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J. Biol. Chem., Vol. 277, Issue 29, 26364-26371, July 19, 2002

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Calcium Oscillations Trigger Focal Adhesion Disassembly in Human U87 Astrocytoma Cells^*

Gregory Giannone, Philippe Rondé^§, Mireille Gaire, Jacques Haiech, and Kenneth Takeda^¶

From the Laboratoire de Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Université Louis Pasteur de Strasbourg, 67401 Illkirch, France

Received for publication, April 23, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrin-associated intracellular Ca²⁺ oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNK) inhibits the Ca²⁺-sensitive component of serum-dependent migration. We investigated how integrin-associated Ca²⁺ signaling might be coupled to focal adhesion (FA) dynamics by visualizing the effects of Ca²⁺ spikes on FAs using green fluorescent protein (GFP)-tagged FAK and FRNK. We report that Ca²⁺ spikes are temporally correlated with movement and disassembly of FAs, but not their formation. FRNK transfection did not affect generation of Ca²⁺ spikes, although cell morphology was altered, with fewer FAs of larger size and having a more peripheral localization being observed. Larger sized FAs in FRNK-transfected cells were not disassembled by Ca²⁺ spikes, providing a possible explanation for impaired Ca²⁺-dependent migration in these cells. Stress fiber end movements initiated by Ca²⁺ spikes were visualized using GFP-tagged myosin light chain kinase (MLCK). Ca²⁺-associated movements of stress fiber ends and FAs had similar kinetics, suggesting that stress fibers and FAs move in a coordinated fashion. This indicates that increases in Ca²⁺ likely trigger disassembly of adhesive structures that involves disruption of integrin-extracellular matrix interactions, supporting a key role for Ca²⁺-sensitive inside-out signaling in cell migration. A rapid increase in tyrosine phosphorylation of FAK was found in response to an elevation in Ca²⁺ induced by thapsigargin, and we propose that this represents the initial triggering event linking Ca²⁺ signaling and FA dynamics to cell motility.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell migration is a cyclic process involving initial protrusion of the leading edge, formation of adhesive sites, contraction of the cell body, and release of adhesive sites at the cell rear (1). Adhesive sites are dynamic membrane structures that vary in size and composition during migration. Integrins, actin stress fibers (SFs)¹ and other structural proteins, and regulatory signaling molecules cluster at focal adhesions (2). Focal adhesions (FAs) serve as points of traction for contractile forces underlying forward cell movement and their dynamics are finely regulated. For example, FAs are highly motile in stationary fibroblasts but are largely stationary in migrating fibroblasts, thereby transducing contractile forces into movement (3). This suggests the existence of a molecular clutch that couples cytoskeleton-mediated traction and cell contraction.

Focal adhesion kinase (FAK) is activated and localized at FAs upon cell adhesion to the extracellular matrix (ECM; Refs. 4 and 5). Given the abundance of FAs and the reduced migration of fibroblasts from FAK null mice (6), FAK is likely involved in FA remodeling during migration. FAK-related non-Kinase (FRNK), the non-catalytic C-terminal portion of FAK containing the FA targeting sequence, is also expressed as a separate dominant negative protein (7). The differential expression of FAK and FRNK is transcriptionally regulated, each of these proteins having distinct promoters within the FAK gene (8). Although the function of endogenous FRNK is not clear, FRNK has been used to alter signaling via endogenous FAK. When overexpressed in cells, FRNK acts as a negative regulator of FAK activity, inhibiting phosphorylation of FAK and different FAK-related processes, including cell cycle progression (9, 10), cell spreading on fibronectin (7, 11), and migration (12, 13). This suggests that the inhibitory effects of FRNK in migration might arise from altered FAK localization and phosphorylation.

We and others reported that migration is dependent on Ca²⁺ signaling in astrocytoma (14), smooth muscle cells (15), neutrophils (16), and neurons (17). In cerebellar granule cells, Ca²⁺-dependent migration is correlated with the amplitude and frequency of Ca²⁺ spikes (17), which may regulate different steps during migration. Disruption of integrin-mediated adhesion involves Ca²⁺-sensitive proteins, including calpain (18, 19), myosin light chain kinase (MLCK; Ref. 20), and calcineurin (21). These data indicate that Ca²⁺ signaling may be a component of the molecular clutch regulating transitions between stationary and non-stationary FAs.

To test whether Ca²⁺ signaling affects FA organization, confocal microscopy was used to visualize simultaneously Ca²⁺ levels and dynamics of FAs or actin SFs in U87 cells expressing FA (FAK, FNRK) or cytoskeletal (MLCK) proteins tagged with GFP. We report that Ca²⁺ spikes trigger movement and disassembly of FAs. Although FRNK expression did not suppress Ca²⁺-dependent FA disassembly, more Ca²⁺-insensitive FAs having a larger surface were observed in FRNK cells. Our results provide an explanation linking FA disassembly to a temporally accurate cellular signal.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Cells-- Cell culture media (EMEM), fetal calf serum, HEPES, L-glutamine, penicillin, streptomycin, gentamycin, and trypsin-EDTA were from Invitrogen; Fura Red-AM, BAPTA-AM, and pluronic acid were from Molecular Probes; Matrigel and the monoclonal antibody (mAb) against FAK kinase domain were from Interchim; phalloidin-TRITC, mAb against the FAK C-terminal region (amino acids 1039-1052), and anti-MLCK mAb were from Sigma; the anti-Tyr³⁹⁷-phosphorylated FAK Ab was from BIOSOURCE; secondary horseradish peroxidase-conjugated Abs were from Promega; the FITC-labeled goat anti-mouse (GAM-FITC) Ab was from Zymed Laboratories Inc.. The human astrocytoma U87 cell line was obtained from the ATCC. Cells were maintained at 37 °C in a humidified incubator gassed with 5% CO₂ in air on type I collagen (0.06 mg/ml)-coated plastic dishes in EMEM supplemented with 10% heat-inactivated fetal calf serum, 0.6 mg/ml glutamine, 200 IU/ml penicillin, 200 IU/ml streptomycin, and 0.1 mg/ml gentamycin.

Plasmids and Transfection-- Fluorescent FA-targeted protein was made by fusion of FAK (human T lymphocyte pCDM8-FAK plasmid; Ref. 40) next to the 3' end of "yellow Cameleon-2" (pcDNA3-Ycam2 plasmid; Ref. 22). To allow fusion of FAK in continuity with the Ycam2 reading frame, the stop codon next to EYFP was replaced by a tyrosine codon (QuikChange, Stratagene). FAK cDNA was amplified by PCR using a 5' primer containing a MfeI site and a 3' primer containing a NheI site. The FAK PCR product was digested with MfeI and NheI and cloned in the corresponding compatible sites, EcoRI and XbaI, located in the multiple cloning site of the newly mutated pcDNA3-YCam2 vector, adjacent to EYFP, to give FAK-Ycam. To create FRNK-Ycam, the FRNK domain was amplified by PCR using pCDM8-FAK as template, a forward primer with an EcoRI site at the 5' end of the glutamic acid codon 681 relative to the FAK start codon, and the same reverse primer as for FAK amplification, adding a NheI site. The PCR product was digested with EcoRI and NheI and cloned in-frame with the EYFP coding sequence in the EcoRI/XbaI compatible sites of the mutated pcDNA3-Ycam2 vector. The MLCK-210-GFP construct (24) was in pEGFP vector (CLONTECH). All constructs were verified by sequencing. The plasmids were isolated (JetStar, Genomed) before transfection by electroporation. Cells (5 × 10⁶) were resuspended at 10⁸ cell/ml in EP buffer (in mM: 50 K₂HPO₄, 20 CH₃CO₂K, 20 KOH, pH 7.4). Plasmidic DNA (2 µg of construct-encoding plasmid, 8 µg of pBluescript) was diluted in 100 µl of EP buffer; 4 µl of 1 M MgSO₄ were added and incubated with 50 µl of cell suspension for 20 min at room temperature. The cell/DNA mixture was electroporated in a 0.4-cm cuvette (Bio-Rad Gene Pulser; 500 microfarads, 240 V). Cells were then placed in 10 ml of EMEM with 10% fetal calf serum in 80-mm² dishes. Cells were selected 24 h later using 800 µg/ml G418 (Sigma) and maintained with 400 µg/ml G418. Cells were sorted to obtain >80% expressing cells using a flow cytometer (FACStar, Becton-Dickinson) before use.

Migration Assay-- A wound-healing migration model was used, as described previously (15). FAK-Ycam or FRNK-Ycam cells (2 × 10⁵ cells/ml) were grown to confluence in Matrigel-coated (178 µg/ml) Petri dishes. After 24-h serum starvation, a rectangular lesion was made, cells were rinsed and incubated with medium with or without the tested compound. After 24 h of migration, three fields at the lesion border were acquired using a CCD camera (Panasonic) on an inverted microscope (Olympus IMT2, 10× phase objective). In each field, migration distance for the 10 most mobile cells was measured using Image Tool software (available by FTP from maxrad6.uthscsa.edu). For BAPTA experiments, cells were first loaded (45 min) with 20 µM BAPTA-AM in the incubator prior to creation of lesions.

Measurement of Intracellular Calcium and Dynamics of Focal Adhesions or Stress Fibers-- Intracellular Ca²⁺ was measured using Fura Red by confocal microscopy (Bio-Rad 1024, krypton-argon laser 488 nm; Nikon Eclipse TE300, 40× oil-immersion CFI Plan-Fluor n.a. 1.3 objective) alone or simultaneously with FA or SF dynamics. Cells (5 × 10⁴ cells/ml) expressing FAK-Ycam, FRNK-Ycam, or MLCK-GFP were grown to subconfluence 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 (41). After 48 or 72 h, cells in culture medium were loaded with 10 µM Fura Red-AM and 0.03% pluronic acid (45 min) in the incubator and then washed two times with Ringer solution (in mM: 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, 11 glucose, pH 7.4). Imaging of single cells in Ringer solution was done at 30 °C, at 585 ± 10 nm for Ca²⁺ alone, at 1-s intervals, usually for 15 min. For simultaneous measurements of Ca²⁺ (at 585 nm) and FA (Ycam constructs) or SF (MLCK-GFP) dynamics, images at 522 ± 16 nm were acquired simultaneously every 10 s (EYFP) and 15 s (EGFP), usually for 30 min. Images were taken at the bottom cell surface. NIH Image software was used to assess FA surface areas and the dynamics of FAs and SFs. For FA size distributions, FAK-Ycam and FRNK-Ycam cells having similar expression levels were used. Fluorescent FAs were selected by thresholding using NIH Image. The same threshold was applied to all cells, corresponding FA pixel areas were calculated and converted to µm². This analysis was done to determine the size of both immobile and motile, Ca²⁺-sensitive FAs. FA movement and disassembly triggered by a Ca²⁺ spike were visualized using a color overlay representation of three sequential images (3).

Immunoblotting-- FAK-Ycam, FRNK-Ycam, or MLCK-GFP cells were plated at low density on Matrigel for 2 days and then treated or not with 1 µM thapsigargin. At the indicated time, cultures were washed with cold PBS and then incubated with lysis buffer (1% Triton-X100, 1% SDS, 100 mM NaF, 1 mM Na₃VO₄, 10 mM NaP₂O₇, in PBS, supplemented with an anti-protease mixture; Complete, Roche Molecular Biochemicals). Cell lysates were solubilized in Laemmli's buffer (5% glycerol, 2.5% -mercaptoethanol, 1% SDS, 0.005% bromphenol blue, 50 mM Tris-HCl, pH 6.8) at 95 °C and resolved by SDS-PAGE (usually 5% polyacrylamide, but 8% for the blot used to separate FRNK and FAK) and then transferred to polyvinylidene difluoride membranes (Millipore). After blocking overnight at room temperature with 0.1% casein in PBS, 0.3% Tween, membranes were incubated 1.5 h with different Abs: anti-FAK kinase (immunogen corresponding to the kinase domain of FAK, amino acids 354-533) used at 1/1000 V/V dilution; anti-FAK Ct, (immunogen corresponding to the C-terminal region of FAK, amino acids 1039-1052) used at 1/4000; anti-Tyr³⁹⁷-phosphorylated FAK (1/4000) or anti-MLCK (1/10000), followed by 1-h exposure to either anti-rabbit IgG coupled to horseradish peroxidase (1/60,000) or anti-mouse IgG coupled to horseradish peroxidase (1/60,000). Specific staining was revealed using ECL kits (Amersham Biosciences). Blots were analyzed by densitometry. Band intensities of FAK and phosphorylated FAK were determined as (OD phosphorylated FAK/OD total FAK) × 100. For each condition, blots from four different experiments were analyzed.

Immunostaining-- FAK-Ycam, FRNK-Ycam, or MLCK-GFP cells were rinsed with PBS and fixed 15 min with 3% paraformaldehye at room temperature. After three washes with PBS, cells were treated for 10 min with 0.2% Triton X-100 in PBS, 0.2% 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 1 µg/ml phalloidin-TRITC, the anti-MLCK mAb K36 (1/10000), or with the anti-FAK mAb (1/1000) in PBS, 0.2% BSA. For actin staining, the incubation buffer was removed and replaced by PBS, 0.2% BSA. For MLCK or FAK staining, cells were washed three times and incubated 1 h with a FITC-labeled goat anti-mouse Ab (GAM-FITC, 1/100). Fluorescence was observed using a confocal microscope and a 40× objective, as above. FITC and TRITC were excited at 488 and 568 nm, respectively, and fluorescence was collected at 522 and 585 nm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human U87 Astrocytoma Cells Expressing FRNK-Ycam Have Impaired Calcium-dependent Migration-- We reported that migration of U87 cells is associated with Ca²⁺ oscillations (14), as intracellular Ca²⁺ buffering by BAPTA partly inhibits serum-dependent migration. To analyze the role of FAK in Ca²⁺-dependent migration, U87 cells were transfected with FAK or the dominant negative FRNK, fused to the fluorescent yellow Cameleon-2 (Ycam) tag, a fluorescence resonance energy transfer-based Ca²⁺ sensor containing CFP and EYFP (22). Expression of FAK-Ycam or FRNK-Ycam did not alter endogenous FAK levels compared with controls (Fig. 1A, left). FRNK-Ycam expression (Fig. 1A, right) was deduced by subtracting the endogenous FAK band from the band representing endogenous FAK plus FRNK-Ycam. Expression levels of FAK-Ycam and FRNK-Ycam were 1.5-2 times that of endogenous FAK. Compared with controls, serum-independent migration was unchanged in FAK- and FRNK-transfected cells, while serum-dependent migration decreased by 25% only in FRNK-transfected cells (Fig. 1B, left), in agreement with previous studies (12). Buffering of intracellular Ca²⁺ with BAPTA did not affect serum-independent migration in control and transfected cells, but inhibited serum-dependent migration by 33% in FAK-Ycam cells. The lack of effect of BAPTA on serum-dependent migration of FRNK-transfected cells together with the clear inhibition by BAPTA in FAK-transfected and control cells indicate that the migration component inhibited by FRNK expression is mainly Ca²⁺-dependent.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Effects of FRNK-Ycam and FAK-Ycam expression in U87 astrocytoma cells on endogenous FAK expression, migration, and generation of calcium spikes. A, immunoblots of FAK-Ycam (199 kDa), FRNK-Ycam (117 kDa), and endogenous FAK (125 kDa) using Abs against kinase (left) or C-terminal (right) FAK domains. The right-most panel shows endogenous FAK plus FRNK-Ycam. B, migration measured 24 h after lesion in a wound-healing model (left). Serum-dependent migration in FRNK-Ycam cells was inhibited (*) compared with control, while BAPTA caused inhibition of serum-dependent migration () only in FAK-Ycam cells (n = 6, 180 cells/condition; p < 0.05, Student's unpaired t test). The number of Ca2+ spikes/15 min (right) was the same (one-way ANOVA) for FAK-Ycam (589 cells from four separate dishes) and FRNK-Ycam (505 cells from four separate dishes) cells. C, distribution of endogenous FAK at FAs was not altered in FRNK-Ycam cells. Endogenous FAK localization in a FRNK-Ycam cell with an anti-FAK Ab/TRITC-labeled secondary Ab (left); FRNK-Ycam was detected by EYFP fluorescence (right).

U87 Astrocytoma Cells Expressing FRNK-Ycam Have Unaltered Calcium Signaling-- We found that Ca²⁺ spikes in U87 cells occur only in the presence of serum and are blocked by inhibitory antibodies against ₁ and ₃ integrin subunits, with the generation of such Ca²⁺ spikes depending on activation of phospholipase C.² The FAK tyrosine 397 autophosphorylation site interacts with and activates phospholipase C, representing a possible link between integrins and Ca²⁺ signaling (23). Since FRNK contains the focal adhesion targeting sequence that is necessary and sufficient for FAK recruitment to FAs but lacks the phospholipase C-interacting autophosphorylation site, FRNK-Ycam expression might inhibit Ca²⁺ oscillations. However, the frequency of Ca²⁺ spikes over 15 min in cells expressing FAK-Ycam or FRNK-Ycam was similar (Fig. 1B, right) and not different compared with controls (not shown). For most FRNK-Ycam cells, the distribution of endogenous FAK at FAs, evaluated using an Ab against the FAK kinase domain, was unaltered (Fig. 1C, left), as found previously (7). This suggests that sufficient endogenous FAK is expressed in most FRNK-transfected cells (Fig. 1C), perhaps accounting for the unaltered generation of Ca²⁺ spikes (23). Thus, the Ca²⁺-dependent migration defect of FRNK-Ycam cells is not due to decreased Ca²⁺ signaling.

Single Calcium Spikes Trigger FA Movement and Disassembly-- Since differences in Ca²⁺ signaling do not underlie impaired migration of FRNK-Ycam cells, possible effects of Ca²⁺ spikes on FAs were investigated by simultaneously measuring Ca²⁺ variations (using Fura Red) and FA dynamics (using EYFP fluorescence of FAK-Ycam and FRNK-Ycam) in migrating cells. Color overlays of three sequential images (Fig. 2A) distinguish immobile FAs (black) versus motile FAs (rainbow). Most FAs were immobile in FAK-Ycam cells irrespective of Ca²⁺ oscillations. Motile FAs were present in oscillatory and non-oscillatory cells, being often localized at one edge of a migrating cell (Fig. 2A). FA movements were linear and usually resulted in FA disassembly, with EYFP fluorescence decreasing or disappearing (Fig. 2B), while immobile FAs never disassembled. In many cases, FA movement and subsequent disassembly was triggered by a Ca²⁺ spike (Fig. 2B). In oscillatory FAK-Ycam cells, such FA disassembly was temporally correlated with Ca²⁺ spikes for 64% (37/58) of motile FAs. For 19% of motile FAs (11/58), the Ca²⁺ oscillation frequency was too high to determine clearly a correlation, and for the remaining 17% (10/58), no correlation was found. This indicates that Ca²⁺ spikes were responsible for the dynamics of a subset of FAs. Somewhat surprisingly, FA disassembly in oscillatory FRNK-Ycam cells (Fig. 2, C and D) was also temporally correlated with a Ca²⁺ spike for 50% (35/70) of motile FAs; the Ca²⁺ oscillation frequency was too high to establish a correlation for 30% of FAs (21/70), and there was no correlation for the remaining 20% (14/70).

View larger version (55K): [in this window] [in a new window]   Fig. 2.   Calcium spikes trigger movement and disassembly of FAs. A and C, overlay of three sequential images (blue, red, and green, taken at times i, ii, and iii indicated in B and D) of EYFP emission in a FAK-Ycam cells (A and B) and a FRNK-Ycam cell (C and D) having Ca2+ oscillations (red traces in B and D). Immobile FAs are black and motile FAs appear as rainbows (blue to green). The boxed areas (left panels, A and C) are shown enlarged on the right. B and D, simultaneous measurement of Ca2+ spikes (red traces, Fura Red at 585 nm) and FA dynamics (green trace, EYFP at 522 nm), from the encircled region of interests in A and C).

In FAK-Ycam (Fig. 3; 45 FAs, n = 19 cells) and FRNK-Ycam cells (not shown; 30 FAs, n = 10 cells), we found no clear link between oscillatory (Fig. 3, A and B) versus non-oscillatory (Fig. 3, C and D) Ca²⁺ behavior and formation of FAs (evaluated as EYFP fluorescence increases). For FAK-Ycam cells, 49% (22/45) of newly formed FAs were seen in oscillatory cells, while 51% (23/45) were not, as was also the case for FRNK-Ycam cells (not shown). Thus, unlike FA disassembly, Ca²⁺ spikes and FA formation were not obviously correlated.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   FA formation in FAK-Ycam cells is not temporally correlated with calcium spikes. Sequential, three-color overlay from typical cells with (A) or without (C) Ca2+ spikes. Immobile FAs appear white, moving FAs appear as rainbows (blue to green), and newly forming FAs appear as a single color (yellow or green). The boxed area (left panels) is enlarged in the right panels. B and D, simultaneous measurement of Ca2+ spikes (red trace, Fura Red at 585 nm) and FA dynamics (green trace, EYFP at 522 nm, from encircled regions of interest in A and C; i, ii, and iii indicate when the three-color images were taken).

Preferential Calcium-dependent Disassembly of Small FAs in U87 Cells Expressing FRNK-Ycam-- Strikingly, the number, size, and localization of FAs were different in FNRK-Ycam compared with FAK-Ycam cells (Fig. 4, A and B). FRNK-Ycam cells had 3-fold fewer FAs (25 ± 3/cell, n = 4 cells), which were larger (Fig. 4, C and D) and more peripherally located compared with FAK-Ycam cells (74 ± 10 FAs/cell, n = 4). Morphometric analysis (ratio of longest to shortest cell lengths) showed that FRNK-Ycam cells were more elongated (3.0 ± 0.2, n = 51) than FAK-Ycam cells (2.3 ± 0.1, n = 69), suggesting that they are more strongly held to the ECM. Comparison of the Ca²⁺ dependence of FA dynamics revealed that the size distribution of motile Ca²⁺-sensitive FAs (Fig. 4E) mirrors that of all FAs in FAK-Ycam cells (Fig. 4C). In contrast, in FRNK-Ycam cells, the size distribution of motile, Ca²⁺-sensitive FAs (Fig. 4F) is not the same as for all FAs (Fig. 4D), with smaller FAs being preferentially disassembled by Ca²⁺ spikes. This may explain impaired Ca²⁺-dependent migration in FRNK-Ycam cells, as larger, immobile Ca²⁺-insensitive FAs would remain attached to the ECM despite oscillatory Ca²⁺ signaling.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Effects of FAK-Ycam and FRNK-Ycam expression on cell morphology and calcium-sensitive FA disassembly. A and B, transfected cells plated for 2 days on Matrigel were fixed, permeabilized, and actin filaments revealed using 1 µg/ml phalloidin-TRITC. FRNK-Ycam cells (B) were more elongated and had fewer, larger and more peripherally located FAs compared with FAK-Ycam cells (A). Size distribution of all FAs in fixed FAK-Ycam (C, n = 295 FAs, 4 cells) and FRNK-Ycam-transfected cells (D, n = 99 FAs, 4 cells) is shown. Size distribution of motile, Ca2+-sensitive FAs in FAK-Ycam (E, n = 30 FAs, 10 cells) and FRNK-Ycam (F, n = 44 FAs, 19 cells) is shown. The distribution in D is different compared with the three other distributions (tested by ANOVA).

Calcium Elevation Rapidly Increases FAK Tyrosine Phosphorylation-- Since FAK regulates FA dynamics (5, 6), and Ca²⁺ spikes trigger FA disassembly, this may be related to FAK activity. Therefore, FAK tyrosine phosphorylation was evaluated after thapsigargin-induced Ca²⁺ elevation (14). Increases in Tyr³⁹⁷ phosphorylation of endogenous FAK and FAK-Ycam were detected within 30 s after 1 µM thapsigargin treatment (Fig. 5A). After 1 min, phosphorylation was maximal (73% increase above control; Fig. 5B). Thus, rapid phosphorylation of FAK in response to Ca²⁺ increases provides a possible link between FA movements and Ca²⁺ spikes, supporting that these are regulatory events in migration.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Thapsigargin induces rapid tyrosine phosphorylation of FAK. A, FAK-Ycam cells were incubated with 1 µM thapsigargin for the indicated times before addition of lysis buffer. After separation by SDS-PAGE and transfer to membranes, blots were probed with Abs against FAK and Tyr397-FAK. B, mean ± S.E. (n = 3) of the percentage of phosphorylated Tyr397-FAK/total FAK in control cells and after 1-min thapsigargin. *, p < 0.05, paired t test.

Calcium-induced FA Disassembly Correlates with Retraction of Stress Fibers-- As FA disassembly involves disruption of ECM-integrin and/or SF-integrin interactions, we simultaneously followed the dynamics of SF ends and Ca²⁺ signals using MLCK-GFP-transfected cells (Fig. 6). No differences were found in the localization, distribution, and expression of MLCK in MLCK-GFP cells compared with controls (Fig. 6, A and B), as reported previously (24). Moreover, the MLCK-GFP construct used here has the same activity in phosphorylating the 20-kDa regulatory light chain kinase when compared with endogenous MLCK (25). Evaluation of SF end dynamics (using MLCK-GFP fluorescence; Fig. 6C) and Ca²⁺ variations (using Fura Red) revealed that SF end movements were triggered by a Ca²⁺ spike (Fig. 6D).

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Calcium spikes trigger disassembly of SF-associated FAs in MLCK-GFP-transfected cells. A, immunostaining with an anti-MLCK Ab in a control cell (left) and in a MLCK-GFP cell (right). B, Western blot analysis of control and transfected cells using a MLCK-specific Ab; the 259-kDa band corresponds to MLCK-GFP. C, SF architecture (EGFP, 522 nm; left) in a MLCK-GFP cell displaying Ca2+ oscillations. Right panels are sequential enlarged images at times i, ii, and iii (see D) showing SF retraction (boxed region, left panel). D, simultaneous measurement of Ca2+ spikes (red trace, Fura Red) and dynamics of the SF end (green trace, EGFP, encircled region of interest in C).

The average latency between a Ca²⁺ spike and SF end movement was 28 ± 6 s, with a rate of movement of 0.25 ± 0.04 µm/min (n = 21 SFs, 6 cells; Fig. 7). These SF kinetic parameters were compared with those for motile Ca²⁺-sensitive FAs in FAK-Ycam and FRNK-Ycam cells (Fig. 7C). The latency between Ca²⁺ spikes and the onset of FA movement was 33 ± 5 s in FAK-Ycam cells (n = 21 FAs, 8 cells) and 31 ± 5 s in FRNK-Ycam cells (n = 35 FAs; 14 cells), with a FA speed of 0.18 ± 0.02 and 0.22 ± 0.02 µm/min, respectively. These kinetic parameters were not different, suggesting that Ca²⁺ spikes trigger coordinated movement of SF-associated FA complexes. This implies that during cell migration, Ca²⁺ signaling results in disruption of ECM-integrin interactions rather than a decrease in FA-cytoskeletal interactions.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Kinetic parameters of calcium-sensitive FA and SF dynamics. A, time lapse three-color overlay of FA movement showing a circular region of interest of diameter x. B, schematic illustration showing how latency (tL) between Ca2+ spikes (red) and FA movements (green) and the time for disappearance of FAs (tD) were determined. Rate of movement was estimated using tD/x. For SF movement, x was the mean diameter of Ca2+-sensitive motile FAs. C, no significant differences were found (ANOVA) among latencies (tL) and speed (tD/x) of FA disassembly in FAK-Ycam versus FRNK-Ycam cells, nor for SF end movements in MLCK-GFP cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We investigated the implication of intracellular Ca²⁺ elevations in FA dynamics during migration of U87 astrocytoma cells. During oscillatory Ca²⁺ signaling, single Ca²⁺ spikes triggered FA disassembly and subsequent cell edge retraction. In FRNK-Ycam cells, smaller sized FAs were more sensitive to Ca²⁺-triggered disruption compared with large FAs, consistent with FA stability being a limiting factor in motility. FAK phosphorylation was rapidly induced by a Ca²⁺ increase, indicating that FAK is a Ca²⁺ target during migration. Analysis of FA and SF kinetic parameters suggests that Ca²⁺ signaling coordinates disruption of ECM-integrin interactions at FAs.

Cell migration includes phases of protrusion, adhesion, and retraction (1), involving, respectively, formation, strengthening, and disassembly of focal contacts. FAK plays an important role in the dynamics of cell adhesion, but is not required for FA formation beneath lamellipodia of migrating cells (26). FAK aggregates to clustered integrin receptors with or without ligand occupancy (27), consistent with FAK localization to FAs being an early or late event in the cascade of interactions, respectively, leading to formation or disruption of integrin-cytoskeletal linkages. This justifies our choice of FAK-Ycam (22) as a probe for FA dynamics, indirectly allowing detection of clustered integrins. FAK-Ycam and FRNK-Ycam were localized to newly forming, punctate structures in protrusive areas, further supporting their use to follow indirectly integrin association/dissociation in FAs. As suggested previously (28), these punctate structures very probably are focal complexes, putative precursors of FAs.

FAK tyrosine kinase activity is involved in the regulation FA turnover (29). In FAK-deficient cells, reduced motility is accompanied by an increased number of FAs (6). FRNK expression inhibits integrin-stimulated migration and phosphorylation of endogenous FAK and other FA components such as paxillin and tensin (7, 11, 13). Thus, FAK phosphorylation likely governs FA dynamics and, hence, motility. In FAK-Ycam cells, FA morphology was similar to controls, and as found in previous studies using FAK-transfected U87 cells (12), migration was unchanged, unlike in Chinese hamster ovary cells where migration increased after FAK overexpression (30). Expression of FRNK-Ycam induced elongated morphology and a sparse and peripheral distribution of enlarged FAs. Much evidence supports that defective FAK signaling leads to enlarged FAs. Cells expressing a kinase-deficient mutant of Src have larger FAs and reduced migration (31). The interaction and activation of Src occurs at the FAK autophosphorylation site, which is absent for FRNK (11). Src kinase activity weakens integrin-cytoskeletal linkages (32) and may stimulate FA turnover by favoring lateral diffusion of integrins away from FAs. Thus, reduced FA dynamics leading to enlarged FAs is consistent with decreased migration of FRNK-Ycam cells. The disassembly of large FAs in FRNK-Ycam cells was Ca²⁺-insensitive, possibly because for this subset of FAs, endogenous FAK levels were small compared with FRNK levels. Our data support that FA size affects migration by also reflecting the Ca²⁺ sensitivity of FAs and, hence, their remodeling.

FA disassembly involves disruption of ECM-integrin and/or integrin-cytoskeleton interactions, which are regulated by several calciproteins. For instance, contractile force activation via Ca²⁺/calmodulin-dependent MLCK may strengthen integrin-cytoskeletal linkages (26) and is necessary for Ca²⁺-dependent migration of neutrophils (20). Conversely, integrin-cytoskeletal linkages are disrupted by calpain, a Ca²⁺-dependent protease, and calpain inhibition or disruption mimics the effects of FRNK expression on FA morphology (18, 19). Calreticulin appears to be essential for integrin-mediated Ca²⁺ signaling and adhesion (33, 34). Calcineurin, a protein phosphatase, is involved in integrin recycling to the front of migrating neutrophils (21), probably via affinity modulation. We show that Ca²⁺ spikes trigger FA disassembly and propose that ECM-integrin linkages are disrupted, given the identical kinetic parameters for FA and SF dynamics, which suggests coordinated FA/SF movement. In agreement, in migrating fibroblasts, FAs move with a similar speed of 0.12 ± 0.08 µm/min and remain associated with SFs in retractile edges (3). Our observation of linearly disassembling FAs suggests that FAK-Ycam and FRNK-Ycam remained linked to SF ends until complete disassembly. However, because integrin-cytoskeleton and integrin-ECM interactions are intimately related, linkage perturbations initiated at either side of these molecular complexes could be transmitted via modulation of integrin affinity/avidity to both intracellular and extracellular partners. For example, integrin ligation initiates recruitment of a cytoskeletal actin component (27) and linkage with the forward moving actin cytoskeleton (35). Conversely, detachment of integrins from the actin cytoskeleton may induce decreased integrin affinity/avidity for their ECM ligands (36, 37).

The cell migration process implies asymmetric signal transduction that directs cell polarization (1). In U87 astrocytoma cells, we observed spatially localized disassembly and formation of FAs, illustrating this asymmetry. Since FA disassembly but not formation was temporally correlated with Ca²⁺ spikes, Ca²⁺ signaling and/or regulatory Ca²⁺ target proteins may be spatially restricted to discrete subcellular compartments (38, 39). We are currently investigating compartmentalized Ca²⁺ signaling in migrating cells using our Ycam constructs as local detectors of Ca²⁺ near FAs.

That a single Ca²⁺ spike is sufficient to trigger FA disassembly agrees with initiation of a regenerative process leading to irreversible FA disruption. The Ca²⁺-triggered event might transiently increase FAK signaling, perhaps followed by a Ca²⁺-independent process. This implies a latency between the Ca²⁺ spike and cell edge retraction resulting from FA disassembly. Consistent with our data, Ca²⁺ elevation and increased migration speed were positively correlated in neutrophils (16), but since there was a 20-s delay between Ca²⁺ elevation and increased motility, Ca²⁺ was proposed not to be the immediately causal signal. However, we show that FA disassembly begins 30 s after a Ca²⁺ spike, and once triggered, several minutes are required for complete disassembly. This supports that Ca²⁺ elevation is a proximal signal leading to increased motility. Finally, rapid thapsigargin-induced FAK Tyr³⁹⁷ phosphorylation agrees with Ca²⁺-dependent FAK activation being an initial event associated with FA disassembly.

	ACKNOWLEDGEMENTS

We thank R. Y. Tsien (University of California at San Diego), S. B. Kanner (Bristol Myers Squibb), and A. Bresnick and D. M. Watterson (Northwestern University) for generously providing Y-Cam-2, FAK, and MLCK-GFP plasmids, respectively.

	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 Assocation pour la Recherche Contre le Cancer, and the Association Régionale pour l'Enseignement et la Recherche Scientifique en Champagne-Ardenne.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Columbia University, Dept. of Biological Sciences, P. O. Box 2408, Sherman Fairchild Center, 1212 Amsterdam Ave., New York, NY 10027.

^§ Recipient of a Fellowship from the Ligue Nationale Contre le Cancer.

^¶ To whom correspondence should be addressed: Pharmacologie et Physicochimie, UMR CNRS 7034, Université Louis Pasteur, BP 24, 67401 Illkirch, France.

Published, JBC Papers in Press, May 14, 2002, DOI 10.1074/jbc.M203952200

² P. Rondé, G. Giannone, A. Scherberich, A. Beretz, J. Haiech, and K. Takeda, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: SF, stress fiber; FA, focal adhesion; FAK, focal adhesion kinase; FRNK, focal adhesion-related non kinase domain; ECM, extracellular matrix; MLCK, myosin light chain kinase; Ca²⁺, calcium; Ab, antibody; mAb, monoclonal antibody; GFP, green fluorescent protein; YFP, yellow fluorescent protein; EMEM, Eagle's minimum essential medium; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester); TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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