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Originally published In Press as doi:10.1074/jbc.M701011200 on March 27, 2007

J. Biol. Chem., Vol. 282, Issue 22, 16585-16598, June 1, 2007
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Calreticulin Affects Fibronectin-based Cell-Substratum Adhesion via the Regulation of c-Src Activity*Formula

Sylvia Papp{ddagger}1, Marc P. Fadel{ddagger}, Hugh Kim§, Christopher A. McCulloch§, and Michal Opas{ddagger}2

From the {ddagger}Department of Laboratory Medicine and Pathobiology and §Canadian Institutes of Health Research Group in Matrix Dynamics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Received for publication, February 2, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calreticulin, a Ca2+-binding protein of the endoplasmic reticulum (ER),3 has been shown to be involved in a great number of cellular processes (1). It is an important chaperone, working in conjunction with calnexin and protein disulfide isomerase; it affects intracellular Ca2+ homeostasis via its Ca2+ storage capacity and its effects on both the SERCA pumps and inositol 1,4,5-trisphosphate receptors (2, 3). Calreticulin has been shown to affect cell adhesion via the induction of vinculin and N-cadherin expression and its involvement in beta-catenin-associated pathways (4, 5). Finally, calreticulin overexpression causes a decrease in total cellular tyrosine phosphorylation levels (5, 6).

Fibronectin is a large glycoprotein which is secreted by the cell into the extracellular matrix (ECM) as a soluble dimer that, via cellular interactions, is deposited as a fibrillar meshwork that is bound to the surface of cells. Fibronectin affects the formation and the stability of cell-substratum adhesions, and conversely, cell-substratum adhesions may affect fibronectin matrix deposition (7, 8). Fibronectin assembly cannot proceed without the presence of cells (9), which implies intracellular inside-out signaling pathways that are crucial for fibronectin fibrillogenesis (10). Fibronectin matrices are essential for embryonic development, wound healing, and tumorigenesis (11) and as such are both spatially and temporally regulated (11). Thus, it is crucial to discern the mechanisms by which a fibronectin matrix is deposited and regulated.

Fibronectin is bound to and regulated by cells at specific sites of cell-substratum adhesions that serve to link the ECM to the actin cytoskeleton via transmembrane integrin heterodimers (12). Forces due to integrin binding to ECM ligands cause the bundling of the actin cytoskeleton into tension-generating stress fibers. This tension also causes the clustering of integrins and associated cytoskeletal proteins, such as vinculin, paxillin, talin, c-Src, and focal adhesion kinase (FAK), into strong cell substratum adhesions termed focal contacts (or focal adhesions) (8, 12-14). FAK plays a central role in cell-matrix adhesion and is also a substrate for Src (15).

Ezzel et al. (16) have shown that the ability of cells to spread out on a substratum correlates directly with the efficacy with which vinculin is coupled to focal contacts. Thus, calreticulin-overexpressing cells, which possess the greatest amount of vinculin, are well spread on the substratum, whereas the calreticulin underexpressers with the least amount of vinculin are poorly spread (4).

Tyrosine phosphorylation has also been shown to regulate cell-substratum adhesion (17-20); thus, the aim of this study was to investigate the role of protein tyrosine kinases, particularly c-Src, in the regulation of cell-substratum adhesion in cells differentially expressing calreticulin. c-Src is an important regulatory molecule of focal contacts and belongs to a family of nonreceptor protein-tyrosine kinases, including the ubiquitous kinases Yes and Fyn (21, 22). To determine how calreticulin, a Ca2+-binding ER-resident protein, influences cell spreading, fibronectin matrix deposition, and cell-substratum adhesion, we examined the activity of c-Src in cells differentially expressing calreticulin on both uncoated and fibronectin-coated surfaces. This study presents novel findings on the role of calreticulin in fibronectin matrix deposition and the regulation of c-Src activity via its influence on releasable Ca2+ from the ER.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Mouse L fibroblasts differing in calreticulin expression used for this study have been extensively characterized and described elsewhere (4, 6, 23, 24). Sense and antisense cDNA were used to generate stable cell lines either over- or underexpressing calreticulin, respectively (23). Calreticulin-overexpressing cells displayed approximately twice as much calreticulin as control L fibroblasts, as determined by Western blot analysis. Calreticulin-underexpressing cells displayed approximately half as much calreticulin as control L fibroblasts. L fibroblasts were maintained in antibiotic free, high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 µg/ml Geneticin.

RNA Isolation and Northern Blotting—RNA extraction and Northern blotting were performed as described previously (23). The blots were normalized by probing with human glyceraldehde 3-phosphate dehydrogenase cDNA.

Western Blotting—Protein samples from cellular lysates (40,000 cells/lane and 2 µg/lane for molecular weight markers) were subjected to SDS-PAGE and Western blotting as described previously (24). Primary antibodies were used at the following dilutions in TBST: anti-actin, 1:500 (Sigma); anti-calreticulin, 1:300 (25-27); anti-FAK (Tyr(P)397), 1:1000 (BioSource International); anti-fibronectin, 1:1000 (Sigma); anti-{alpha}5 and -beta1 integrins, 1:500 (gift from Dr. B. Chan); anti-Src, 1:1000 (Upstate Cell Signaling); anti-Src (Tyr(P)418), 1:1000 (BioSource International); anti-active Src, 1:1000 (BioSource International); and anti-vinculin, 1:500 (Sigma). All horseradish peroxidase-conjugated secondary antibodies were used at a dilution of 1:10,000 (Jackson ImmunoResearch Laboratories). Isolation of the ECM fractions and identification of fibronectin in the ECM was carried out as previously described (28). Briefly, the ECM was isolated by sequential washes with 1) PBS; 2) 3% Triton X-100 in PBS; 3) 50 mM Tris, pH 7.4, 10 mM MnCl2, 1 M NaCl; 4) 2% deoxycholate in 50 mM Tris, pH 8.8, 10 mM EDTA. All washes were carried out at room temperature for 3 min in the presence of 1 mM phenylmethylsulfonyl fluoride. The material remaining after the washes was considered to be ECM and was scraped in 1% SDS and boiled for 5 min prior to loading.

Sample Preparation for Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS)—Aliquots of conditioned media were separated by SDS-PAGE. Proteins were visualized using Bio-Rad silver stain. For protein identification by MALDI-TOF MS, the band of interest was excised from the gel and placed in 1% acetic acid. Gel fragments were then washed with water, incubated in acetonitrile for 15 min at room temperature, and reduced in 100 mM ammonium bicarbonate containing 10 mM dithiothreitol for 30 min at 50 °C. After a second acetonitrile wash, the gel fragments were alkylated with 100 mM ammonium bicarbonate containing 55 mM iodoacetamide for 20 min at room temperature in the dark and washed in 100 mM ammonium bicarbonate. After a final acetonitrile wash, the gel fragments were dried by SpeedVac for 1 min. For trypsin digestion, the gel fragments were rehydrated in digestion buffer (50 mM ammonium bicarbonate, 5 mM CaCl2, and 12.5 ng/µl sequencing grade trypsin) (Promega) for 45 min on ice, and then incubated overnight at 37 °C. To extract tryptic fragments, after brief centrifugation, the liquid fraction was transferred to a fresh tube containing 100 mM ammonium bicarbonate for 30 min at 37 °C. After a second extraction, the liquids were pooled and purified by repeated passage through a ZipTipC18 (Millipore). The sample was eluted with 65% acetonitrile, 1% acetic acid and analyzed by MALDI-TOF MS by the Mass Spectrometry Laboratory, University of Toronto. Protein identification was performed using the ProFound data mining software (available on the World Wide Web at prowl.rockefeller.edu).

Fluorescence-activated Cell Sorting—Cells were trypsinized, rinsed with PBS, and fixed with 3.7% formaldehyde in PBS for 10 min. After rinsing, the cells in suspension were incubated with anti-{alpha}5 or -beta1 integrin primary antibodies for 30 min at room temperature under gentle agitation. The cells were then rinsed with PBS and incubated for 30 min at room temperature with secondary antibodies. The cells were rinsed with PBS, resuspended at equal densities, and analyzed using an EPICS Elite Cell Sorter (Beckman-Coulter).

Fibronectin Patterned Substrata—Coverslips were coated with 10 µg/ml fibronectin from Sigma in PBS for 90 min at 37 °C and then rinsed in PBS. The coated surface was then scratched with a 1-ml pipette tip to remove fibronectin, creating areas of fibronectin carpet next to areas without.

Immunolabeling and Microscopy—For immunolocalization, cells on coverslips were fixed in 3.7% formaldehyde in PBS for 10 min. After washing (three times for 5 min) in PBS, the cells were permeabilized for 2 min with 0.1% Triton X-100 in buffer containing 100 mM PIPES, 1 mM EGTA, and 4% (w/v) polyethylene glycol 8000 (pH 6.9) and then washed three times for 5 min in PBS and incubated with primary antibodies for 30 min at room temperature at the following dilutions in PBS: anti-fibronectin, 1:50, anti-{alpha}5 integrins, 1:50, anti-vinculin, 1:20; Texas Red phalloidin, 1:10. After washing in PBS, the cells were stained with appropriate fluorescent secondary antibodies (1:50 in PBS) for 30 min at room temperature. After the final wash, slides were mounted in Vinol 205S, which contained 0.25% 1,4-diazabicyclo-(2,2,2)-octane and 0.002% p-phenylenediamine or in Immuno Fluore (ICN Biochemicals). A Bio-Rad MRC-600 confocal fluorescence microscope equipped with a krypton/argon laser was used for fluorescence and phase-contrast microscopy.

Tyrphostins, Radicicol, and Calcium Treatments—Before any treatments, equal numbers of cells were plated and grown overnight (12 h). Treatment times listed are those that follow this overnight attachment and growth. Cells were treated with 100 µM tyrphostin A1, A23, A25, or A63 for 24 h. The cells were then fixed and stained for immunofluorescence imaging as described above. For c-Src inhibition experiments, cells were treated with 5 µM radicicol (Sigma) overnight (12 h). The next day, cells were washed three times with PBS and replaced with fresh growth medium for 5 h. The cells were then collected either for Western blotting or fixed and stained for immunofluorescence imaging. Thapsigargin was used to reduce the concentration of Ca2+ in the ER. Cells were treated with 1 µM thapsigargin for 30 min at 37 °C and immediately collected for Western blotting of Src Tyr(P)418. For Western blotting of fibronectin and immunofluorescence of fibronectin deposition, cells were treated with 1 µM thapsigargin or 1 µM ionomycin for 2 h, followed by overnight growth in regular growth medium.


Figure 1
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  		FIGURE 1.
A, Northern blot of fibronectin (fn) mRNA in L fibroblasts differentially expressing calreticulin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. over, overexpressers; under, underexpressers; control, mock-transfected control cells. B, Western blot of intracellular fibronectin protein levels in L fibroblasts. Cells overexpressing calreticulin contain the greatest amount of intracellular fibronectin, whereas calreticulin underexpressers have the least. Mock-transfected control cells have an intermediate amount of intracellular fibronectin. Actin was used as a loading control. B', identification of fibronectin protein in the matrix of L fibroblast cultures differentially expressing calreticulin. Western blotting reveals that calreticulin-overexpressing cells deposit greater levels of fibronectin protein into the matrix compared with calreticulin-underexpressing cells or control cells. C and C', silver stain of proteins from conditioned media from calreticulin-overexpressing (O), calreticulin-underexpressing (U), and control cells (C). The time in culture of each sample is indicated in hours. Fibronectin (F) was loaded as a positive control. Molecular weight markers were loaded in the first lane in each gel. The gel in C was exposed 10 times longer than in C'. D, Western blotting of the gel in C' for fibronectin confirms the identity of the high molecular weight bands.

 
Calcium Measurements—For measurements of intracellular Ca2+, cultured L cells, either in suspension or adherent to tissue culture dishes, were loaded with fura-2-AM (Molecular Probes) at a concentration of 3 µM, for 30 min at 37 °C. A Nikon inverted fluorescence microscope (Diaphot) optically interfaced to a dual wavelength excitation ratio fluorimeter (Photon Technology International) was used to estimate changes in intracellular [Ca2+], based on changes in fluorescence intensity at alternating 346/381 nm excitations. For bradykinin stimulation experiments, bradykinin (Sigma) was reconstituted in dimethyl sulfoxide and added to fura-2-loaded cells at a concentration of 2 µM to stimulate calcium release from intracellular stores. For the experiments using L cells plated on fibronectin, fura-2-loaded cells were trypsinized and suspended in a buffer solution containing HEPES, NaCl, KCl, glucose, MgSO4, Na2HPO4, and CaCl2. Cells were then seeded onto 32-mm diameter coverslips that were precoated with 10 µg/ml fibronectin (Sigma). Intracellular [Ca2+] was measured at 5, 15, and 30 min postplating. As a control, the experiment was performed with fura-2-loaded cells seeded onto untreated glass coverslips.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
L Fibroblasts Differentially Expressing Calreticulin Also Differ in Their Ability to Synthesize Fibronectin—Three stably transfected cell lines of mouse L fibroblasts were previously generated and extensively characterized (4, 6, 23, 24). Cells with increased calreticulin expression level (overexpressers) or reduced calreticulin level (underexpressers) or mock-transfected cells with unchanged calreticulin level (control), as determined by Western blot analyses (23), were used in this study. Our previous studies showed that calreticulin influences the expression of vinculin, an intracellular component of focal contacts; thus, we examined here whether the extracellular face of focal contacts may also be affected by calreticulin. Our particular focus was on fibronectin, a major component of integrin-based cell substratum adhesions. Interestingly, we found that cells differentially expressing calreticulin differed in their ability to synthesize fibronectin. Using Northern blotting of total cellular lysates of L fibroblasts grown for 24 h, we found that calreticulin overexpressers had greater levels of fibronectin mRNA in comparison with either calreticulin underexpressers or control cells (Fig. 1A). Furthermore, calreticulin overexpressers contained the most intracellular fibronectin protein, whereas the underexpressers had the least amount and control cells were intermediate (Fig. 1B). The increased fibronectin protein in calreticulin overexpressing L fibroblasts was accompanied by an increase in the deposition of extracellular fibronectin into the matrix (Fig. 1B').

Next, we determined whether calreticulin's effects were specific for fibronectin or if the secretion of other proteins was also affected. Conditioned media from the L fibroblast cell lines were collected at various time points and subjected to SDS-PAGE. The proteins were revealed by silver staining. Overall, there were no changes in the bulk outflow of proteins from cells differentially expressing calreticulin (Fig. 1, C and C'). However, there were differences in the secretion of a high molecular weight protein that migrated at the same mobility as fibronectin-positive controls, such that secretion was increased in calreticulin overexpressers and decreased in calreticulin underexpressers. Control cells secreted an intermediate amount of this protein (Fig. 1, C and C'). To identify this unknown protein, the indicated band (Fig. 1C, arrowhead) was excised from the gel and analyzed by MALDI-TOF MS as outlined under "Experimental Procedures." The tryptic fingerprint was compared against a data base using the ProFound software. The first ranked retrieved protein from the list was fibronectin, with a molecular mass of 276.15 kDa. Of the 26 peptides measured, eight were found in the data base with sequence coverage of 5% (supplemental Table 1). Proteins from SDS-PAGE of conditioned media were also transferred to nitrocellulose before silver staining. Western blotting confirmed the identity of the secreted high molecular weight protein to be fibronectin (Fig. 1D).


Figure 2
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  		FIGURE 2.
Visualization of fibronectin matrix deposition in crowded L fibroblasts overexpressing (over) or underexpressing (under) calreticulin and in mock-transfected (control) cells. The right column is a x3 magnification of the outlined field in the adjacent image. For calreticulin-overexpressing cells, there is a greater amount of fibronectin deposited, with thicker and more extensive fiber formation. Contrast this with calreticulin-underexpressing cells, which deposit much less fibronectin and have only sparse patches of fibrils.

 


Figure 3
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  		FIGURE 3.
A, focal contact formation in sparse L fibroblasts. Cells either underexpressing (under) or overexpressing (over) calreticulin and mock-transfected control cells (control) were double-labeled with fluorescently tagged phalloidin to show the F-actin cytoskeleton and with vinculin to reveal focal contacts. The calreticulin-overexpressing cells exhibit the most numerous and prominent focal contacts, whereas calreticulin underexpressers form very few focal contacts. B, quantification of the number of vinculin-containing focal contacts in L fibroblasts. The graphs represent an average count of 50 cells. Note that the calreticulin-overexpressing cells have a significantly greater number of vinculin-positive contacts compared with control, whereas underexpressers have significantly fewer. *, p < 0.01.

 
Taken together, these results show that calreticulin-overexpressing L cells have increased fibronectin mRNA and protein and secrete more fibronectin, which leads to a greater accumulation of it in the matrix. Importantly, this trend is also seen in HEK293 cells containing the calreticulin gene driven by a doxycycline-sensitive promoter following calreticulin induction, as well as in mouse embryonic fibroblast cell lines with normal calreticulin levels compared with calreticulin-null cells.4 In all cases, there is greater fibronectin expression in cells with greater calreticulin abundance.


Figure 4
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  		FIGURE 4.
A, fluorescence-activated cell sorting analysis reveals no difference in the amount of {alpha}5beta1 integrins at the cell surface of crowded L fibroblasts differentially expressing calreticulin. The data are presented as the mean increase in surface fluorescence over background (cells labeled with secondary antibodies only) of three independent experiments ± S.D. B, localization of {alpha}5 integrins in L fibroblasts differentially expressing calreticulin. Although there is no difference in total protein levels, there is a striking difference in the localization of {alpha}5 integrins in these cells. In calreticulin-overexpressing cells (over), {alpha}5 integrins are clustered in prominent focal contact-like patches, which are far less numerous in calreticulin underexpressers (under). Mock-transfected control cells (control) show an intermediate amount of {alpha}5 integrins.

 
L Fibroblasts Overexpressing Calreticulin Assemble an Elaborate Fibronectin Matrix, whereas Calreticulin Underexpressers Exhibit Poor Fibronectin Deposition—We next examined if the relationship between the level of calreticulin expression and deposition of fibronectin into the ECM led to differential fibronectin matrix assembly. Using indirect immunofluorescence with anti-fibronectin antibodies on nonpermeabilized cells, we showed that crowded calreticulin overexpressers assembled an extensive, weblike fibronectin matrix after overnight growth in culture (Fig. 2). High magnification reveals extensive, thick fibronectin fibrils. Crowded L fibroblasts underexpressing calreticulin assembled a poor fibronectin matrix, which consisted of a loose arrangement of thin fibrils and patches. The fibronectin matrix assembled by control cells was intermediate between that of overexpressers and underexpressers (Fig. 2).

Focal Contact Formation and Differential Localization of the Fibronectin Receptor in L Cells—Since the deposition and organization of a fibronectin matrix is cell-dependent and, more specifically, is remodeled by focal contacts, we next analyzed the adhesive architecture of cells. L fibroblasts double-labeled with fluorescently tagged phalloidin to reveal the F-actin cytoskeleton and an antibody to vinculin to show focal contacts were examined. Increased focal contact formation and a more robust stress fiber-containing actin cytoskeleton are evident in calreticulin-overexpressing cells (Fig. 3A). The calreticulin underexpressers are poorly spread and have very few focal contacts and no stress fibers. Quantification of the number of vinculin-containing focal contacts per cell reveals a significantly greater number of focal contacts in calreticulin overexpressers compared with control cells (Fig. 3B). Calreticulin underexpressers contain only a slight number of focal contacts, which are significantly lower than those in control cells (Fig. 3B).

The fibronectin receptor in L fibroblasts is the {alpha}5beta1 integrin heterodimer, and the extracellular domain of the {alpha}5 subunit is responsible for ligand binding (11). We next wanted to determine if the difference in fibronectin matrix assembly in L cells was due to differential fibronectin receptor expression at the cell surface, since changes in calreticulin expression do not affect the total protein levels of {alpha}5beta1 integrins in these cells (4, 6). Fluorescence-activated cell sorting analysis revealed no differences in the surface expression of {alpha}5 and beta1 integrins (Fig. 4A). However, the observed difference in fibronectin matrix deposition in L fibroblasts could result from differential receptor localization. Thus, immunolocalization of the {alpha}5 integrin subunit was performed, and distinct differences were detected in its localization to the cell surface in each L fibroblast cell line. Crowded calreticulin-overexpressing cells formed abundant {alpha}5 integrin-rich patches, which had the appearance of focal contacts (Fig. 4B). In calreticulin underexpressers, {alpha}5 integrin-positive patches were very scarce, and there was little focal contact formation (Fig. 4B). Control cells had an intermediate morphology with respect to {alpha}5 integrin and vinculin-containing focal contacts (Figs. 3A and 4B).

Preformed Fibronectin Induces Cell Spreading and Increased Focal Contact Formation, with the Greatest Changes Occurring in Calreticulin Underexpressers—Since the calreticulin-underexpressing cells were unable to assemble a robust fibronectin matrix and showed poor focal contact formation, we asked if an exogenous preadsorbed fibronectin matrix would rescue the poor adhesive phenotype of these cells. Cells were grown overnight on precoated fibronectin substrata, and were imaged by confocal microscopy following double label immunolocalization for vinculin and fibronectin. This time of growth in vitro allowed for imaging of both preformed fibronectin matrix remodeling and initial stages of fibronectin matrix deposition on glass. To visualize changes in cellular morphology and adhesiveness due to fibronectin, we imaged the cells at the interface between the fibronectin carpet and fibronectin-free bare glass, positioned so as to fit into a single microscopic field of view.


Figure 5
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  		FIGURE 5.
A, cell-substratum adhesions in crowded L fibroblasts overexpressing calreticulin (over) and underexpressing calreticulin (under), imaged at the interface between the fibronectin carpet and bare glass as marked. Cells were immunostained for vinculin and fibronectin. On glass, calreticulin overexpressers make many vinculin-positive focal contacts and deposit an extensive fibronectin matrix. On fibronectin carpets, calreticulin overexpressers are induced to become even more spread. In contrast, calreticulin underexpressers on glass make barely any vinculin-positive contacts. These cells also do not deposit fibronectin fibrils but have fibronectin aggregates on their dorsal surface. On fibronectin carpets, calreticulin underexpressers spread extensively via numerous vinculin-positive contacts, which co-align with some fibronectin fibrils. B, quantification of the number of vinculin-containing focal contacts in L fibroblasts plated either on glass or on fibronectin. The graphs represent an average count of 50 cells. Both calreticulin over- and underexpressing cells on fibronectin have a significantly greater number of vinculin-positive contacts compared with cells on glass, with the increase in the number of contacts being truly remarkable in the underexpressers. *, p < 0.05; **, p < 0.01.

 
On glass, calreticulin-overexpressing cells were well spread, made many vinculin-positive focal contacts, and deposited fibronectin, whereas the calreticulin underexpressers were poorly spread with few, if any, focal contacts (Figs. 3A and 5A). In stark contrast, calreticulin underexpressers on fibronectin carpets spread extensively, and vinculin staining revealed a dramatic increase in the number of vinculin-containing focal contacts in calreticulin underexpressers on fibronectin versus on glass (Fig. 5A). Quantification of the vinculin-containing focal contacts are shown in Fig. 5B. These results show that L fibroblasts differentially expressing calreticulin are induced to spread by a preformed fibronectin carpet, with the degree of induction in the case of calreticulin underexpressers being truly dramatic. It should be noted that, since culturing cells on fibronectin carpets had no effect on the level of fibronectin expression (data not shown), the observed effects are due to the exogenous fibronectin matrix itself.


Figure 6
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  		FIGURE 6.
A, [Ca2+]c measurements of L fibroblasts either overexpressing (over), underexpressing (under), or with unchanged calreticulin (control). There is no difference in resting calcium levels between the cell lines, although the underexpressers exhibit slightly higher basal calcium levels. B, bradykinin (2 µM) induces a much larger calcium release from stores in the overexpressers compared with the underexpressers.

 
Fibronectin Expression and Deposition Is Dependent on Calreticulin and Ca2+—Next, we investigated the mechanism by which calreticulin was affecting fibronectin gene expression and deposition. Since calreticulin is a known regulator of Ca2+ signaling from the ER (29), we first examined the levels of calcium in cells at rest and following stimulation with bradykinin, followed by an examination of the effects of [Ca2+]ER on fibronectin. Fig. 6A shows that resting levels of calcium in the cytosol ([Ca2+]c) are slightly higher in calreticulin underexpressers than in overexpressing or control cells although not significantly so. Importantly, the amount of calcium released from the ER via bradykinin stimulation is the greatest in calreticulin-overexpressing cells (Fig. 6B). Calreticulin underexpressers show the lowest amount of Ca2+ released following bradykinin stimulation, whereas control cells are intermediate (Fig. 6B).


Figure 7
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  		FIGURE 7.
A, Western blot of total fibronectin (both intra- and extracellular) following thapsigargin (TG) and ionomycin (iono) treatments. Cells were scraped along with the matrix to obtain this blot. Note the decrease in fibronectin (fn) levels after thapsigargin treatment and the increase in fibronectin levels following ionomycin treatment. Actin was used as a loading control. B, immunofluorescence of fibronectin deposited by L fibroblasts, which follows the pattern seen in the Western blot in A.

 
To examine the effects of altering Ca2+ on fibronectin expression and deposition, we treated L fibroblasts with either thapisgargin, to effectively reduce [Ca2+]ER, or with ionomycin in regular Ca2+-containing growth medium, thus effectively increasing [Ca2+]c. Fig. 7A shows that Ca2+ depletion of the ER with thapsigargin causes a decrease in the expression level of fibronectin in all cell lines. Furthermore, the amount of fibronectin deposited into the ECM is also decreased after lowering of [Ca2+]ER with thapsigargin (Fig. 7B).

It has previously been shown by Nakamura (30) that calreticulin-deficient cells, which have low levels of [Ca2+]ER, exhibit impaired agonist-induced Ca2+ release from the ER. This is in agreement with our current data, which show that calreticulin underexpressers are also impaired in bradykinin-stimulated Ca2+ release as compared with control cells. Thus, we hypothesized that this decreased Ca2+ release may be affecting fibronectin gene expression and matrix deposition. Thus, we also performed Western blotting and immunofluorescence localization of fibronectin following ionomycin treatment. As revealed by Western blotting, fibronectin levels increased following ionomycin treatment (Fig. 7A), which was accompanied by increased fibronectin deposition and fibrillogenesis in all cell lines (Fig. 7B). Thus, altering [Ca2+] has profound effects on both the expression and deposition of fibronectin.


Figure 8
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  		FIGURE 8.
A, immunofluorescence of calreticulin-underexpressing L fibroblasts treated with 100 µM tyrphostins for 24 h. Double labeling was used to visualize the actin cytoskeleton with phalloidin (red) and to visualize fibronectin matrix with anti-fibronectin antibodies (green). Note the dramatic increase in cell spreading and fibronectin deposition following tyrphostin treatments. B, Western blotting of Tyr(P)418 c-Src (Src pY418) in L fibroblasts differentially expressing calreticulin. Src Tyr(P)418 levels, indicative of c-Src activity, are highest in L fibroblasts underexpressing calreticulin (under) and lowest in calreticulin overexpressers (over) and show moderate levels in mock-transfected control cells (control). C, total c-Src protein levels as visualized by Western blotting. There is no difference between total c-Src levels regardless of calreticulin expression level. D, Western blotting of tyrosine 397-phosphorylated FAK (FAK pY397) reveals that active FAK levels are greatest in calreticulin overexpressers and lowest in calreticulin-underexpressing L fibroblasts. Actin was used as a loading control for all Western blotting experiments.

 
c-Src Activity Is Greatest in L Fibroblasts Underexpressing Calreticulin—We wanted to further investigate the mechanism by which fibronectin and focal contacts were being affected by calreticulin. We have previously shown that poorly spread cells that underexpress calreticulin have elevated levels of total cellular phosphotyrosine in comparison with well spread calreticulin overexpressers (6), thus implicating tyrosine kinases in the regulation of cell spreading and cell-substratum adhesion. Would the general inhibition of tyrosine kinases by tyrphostins revert the poorly adhesive phenotype of calreticulin underexpressers? To test this, cells were treated with tyrphostins for 24 h and then double-labeled for actin and fibronectin. Fig. 8A shows that a general inhibition of tyrosine kinases in calreticulin-underexpressing cells induces dramatic cell spreading and fibronectin deposition. Much lesser effects of tyrphostins were noted in calreticulin overexpressers, and tyrphostin 63 (an inactive control) was without effect (data not shown).


Figure 9
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  		FIGURE 9.
A, Western blotting of tyrosine 418-phosphorylated c-Src (Src pY418) reveals that active Src levels are lower in L fibroblasts plated on fibronectin (FN) compared with cells plated on glass. over, calreticulin overexpressers; under, calreticulin underexpressers; control, mock-transfected control cells. B, Western blotting of total c-Src levels revealed that there are no changes in the amount of Src synthesized in cells plated on fibronectin or on glass. Actin was used as a loading control.

 
We next investigated the activity of a major regulator of focal adhesion turnover, the nonreceptor tyrosine kinase, c-Src, in L cells differentially expressing calreticulin. We employed antibodies against the c-Src Tyr(P)418 residue, which has been previously characterized (31). Src contains an autophosphorylation site at Tyr418 (in humans and mice; Tyr416 in chicken), which renders the kinase active when phosphorylated (32, 33). Here we show that calreticulin-underexpressing cells contained the highest levels of active c-Src (Fig. 8B), whereas calreticulin overexpressers exhibited the lowest levels of Tyr(P)418, and control cells had intermediate levels. Total c-Src levels remained the same in all cell lines, regardless of calreticulin expression (Fig. 8C). Interestingly, the levels of active FAK, as determined by Western blotting against the FAK Tyr(P)397 residue, were found to be highest in calreticulin-overexpressing cells, with the underexpressers exhibiting the lowest amounts of active FAK (Fig. 8D). In addition, when the cells were plated on a preformed fibronectin carpet, Tyr(P)418 levels were found to be diminished in all cell lines (Fig. 9A), which was not paralleled by a change in total c-Src levels (Fig. 9B). This indicates that c-Src activity is subject to regulation by outside-in signaling from the matrix.

Inhibition of c-Src Improves the Poorly Adhesive Phenotype of Calreticulin Underexpressers—Since Src activity was greatest in poorly spread cells, we next treated L fibroblasts with a c-Src inhibitor, radicicol, to determine if it would rescue the poorly adhesive phenotype of the calreticulin underexpressers. Radicicol has previously been shown to reduce the autophosphorylation of v-Src and c-Src, thus effectively inhibiting/reducing Src kinase activity (34, 35). We also show here that radicicol indeed decreases the levels of active Src as well as of active FAK, as seen by Western blotting (Fig. 10, A and B, respectively). Fig. 10C shows phase-contrast images of L fibroblasts following radicicol treatment. All cell lines, but most notably the underexpressers, exhibited increased cell spreading following a 12-h radicicol treatment. This increased cell spreading was paralleled by increased focal contact formation (Fig. 11B), as assessed by vinculin immunofluorescence (Fig. 11A), which was most dramatic in the calreticulin underexpressers. There was no change in the level of vinculin protein following radicicol treatment (Fig. 11C); thus, the increased focal contact formation following c-Src inhibition is not a result of the induction of vinculin gene expression but rather a redistribution of vinculin from free cytosolic pools to focal contacts. Concomitant with increased focal contact formation, fibronectin deposition into the ECM was also increased following radicicol treatment, as assessed by Western blot (Fig. 12A) and immunofluorescence (Fig. 12B). The levels of intracellular fibronectin remained unchanged following radicicol treatment (Fig. 12A). Similar results were obtained using PP2, another inhibitor of c-Src (data not shown).


Figure 10
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  		FIGURE 10.
A, Western blotting of active Src levels shows that a 12-h radicicol treatment (5 µM) diminishes the levels of active Src in L fibroblasts differentially expressing calreticulin. B, Western blotting of tyrosine 397-phosphorylated FAK (FAK pY397) reveals that active FAK levels are also reduced following radicicol treatment. Actin was used as a loading control. C, phase-contrast images of L fibroblasts following radicicol treatment. All cell lines become increasingly spread on the substratum, with the most dramatic change occurring in the calreticulin underexpressers, which normally exhibit a round and poorly adhesive phenotype. over, calreticulin overexpressers; under, calreticulin underexpressers; control, mock-transfected control cells.

 
c-Src Activity Is Ca2+-sensitive—Since we found fibronectin gene expression and deposition to be Ca2+-sensitive and found that fibronectin affects c-Src activity, we hypothesized that calreticulin, via its effect on total [Ca2+]ER, may control the activity of c-Src. To test this hypothesis, we exposed L cells differentially expressing calreticulin to thapsigargin for 30 min to deplete [Ca2+]ER. Interestingly, the levels of c-Src Tyr(P)418 substantially increased following thapsigargin treatment, with the biggest increase seen in the calreticulin-overexpressing cells (Fig. 13A). In order to test the hierarchy of interaction between Src and Ca2+ signals, we measured [Ca2+]c in the presence of radicicol. Intriguingly, radicicol treatment increased [Ca2+]c in all cell lines tested, with the degree of increase being greatest in the calreticulin underexpressers (Fig. 13B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The novel findings of this study are the identification of the fibronectin gene as being induced by calreticulin and the regulation of c-Src activity by calreticulin, both of which affect cell-substratum adhesion. Interestingly, both effects are Ca2+-sensitive. Overexpression of calreticulin in L fibroblasts induces fibronectin mRNA and protein, which impinges on extracellular fibronectin matrix formation. The regulation of fibronectin gene activity is complex (28, 36, 37), since fibronectin gene expression is sensitive to cAMP, glucocorticoid, and vitamin D signaling pathways (28, 37, 38). All of these may be affected by the calreticulin expression level (1).

We show here that fibronectin expression is sensitive to intracellular Ca2+ levels. Thapsigargin treatment reduced fibronectin expression in all L fibroblast cell lines, whereas ionomycin treatment had the opposite effect. Since Ca2+ was present in the medium, thapsigargin effectively reduced [Ca2+]ER, whereas ionomycin increased cytosolic [Ca2+]. The present data showing the dependence of agonist-induced Ca2+ release from the ER on calreticulin abundance are in accordance with a number of studies showing that total [Ca2+]ER is related to the level of calreticulin expression, and so is the content of Ca2+ stores releasable with bradykinin or thapsigargin (4, 24, 30, 39-41). The effects of calreticulin on gene expression and cell adhesiveness, therefore, are indirect, with Ca2+ being a regulatory molecule in these events. In fact, it has been postulated that calreticulin may be a centrally located connector molecule in a signaling network in the lumen of the ER (29). Calreticulin is uniquely endowed for such regulation, because its Ca2+ binding may (i) regulate the free [Ca2+] within the ER lumen, (ii) regulate the function of Ca2+ transport molecules such as SERCA2b and/or inositol 1,4,5-trisphosphate receptor, (iii) affect the function of the store-operated Ca2+ channels, and finally (iv) affect the transcriptional activity of several genes.


Figure 11
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  		FIGURE 11.
A, immunofluorescence staining shows increased focal contact formation in L fibroblasts following radicicol treatment (5 µM), as assessed via vinculin staining (green). The actin cytoskeleton is revealed with fluorescently tagged phalloidin (red). B, quantification of the number of vinculin-containing focal contacts in L fibroblasts either untreated or following radicicol treatment. The graphs represent an average count of 50 cells. Both calreticulin over- and underexpressing cells and control cells have a significantly greater number of vinculin-positive contacts following radicicol treatment compared with untreated cells, with the increase in the number of contacts being truly remarkable in the underexpressers. over, calreticulin overexpressers; under, calreticulin underexpressers; control, mock-transfected control cells. *, p < 0.05; **, p < 0.01. C, Western blot of vinculin following radicicol treatment in L fibroblasts. Vinculin levels remain unchanged following radicicol treatment. Actin was used as a loading control.

 
It must be noted here that other groups have found calreticulin in residences besides the ER, such as the cytosolic fraction, the nucleus, or the cell surface. However, we have not found calreticulin in the cytosol or, more specifically, in focal contacts or integrin clusters (4), and neither did the group of Pozzan (40). Dedhar's group (42) has found that calreticulin binds to the GFFKR sequence of integrins, but this has only been shown biochemically and has never been shown within an intact cell. Furthermore, our previous data show that cytosolically targeted calreticulin has no effect on cell morphology or cell adhesion (24). Last, although the group of Murphy-Ullrich (43) has localized calreticulin to the extracellular aspect of the cell surface of endothelial cells, we have never localized calreticulin to the cell surface of L fibroblasts used in the present study (data not shown). We therefore hypothesize that ER-resident calreticulin acts as a Ca2+ sensor inside the ER and thereby regulates gene expression by participating in an ER to nucleus signaling pathway (29, 44). This may also be the case for vinculin, whose expression was previously shown by us to be regulated by calreticulin (4, 5). Induction of vinculin in L fibroblasts overexpressing calreticulin causes more effective assembly of stress fibers and focal contacts than in underexpressing or control cells (4, 5). Indeed, increased vinculin expression has been correlated with increased focal contact formation, increased stress fiber formation, and enhanced cell spreading (45). Although the expression of vinculin is not affected by the increased cell spreading following c-Src inhibition, there must be a net movement of vinculin from cytosolic pools to focal contacts, thus enhancing the adhesive ability of calreticulin underexpressers. Therefore, calreticulin may also participate in an ER-to-focal contact signaling pathway. This signaling pathway is likely to affect both cytosolic focal contact components, such as c-Src and vinculin, and extracellular components, such as fibronectin, explaining the effects of calreticulin on fibronectin deposition.


Figure 12
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  		FIGURE 12.
A, Western blot of intracellular (fn cellular) and extracellular fibronectin in the matrix (fn ECM) following radicicol treatment of L fibroblasts reveals an increase in fibronectin deposited into the matrix in all cell lines. Cellular fibronectin levels remain unchanged. B, increased fibronectin deposition in L fibroblasts following radicicol treatment. Immunofluorescence staining of unpermeabilized L fibroblasts using anti-fibronectin antibodies reveals increased fibronectin deposition in all cell lines following radicicol treatment. over, calreticulin overexpressers; under, calreticulin underexpressers; control, mock-transfected control cells.

 
L fibroblasts overexpressing calreticulin may exhibit better fibronectin matrix assembly due to their increased number of focal contacts. Both {alpha}5 and beta1 integrin subunits of the fibronectin receptor must be present and functional for fibronectin matrix deposition to occur (11, 46). Calreticulin overexpressers displayed many, large {alpha}5-positive staining patches at the cell surface, whereas calreticulin-underexpressing cells had very few, small {alpha}5-positive patches. Since all cells had similar abundance and surface expression of {alpha}5beta1 integrins, the difference in surface distribution indicates differential stabilization of integrins within focal contacts. Mechanical force generated by stress fibers recruits vinculin to focal contacts (47), thereby stabilizing its components (16, 48). Increased accumulation of vinculin in focal contacts in these cells may serve as a more efficient nucleation site for {alpha}5beta1 integrin clustering. This would account for more effective fibronectin fibrillogenesis. In a positive feedback, fibronectin binding itself could stabilize the superstructure of focal contacts (49, 50). Furthermore, integrin engagement and clustering is reciprocally related to Src activity (51, 52), thus linking Src to focal contact formation and fibronectin matrix deposition.


Figure 13
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  		FIGURE 13.
A, Western blot of tyrosine-phosphorylated c-Src (Src pY418) and total c-Src following thapsigargin (TG) treatment. Treating L fibroblasts with 1 µM thapsigargin for 30 min increases the levels of active c-Src as assessed using an anti-Src Tyr(P)418 antibody, whereas the levels of total c-Src remain unchanged. Actin was used as a loading control. B, cytosolic calcium [Ca2+]c measurements of L fibroblasts before and after a 12-h radicicol treatment (5 µM). Radicicol treatment causes an increase in [Ca2+]c in all cell lines.

 
On a preformed fibronectin matrix, all cell lines were induced to spread and to form stress fibers and many focal contacts. This cell area increase was particularly dramatic in calreticulin-underexpressing cells, which formed many vinculin-positive focal contacts. Induction of focal contacts could further facilitate the increase of fibronectin deposition and matrix remodeling in L fibroblasts plated on fibronectin, since focal contacts play a role in fibronectin fibrillogenesis (8, 53, 54). Focal contacts have been subdivided into "classical" focal contacts and "fibrillar adhesions" that coexist in dynamic and interchangeable states (55). Both classical focal contacts and fibrillar adhesions take part in fibronectin matrix remodeling; however, under the conditions in the present study, they would be hardly distinguishable, especially in the calreticulin overexpressers with low Src activity, since focal contacts and fibrillar adhesions are thought to be segregated by Src (56).

Tyrosine phosphorylation of focal contact components has also been shown to be an important regulator of focal contacts (17-20). In fact, the majority of a cell's phosphotyrosine detectable by immunofluorescence is in focal contacts (57). What is the mechanism by which calreticulin affects tyrosine phosphorylation in L fibroblasts? Calreticulin-underexpressing cells exhibited the highest levels of c-Src Tyr(P)418, indicative of catalytically active c-Src, as well as the lowest levels of active FAK. This is in agreement with previous work that has shown that there is greater recruitment of FAK into focal contacts in cells with no Src activity (56), as is the case here with the calreticulin overexpressers. Furthermore, previous studies have shown that c-Src is an important modulator of focal contact disassembly and turnover (14, 56, 58). The transformation of cells by v-Src causes cell rounding, increased cell migration, and invasiveness (14, 59). Maher (51) showed that the disruption of cell-substrate adhesion led to increased c-Src activity, and increased c-Src activity also correlates with focal adhesion disassembly and cell rounding (60-65). Finally, studies with Src-/- and Src/Fyn/Yes-/- cells showed that these cells exhibit an earlier onset of focal contact formation and an increase in the size of focal contacts (56). Consistent with these studies, we showed here that the calreticulin underexpressers, with the greatest c-Src activity, had a round, poorly adhesive phenotype compared with their calreticulin-overexpressing counterparts, which were well spread and highly adhesive and showed very little c-Src activity. Importantly, total cellular levels of c-Src were the same in all cell lines, indicating that calreticulin does not play a role in c-Src gene regulation. Plating cells on a preformed fibronectin matrix rescued the poorly adhesive phenotype of the calreticulin underexpressers and caused a decrease in c-Src activity as well as FAK activity. A preformed fibronectin matrix, then, may serve as a functional FAK deletion, since FAK-null cells have been shown to exhibit an increased number of cell-substratum contacts (66).


Figure 14
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  		FIGURE 14.
A simplified diagram showing the influence of calreticulin on Ca2+ release from intracellular stores, which then affects focal contact formation and fibronectin synthesis/deposition via Src. BK, bradykinin; BKR, bradykinin receptor; CRT, calreticulin; FN, fibronectin; IP3R, inositol 1,4,5-trisphosphate; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase.

 
Phosphorylation of integrins in adhesion complexes deactivates them, decreasing their interactions with the ECM (67-70), which could lead to cell rounding (51). Such cell rounding was observed here in the calreticulin underexpressers, and the high c-Src activity in these cells may be causing integrin phosphorylation and deactivation, thus accounting for the poor clustering of integrins in this cell line. Maher (51) further showed that reattachment of cells to the ECM leads to the inactivation of c-Src, which reactivates integrins and induces the reassembly of focal contacts. This is consistent with our data, which showed that attachment of L fibroblasts to a preformed fibronectin carpet decreased c-Src activity. Furthermore, c-Src inhibition caused greater deposition of fibronectin into the matrix, and since intracellular fibronectin levels remained unchanged, this indicates that there was accelerated secretion of fibronectin into the matrix, that the cells were more efficiently pulling fibronectin from the culture medium into a matrix, or both. Support for the first model comes from studies with v-Src, which show that the fibronectin gene is repressed in v-Src transformed cells (71, 72) and that this regulation was indirect (73).

Babnigg et al. (74) has previously shown that c-Src regulates the entry of Ca2+ through the plasma membrane via store-operated calcium channels, and it has been modeled that Ca2+ entry through the plasma membrane is a necessary part of normal physiological Ca2+ buffering in cells (75). c-Src, then, is a main connector molecule in the regulation of both focal contacts and Ca2+ homeostasis, two roles that have also been attributed to calreticulin. We therefore hypothesize that c-Src lies downstream of the signaling pathway originating in the lumen of the ER with calreticulin, which via a Ca2+-sensitive mechanism regulates cell-substratum adhesion. However, c-Src (or lack of c-Src activity) is then able to signal back to the ER, since radicicol treatment was able to invoke increased Ca2+ release from the ER. Our novel finding, then, that lowering of [Ca2+]ER increases c-Src activity is exciting and has potential for delineating the pathway by which an ER luminal protein can modulate cell-adhesive structures near the plasma membrane (Fig. 14). Such an ER-to-focal contact regulation has important implications, which would make calreticulin a regulatory molecule during development as well as in cancer. Indeed, previous studies have shown that c-Src exhibits elevated activity in a variety of human cancers (76). By controlling c-Src activity, calreticulin expression could impede the invasiveness of many types of tumors, since c-Src is a critical component of many signaling pathways regulating cell survival and metastasis.


   FOOTNOTES
 
* This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundation of Ontario (to M. O.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1. Back

1 Recipient of a Canada Graduate Scholarship from the CIHR. Back

2 Member of the Heart and Stroke/Richard Lewar Centre of Excellence. To whom correspondence should be addressed: Dept. of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Medical Sciences Bldg., Rm.6326 Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-8947; Fax: 416-978-5959; E-mail: m.opas{at}utoronto.ca.

3 The abbreviations used are: ER, endoplasmic reticulum; [Ca2+]ER, concentration of Ca2+ within the endoplasmic reticulum; [Ca2+]c, concentration of calcium in the cytosol; ECM, extracellular matrix; FAK, focal adhesion kinase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid. Back

4 S. Papp, M. P. Fadel, and M. Opas, unpublished data. Back


   ACKNOWLEDGMENTS
 
L fibroblasts differentially expressing calreticulin as well as the antibody to calreticulin were generously provided by Dr. Michalak. We also thank Dr. Bosco Chan for providing anti-integrin antibodies, Ewa Dziak for excellent help and advice, Dr. Cécile Gauthier-Rouvière for helpful discussion, and Dr. Wolfgang Vogel for valuable comments and critical reading of the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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