in FRNK, an integrin interaction site in the FAK N-terminal domain is absent (39), along with the kinase domain and the Tyr397 autophosphorylation site responsible for recruitment and activation of Src-kinase (40).

In agreement with the predominant signaling hypothesis for FAK, the region between FAT and the kinase domain, as well as the N-terminal domain, are not necessary for adhesion-dependent FAK tyrosine phosphorylation and FAK-dependent paxillin tyrosine phosphorylation, and thus are probably not involved in FAK targeting to integrins and downstream signaling mediated by FAK (41). However, the FAT domain is necessary for FAK and FRNK to promote adhesion-dependent FAK tyrosine phosphorylation and for the dominant-negative function of FRNK (30, 41). Thus, it seems that regions underlying binding capabilities are mainly present in the FAT domain, thus supporting the idea that events linked to FAK tyrosine phosphorylation activity determine kinetics of FAK and FRNK residency at FAs. Both FAT and Tyr397 are required for complete tyrosine phosphorylation of downstream FAK targets like paxillin (20, 21, 41). Formation of Src-kinase/FAK complexes allows subsequent trans/cis-phosphorylation of other FAK tyrosine residues, inducing maximal FAK tyrosine kinase activity (42) and direct or indirect recruitment of FA components (16). Like others (43), we found that transfection of FRNK leads to reduced phosphorylation of endogenous FAK at FAs, as well as reduced global FA tyrosine phosphorylation, probably because of decreased tyrosine phosphorylation of paxillin, p130cas, and tensin (20, 21, 44). Therefore, the faster recovery half-time of FRNK-Ycam at FAs may result from either a reduction in direct docking of FRNK at FA-localized proteins interacting with Tyr397 or a decreased amount of FA-phosphotyrosines and, hence, a reduced number of potential interaction partners (43, 45).

In support of intrinsic differences in FA docking, a C-terminal domain mutation which disrupts paxillin binding to FAK and FRNK affects only FA localization of FRNK but not FAK targeting (22, 30, 37). On the other hand, even if the N-terminal domain is unnecessary for complete integrin-dependent kinase activity of FAK (46) or FAK-dependent tyrosine phosphorylation of paxillin (41), its interaction with integrin (39) may act to stabilize and extend FAK association at FAs. Indeed, the N-terminal domain of FAK has been demonstrated to be involved in the control of FAK activity (15, 47, 48).

Several reports demonstrate that migration speed is increased by Ca2+ signaling, for example in astrocytoma (28), smooth muscle cells (49), neutrophils (10), and neurons (50). Here, we have directly linked local Ca2+ variations to the migration process by showing that Ca2+-dependent FA disassembly is associated with temporally and spatially restricted Ca2+ increases around individual disassembling FAs. The migration process is complex, involving the regulation of cell protrusion and retraction both in space and time (51), characteristics shared by the complex patterns of Ca2+ signaling reported here (Fig. 1). In cerebellar granule cells, Ca2+-dependent migration is correlated with the amplitude and frequency of Ca2+ spikes (50), which may regulate different steps during migration. Migrating fibroblasts in the later stages of wound healing exhibit an increasing gradient of free Ca2+ from the front to the rear (2, 3), supporting a role for spatially restricted Ca2+ variations in the disassembly of FAs. Indeed, this might represent a mechanism by which the cell controls its polarity during migration. Oscillatory Ca2+ signaling may serve to prevent inappropriate FA disassembly, which may occur during sustained Ca2+ elevation, given the massive FA disruption seen for ionomycin-challenged cells.

It is widely held that the frequency and amplitude of Ca2+ signals allow activation of specific Ca2+ targets (54–57). High Ca2+ triggers diffusion of integrins out of FAs in adherent fibroblasts (52). Consistently, Ca2+ increases lead to calpain activation and the liberation of a fraction of {beta}2 integrins, which are tethered to cytoskeletal components (53). The Ca2+ targets involved in FA disassembly might be more sensitive to high levels of Ca2+, because only strong local Ca2+ elevations appear effective in triggering FA disassembly (Fig. 2). However, further studies are needed to explain why all FAs do not disassemble in response to massive ionomycin-induced Ca2+ increases, and to explain why FAs respond to a given Ca2+ spike and not the previous ones. One possibility is that repetitive increases in Ca2+ around individual FAs are necessary to raise permissive signals beyond a putative threshold, thereby triggering disassembly, as has been proposed previously (58) for repetitive targeting of microtubules to individual FAs (which also promotes disassembly). In any case, the data presented here do not permit us to identify precisely the molecular processes underlying the generation of Ca2+ oscillations nor to conclude that FAs are the exclusive sites of the active Ca2+ oscillators which initiate Ca2+ wave propagation. However, in cultured astrocytes, endoplasmic reticulum-associated proteins (calreticulin and type 2 IP3 receptors) and mitochondria have been described as Ca2+ wave-amplification sites (69).

Currently, the precise Ca2+ targets underlying FA disassembly are unknown, but they potentially include calcineurin (5), myosin light chain kinase (8), calpain (6, 33), calreticulin (7, 58–60), and the FAK-related proline-rich tyrosine kinase 2 (Pyk2) (61–63). Ca2+-dependent changes in FAK tyrosine phosphorylation activity have been described (23–25), and we found that Ca2+ elevation rapidly (<1 min) increases FAK Tyr397 phosphorylation (12), whereas Ca2+ buffering with BAPTA reduces FAK Tyr397 phosphorylation at FAs. Because FAK localization to FAs and autocatalytic activation (15, 40) are involved in the subsequent Src activation and recruitment that enhances FAK activity (42), and both FAK (18, 19) and Src (64, 65) activity are linked to FA turnover, FAK may well be one of the Ca2+ targets involved in Ca2+-dependent FA disassembly. In support of this idea, our FRAP experiments show that when [Ca2+] is high, the FA-associated state of FAK-Ycam is prolonged, and the immobile FAK-Ycam fraction at FAs increases. In contrast, neither the recovery half-time nor the immobile fraction of FRNK-Ycam at FAs were Ca2+-sensitive, which is consistent with tyrosine phosphorylation events being necessary for the effect of Ca2+ on FAK molecular dynamics. The Ca2+-insensitive dynamics of FRNK-Ycam strongly support the idea that the Ycam tag does not confer Ca2+-sensitivity to FAK-Ycam dynamics; rather, they support the idea that Ca2+ affects FAK dynamics in a Ycam-independent manner. In further support of this, note that both endogenous FAK and FAK-Ycam in our cells undergo apparently normal Tyr397 autophosphorylation, which is increased by [Ca2+] elevation, and that native and FAK-Ycam-transfected U87 cells have similar rates of migration (12). We directly verified that Ca2+-induced conformational changes of Ycam do not underlie the slower dynamics of FAK-Ycam at FAs after ionomycin treatment (Fig. 5) by showing that FAK-GFP recovery half-times were also significantly increased when intracellular Ca2+ levels were elevated (Fig. 7).

We propose that extended Ca2+-sensitive docking of FAK at FAs favors the turnover of FAs by increasing the probability of affecting FAK targets involved in FA disassembly, for example, RhoA (19) and Src (65). Consistently, extended interaction of {alpha}4 integrin subunits with paxillin is associated with faster FAK activation and increases in FA turnover and migration speed (66). Also, the inability of Pyk2 to maintain migration in FAK-deficient fibroblasts (67) does not involve differences in signaling pathways (16). Rather, the perinuclear distribution of Pyk2 (38), which, unlike FAK, is preferentially localized at FAs, suggests a time-limited co-localization of Pyk2 with its FA-substrates. Indeed, in FAK-deficient fibroblasts, a fusion protein of the N-terminal domain of Pyk2 and the C-terminal domain of FAK promotes both FA localization and migration (68). These data support our hypothesis that an enhanced time residency of FAK at FAs is correlated with increased FA turnover. Given the increases in time residency of FAK-Ycam at FAs and in FAK autophosphorylation when Ca2+ is elevated, we suggest that the prolonged association of FAK with FAs is related to its level of autophosphorylation. We do not know whether Ca2+ modifies other FA components at FAs, leading to a longer residency of FAK at FAs and increased transphosphorylation (15). The inverse possibility is that Ca2+ signaling enhances FAK autophosphorylation at FAs, Tyr397-phosphorylated FAK being able to associate longer with FAs. This may arise because of increased amounts of FA-phosphotyrosines leading to a greater number of potential interaction partners (43, 45) or because of conformational changes induced by phosphorylation.

Our data show that FAK molecular dynamics are fast and tunable by Ca2+. This may help to explain how the cell is capable of rapidly regulating FA stability during cell spreading and migration, in response to both local (FA) and remote (cytosolic) signals. Indeed, the fast flux of FAK between FA and cytosolic compartments raises the possibility that FA-associated FAK may be replaced by FAK having modified activity coming from the cytosol. Finally, our results indicate that regulation of FAK signaling might be achieved by changes in its association time with FA partner components.


   FOOTNOTES
 
* This work was supported in part by the Ligue Nationale Contre le Cancer (Comités du Haut Rhin et du Bas Rhin), the Fondation pour la Recherche Médicale, the Association pour la Recherche Contre le Cancer, and the Association Régionale pour l'Enseignement et la Recherche Scientifique et Technologique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains one figure. Back

§ Recipient of fellowships from the Ministère de la Recherche and the European Advanced Light Microscopy Facility. Present address: Columbia University, Dept. of Biological Sciences, P.O. Box 2408, Sherman Fairchild Center, 1212 Amsterdam Ave., New York, NY 10027. Back

Recipient of a fellowship from the Ligue Nationale Contre le Cancer. Back

** To whom correspondence should be addressed: Pharmacologie et Physicochimie, UMR CNRS 7034, Université Louis Pasteur, BP 60024, 67401 Illkirch, France. Tel.: 33-3-9024-4111; Fax: 33-3-9024-4313; E-mail: kt{at}pharma.u-strasbg.fr.

1 The abbreviations used are: FAs, focal adhesions; FAK, focal adhesion kinase; Ycam, yellow Cameleon-2; FRET, fluorescence resonance energy transfer; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRNK, FAK-related non-kinase domain; FRAP, fluorescence recovery after photobleaching; FCS, fetal calf serum; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; BAPTA, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N, N',N'-tetraacetic acid; FAT, focal adhesion targeting sequence; GFP, enhanced green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank R. Y. Tsien (University of California at San Diego), S. B. Kanner (Bristol Myers Squibb), and L. H. Romer (Johns Hopkins Medical School) for kindly providing plasmids.



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 DISCUSSION
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