Polyamine depletion alters the relationship of F-actin, G-actin, and thymosin beta 4 in migrating IEC-6 cells

Shirley A. McCormack, Ramesh M. Ray, Patrick M. Blanner, and Leonard R. Johnson

Department of Physiology and Biophysics, College of Medicine, University of Tennessee, Memphis, Tennessee 38163


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The cause of reduced migration ability in polyamine-deficient cells is not known, but their actin cytoskeleton is clearly abnormal. We depleted polyamines with alpha -difluoromethylornithine (DFMO) in migrating cells with or without stimulation by epidermal growth factor (EGF) and investigated filamentous (F-) actin, monomeric (G-) actin, and thymosin beta 4 (Tbeta 4), using immunofluorescent confocal microscopy, DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cell interior, increased it in the cell cortex, redistributed G-actin, and increased nuclear staining of Tbeta 4. However, DFMO did not affect the amount of Tbeta 4 mRNA. EGF caused a rapid increase in the staining of F-actin in control cells, but DFMO prevented this response to EGF. Despite the visible changes shown by immunocytochemistry, statistically significant changes in the amount of either actin isoform or of total actin did not occur. We propose that DFMO reduces migration by interfering with the sequestration of G-actin by Tbeta 4 and the association of F-actin with activated EGF receptors.

epidermal growth factor; alpha -difluoromethylornithine; thymosin beta 4 messenger ribonucleic acid; monomer sequestration


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

THE ACTIN CYTOSKELETON IS the structural basis of the changes in cell shape required for migration and proliferation in animal cells (27). These processes require rapid cytoskeletal reorganization that is accomplished by a cycle of polymerization and depolymerization of actin monomers (G-actin) and filaments (F-actin) and their reorganization within the cell. The reorganization is carried out by an array of actin binding proteins in response to extracellular and intracellular signals. Epithelial cells typically form broad lamellipodia when migrating. These structures are protrusions of the cell membrane over dense, short actin filaments arranged with their rapidly polymerizing barbed ends toward the cell membrane. The assembly of new attachment sites on the extended edge of lamellipodia provides a purchase on which the cell contents can move forward propelled by force exerted through actin filaments. [For a commentary on this subject, see Heidemann and Buxbaum (13).] Actin filaments are produced by a combination of monomer sequestration, nucleation, filament severing, and the uncapping of filament ends by specific actin binding proteins (15, 20).

Studies with alpha -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (the rate-limiting enzyme of polyamine biosynthesis), have shown that the polyamines must also be considered in these processes. Polyamines are essential for cell proliferation (36), attachment (32), efficient migration in culture (18), and healing in vivo (38). In IEC-6 cells, a normal rat intestinal crypt cell (28), polyamine deficiency lowers epidermal growth factor (EGF) receptor phosphorylation, changes its distribution within the cell, and inhibits proliferation and migration (17). Inhibitors of polyamine biosynthesis prevent the accumulation of mRNAs that encode major cytoskeletal components in mouse splenocytes (16). Despite these effects, DFMO does not reduce total protein (17) and is not toxic to cells even at a concentration of 10 mM (unpublished data).

The distribution of F-actin has been described by many investigators. However, the distribution of G-actin has received less attention. In cultured cells, most of the G-actin is sequestered by monomer binding proteins (22). Punctate structures thought to represent transient storage of G-actin have been described in the region behind the lamellipodia (4) and in a ring around the nucleus (11). Extracellular ATP has been shown to induce nuclear accumulation of G-actin (19).

Actin binding proteins regulate the polymerization of actin by severing and capping actin filaments and by sequestering actin monomers. Sequestering monomers keeps the monomer level above the critical concentration for actin filament assembly and provides a reserve that can be rapidly mobilized where needed for polymerization when cell migration is stimulated. The beta -thymosins, a family of highly conserved 4.9-kDa polypeptides, comprise the bulk of the actin monomer-sequestering capacity in nonmuscle cells (22). Thymosin beta 4 (Tbeta 4) is the major monomer binding protein in most nonmotile cells and is present at concentrations near 1 × 10-5 M (25). When bound to actin, Tbeta 4 strongly inhibits nucleotide exchange by blocking its dissociation (8) and may alter the rate or location of polymerization through its effect on the actin monomer supply. At the low levels (<20 µM) found in most nonmotile cells, Tbeta 4 sequesters actin monomers at a ratio of 1:1. At high levels such as those present in circulating cells (>200 µM), the Tbeta 4-actin complex can incorporate into the actin filament, reducing the sequestering ability of Tbeta 4 (5). Whether the Tbeta 4-actin complex may reach high levels in particular intracellular areas in nonmotile cells is not known. Other actin monomer binding proteins, profilin in particular, can oppose the action of Tbeta 4, thereby providing a regulating step in the cycle of polymerization and depolymerization (8). Tbeta 4 gene expression has been demonstrated in various cells in which differentiation is occurring: mouse embryonic stem cells, neural and cardiovascular cells (10), embryonic brain tissue (6), and NIH/3T3 cells (41).

Previously, we have found significant inhibition of migration and other responses to EGF in polyamine-deficient IEC-6 cells. These changes were accompanied by marked alterations in F-actin and EGF receptor distribution (17). In the present investigation, we show that cytoskeletal alterations also involve G-actin and Tbeta 4. We propose that these alterations interfere with the regulation of actin polymerization and are responsible, at least in part, for the lowered migration ability of these cells and their failure to respond normally to a stimulus by EGF.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Cell culture and general experimental plan. The IEC-6 cell line (CRL-1592) was purchased from the American Type Culture Collection (ATCC; Manassas, VA). Medium and other cell culture reagents were obtained from GIBCO (Grand Island, NY). Fetal bovine serum (FBS), dialyzed FBS (dFBS), and all other chemicals and biochemicals were obtained from Sigma (St. Louis, MO), except as noted. The cell stock was maintained in DMEM containing 5% heat-inactivated FBS, 10 µg insulin, and 50 µg/ml gentamicin sulfate (DMEM-FBS) in 90% air-10% CO2. Stock was split once a week at 1:30 and used for no more than four passages. Cells were tested for mycoplasma every 6 mo.

Treatment began with plating and lasted for 4 days. Cells were fed on day 2 and serum was removed on day 3. Treatment groups included controls (DMEM-dFBS), DFMO (DMEM-dFBS plus 5 mM DFMO), and DFMO-putrescine (DMEM-dFBS plus 5 mM DFMO supplemented with 10 µM putrescine). DFMO was kindly provided by the Merrell Dow Research Institute of Marion Merrell Dow (Cincinnati, OH). On day 4, cells were removed from about one-third of the monolayer with a razor blade, the medium was changed to remove cellular debris, and the cells were allowed to migrate for 3 h. EGF (Collaborative Research, Bedford, MA) was added in fresh medium at a concentration of 10 ng/ml 2 and 30 min before the end of the 3-h period. Control medium was changed as well. We have shown previously that 5 mM DFMO reduces intracellular putrescine to undetectable levels in 6 h, spermidine to undetectable levels by day 2, and spermine to 30% by day 4 and that 10 µM putrescine is the optimal dose to maintain DFMO-inhibited cell migration and proliferation at control levels (18).

Immunocytochemistry. Cells were plated at 1.2 × 104 cells/cm2 on Matrigel-coated coverslips, treated, wounded, and allowed to migrate for 3 h as above. Matrigel was purchased from Collaborative Research. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, stained for G-actin with Gc globulin (Calbiochem, La Jolla, CA), anti-Gc globulin (Dako, Carpenteria, CA), and donkey anti-rabbit fluorescein-conjugated IgG (Jackson ImmunoResearch, West Grove, PA) for 1 h each, and finally stained for F-actin with rhodamine phalloidin (Molecular Probes, Eugene, OR) for 45 min, mounted, and sealed.

Tbeta 4 was identified by means of a rabbit polyclonal antibody generously provided by Dr. Gregory Evangelatos, head of the Radioimmunochemistry Laboratory of the National Centre for Scientific Research "Demokritos" (Athens, Greece). The second antibody was donkey anti-rabbit FITC-conjugated IgG (Jackson ImmunoResearch).

The selected images were chosen from a confluent area of the migrating edge, which was examined in its entirety. The cells were selected for similar size and density between treatment groups and to show characteristics of the majority of cells present. Slides were in duplicate, and each experiment included all treatments and was repeated three or more times. Identifying numbers on the slides bore no resemblance to labels assigned to the 35-mm dishes containing the coverslips. Treatments were matched later with the finished images. Images were captured by confocal laser scanning microscopy using Bio-Rad MRC-1024 Laser Sharp. The images for F- and G-actin were obtained sequentially by z-series and for Tbeta 4 by z-series only and processed by Adobe Photoshop.

F- and G-actin assay. F- and G-actin were assayed by DNase inhibition according to Heacock and Bamburg (12) with minor modifications. In this assay, actin filaments are bound by activated myosin to prevent depolymerization, immediately separated from unpolymerized actin by centrifugation, extracted, and depolymerized (if polymerized) for quantitation by DNase inhibition. Briefly, a buffer containing 0.5 M KCl, 50 mM K2PO4, and rabbit muscle myosin at an estimated molar ratio to actin of 1:1 (pH 6.2) was added to the cells while on plates. The plates were placed on ice, and the cells were quickly lysed in a buffer consisting of 10 mM Tris, 2 mM MgCl2, 0.2 mM dithioerythritol (DTE), 15% glycerol, and 1.0% Triton X-100 (pH 7.6). An aliquot was removed for the determination of total protein and total actin. The lysate was immediately centrifuged at 12,000 g for 1 min, and the resulting pellet (F-actin) was depolymerized to G-actin in actomyosin extraction buffer containing (in mM) 2 Tris, 0.2 CaCl2, 1.0 ATP, and 0.5 DTE (pH 8.0). The depolymerized pellet (originally the F-actin fraction) and the supernatant (G-actin) were then assayed by DNase inhibition. Fifty-microliter aliquots from each fraction were assayed for protein by the Bradford assay (2). Total actin from the original lysate was assayed in a guanidinium chloride buffer containing 1.5 M guanidinium chloride, 1 M Na acetate, 20 mM Tris, 7 mM CaCl2, and 1 mM ATP (pH 7.5).

Standards were prepared from rabbit muscle actin at a concentration of 1 mg/ml in actin monomer buffer consisting of (in mM) 2 Tris, 0.1 ATP, and 0.2 DTE (pH 7.6). Standard curves for each fraction were run in the appropriate buffer for that fraction. Calf thymus DNA was prepared at a 0.1 mg/ml concentration in DNase buffer consisting of (in mM) 125 Tris, 5 MgCl2, 2 CaCl2, and 3 NaN3 (pH 7.5). DNase 1 from bovine pancreas was used at a concentration of 0.1 mg/ml in DNase buffer. To carry out the reaction, 900 µl of DNA, 20 µl of DNase, and 100 µl of sample or standard plus buffer were added to a cuvette, mixed, and immediately read every 10 s for 90 s. Sample values were calculated from standard curves and expressed as nanograms of actin per microgram protein. The entire experiment was carried out a total of 10 times, but not all samples in each experiment could be used, resulting in individual sample replication of between 4 and 9.

Immunoblotting analysis. The cells were plated in 60-mm dishes at 6.25 × 104 cells/cm2 in duplicate and treated as described above. On day 4, 10 ng EGF/ml medium were added in fresh medium (also containing the treatments) for 2 and 30 min. The cells were extracted and analyzed for total protein by the Bradford method (2). Total protein (50 µg) was separated by electrophoresis on 15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes for Western blotting. Equal loading of protein samples was confirmed by Ponceau S staining of the membrane. The actin band was identified with rabbit polyclonal antiserum (Sigma, St. Louis, MO). A peroxidase-labeled secondary antibody was used for visualization (Sigma) with purified actin as the standard. The experiment was repeated five times.

RNA isolation and Northern blot analysis. Total RNA was extracted with guanidinium isothiocyanate solution and purified by CsCl density gradient ultracentrifugation. The resulting RNA pellet was dissolved in 10 mM Tris · HCl (pH 7.4) containing 5 mM EDTA and 1% SDS.

The purified RNA was precipitated from the aqueous phase with 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol in sequence and dissolved in water. Total RNA (30 µg) was denatured and fractionated electrophoretically on a 1.2% agarose gel containing 3% formaldehyde and transferred by blotting to a nitrocellulose membrane. The blot was prehybridized for 24 h at 42°C with 5× Denhardt's solution, 5× standard saline citrate (SSC), 50% formamide, 25 mM potassium phosphate, and 50 µg/ml denatured salmon sperm DNA. Hybridization was carried out overnight at 42°C in the same solution containing 10% dextran sulfate and DNA probes for Tbeta 4 and alpha -actin that were labeled with a [alpha -32P]dCTP by a standard nick translation procedure. Blots were washed once with 1× SSC-0.1% SDS at room temperature for 10 min, followed by two changes of 0.25× SSC-0.1% SDS, first at 42°C for 25 min and then at room temperature for 10 min. After the final wash, the membrane was autoradiographed with intensifying screens at -80°C. Loading of RNA was monitored by hybridization to the labeled actin probe. The experiment was repeated twice. Murine Tbeta 4 cDNA was a gift from Dr. Grazyna Bozek (Dept. of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL). Alpha actin cDNA was obtained from ATCC.

Statistics. Results were analyzed by ANOVA and unpaired, two-tailed Student's t-test or by nonparametric tests when SD varied widely. Differences were considered significant when P <=  0.05.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

F- and G-actin distribution. The effects of polyamine depletion on the cytoskeletal response to EGF are shown in Figs. 1-3, in which column 1 shows merged images of F-actin (red) and G-actin (green). Cells show areas of yellow where the two coincide. Columns 2 and 3 of Figs. 1-3 show F- and G-actin, respectively. Figures 1-3 depict control cells (A), DFMO-treated cells (B), and DFMO-putrescine-treated cells (C).


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Fig. 1.   Cells were untreated (control) or treated with 5 mM alpha -difluoromethylornithine (DFMO) or with 5 mM DFMO and 10 µM putrescine. Serum was removed on day 3, and cells were wounded 24 h later and allowed to migrate for 3 h. A: control. B: DFMO. C: DFMO-putrescine. Column 1 shows F-actin (red) and G-actin (green) in merged confocal images. Column 2, F-actin, and column 3, G-actin, are separated images of same cells. Representative images from 3 or more experiments. Scale bar, 22 µm.


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Fig. 2.   Treatment, staining, and labeling as in Fig. 1 except 10 ng epidermal growth factor (EGF)/ml medium was added 2 min before end of migration period. A: control. B: DFMO. C: DFMO-putrescine. Column 1 shows F-actin (red) and G-actin (green) in merged confocal images. Column 2, F-actin, and column 3, G-actin, are separated images of same cells. Scale bar, 22 µm.


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Fig. 3.   Treatment, staining, and labeling as in Fig. 1 except EGF was added 30 min before end of migration period. A: control. B: DFMO. C: DFMO-putrescine. Column 1 shows F-actin (red) and G-actin (green) in merged confocal images. Column 2, F-actin, and column 3, G-actin, are separated images of same cells. Scale bar, 22 µm.

In control cells (Fig. 1A), F-actin (column 2) was present in abundant long filaments throughout the cytoplasm, in a strong band on the migrating edge of the cells, and in lamellipodia. G-actin (Fig. 1A, column 3) was distributed diffusely throughout the cytoplasm as well as being concentrated in a wide area at the migrating edge and at the extreme outer edge of lamellipodia. In DFMO-treated cells (Fig. 1B), F-actin (column 2) formed a heavy cortex on the migrating edge and tended to be more pronounced between the cells. Interior stress fibers were dramatically decreased, and lamellipodia were absent. G-actin (Fig. 1B, column 3) was also concentrated on the migrating edge. F- and G-actin distribution in DFMO-putrescine-treated cells (Fig. 1C) resembled that in control cells, although G-actin was less well concentrated on the migrating edge.

Two minutes after EGF was added (Fig. 2A), the density of F-actin filaments (column 2) increased markedly, and prominent attachment areas appeared along the migrating edge. G-actin (Fig. 2A, column 3) was still diffuse but concentrated in a narrow band at the migrating edge. In DFMO-treated cells (Fig. 2B), the cells were slightly separated. F-actin (Fig. 2B, column 2) formed a heavy cortex and strong filaments connected cells. Interior F-actin filaments were few. G-actin staining (Fig. 2B, column 3) decreased at the migrating edge and appeared disorganized and aggregated in cells throughout the monolayer. F- and G-actin distribution in DFMO-putrescine-treated cells (Fig. 2C) resembled that in the controls.

Thirty minutes after EGF (Fig. 3A), F-actin (column 2) and G-actin (column 3) had begun to return to their original appearance. However, an increased number of actin filaments were still visible. In DFMO-treated cells, F-actin (Fig. 3B, column 2) formed a heavy cortex, interior filaments were few, and cells were further separated. G-actin staining (Fig. 3B, column 3) ringed the nucleus in some of the inner cells and had lessened on the migrating edge. F- and G-actin distribution in DFMO-putrescine-treated cells (Fig. 3C) was concentrated on the migrating edge, as in the corresponding controls. G-actin had a slightly spongy appearance in all three treatment groups.

To summarize the effect of DFMO treatment on the response of migrating cells to EGF, it appears that depleting the polyamines by interfering with polyamine synthesis caused actin polymerization to occur primarily at the cell cortex. Supplementation with putrescine corrected the defect.

F- and G-actin concentration. In DFMO-treated cells, F-actin concentrations tended to be higher than in control cells or those supplemented with putrescine (Fig. 4). This was the result of sporadic high values in all 10 experiments, causing a variability that prevented statistical significance. Similar variation did not occur in the concomitant control and DFMO-putrescine-treated groups. F-actin accounted for ~65% of the total actin in the control and putrescine-supplemented groups and for 80% in DFMO-treated groups. These values are within the range reported by Heacock and Bamburg (12) for Chinese hamster ovary (CHO) cells. EGF, whether at 2 or at 30 min, did not affect the concentration of F-actin in any of the three groups. The discrepancy between the assay results and the apparent increases shown by immunocytochemistry could be due to the loss of measurable filaments to depolymerization in the assay. However, this is unlikely since there were no concomitant increases in G-actin. Other possibilities are rapid spikes of polymerization only occasionally captured and increased visibility of previously invisible filaments.


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Fig. 4.   DNase assay of F-actin. Cells were untreated (control) or treated with 5 mM DFMO or with 5 mM DFMO and 10 µM putrescine (PUT) as in Fig. 1, except that 10 ng EGF/ml medium was added for 2 or 30 min before solubilization of monolayer, as indicated. Means ± SE for 4-9 replicates from 10 separate experiments are represented.

G-actin concentration was slightly, but not significantly, lowered in both DFMO-treated groups that received EGF for 2 and 30 min, compared with controls (Fig. 5). Otherwise, G-actin levels varied little from the controls in any group. G-actin accounted for ~30% of total actin in control and DFMO-putrescine-treated cells and for ~20% in DFMO-treated cells. The DFMO-treated cells exposed to EGF had ~16% of total actin as G-actin.


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Fig. 5.   DNase assay of G-actin. Treatments as in Fig. 4. Means ± SE for 4-9 replicates from 10 separate experiments are represented.

Assayed values of total actin for all samples (data not shown) were 83.1 ± 8.9% of the sum of G- and F-actin measured separately. That they were not 100% may be ascribed to differences in the intrinsic ultraviolet absorption of the three buffers required for solubilization of the different actin fractions, extraction efficiency from the pellet, and interfering factors in the whole homogenate needed for measuring total actin. Means ± SD for total actin in the various groups were as follows: controls, 13.4 ± 2.42 pg/cell; DFMO, 11.4 ± 2.24 pg/cell; and DFMO-putrescine, 11.3 ± 0.61 pg/cell. SD are from three experiments carried out in triplicate. The values are in approximate agreement with those reported by others (1, 12, 34) for other cell lines.

Western blot analysis of total actin showed that the changes in actin polymerization revealed by the DNase assay did not involve changes in overall actin concentration (Fig. 6). A strong band at 45 kDa resulted from 50 µg of protein taken from each treatment group. No differences between groups could be measured.


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Fig. 6.   Total actin by Western blotting. Treatments as in Fig. 1, with control (C), DFMO (D), and DFMO-putrescine (DP) groups and either no further additions or 10 ng EGF/ml medium added for 2 or 30 min, as indicated. Actin standard is indicated at right. A representative experiment of 5; 50 µg protein were applied to each lane.

Staining for Tbeta 4 (Fig. 7) was not uniform throughout the entire monolayer in any treatment group and may have reflected the cell cycle stage of small clones. In the majority of control cells (Fig. 7A, column 1), staining was light, punctate in appearance, primarily cytoplasmic, and close to or on the nuclear membrane. The nuclei were not stained. In most DFMO-treated cells (Fig. 7B), both cytoplasmic and nuclear staining of Tbeta 4 was present. Most of the staining was in the nuclei and on the nuclear membrane. After 2 min of EGF treatment (Fig. 7, column 2), staining increased markedly in the cytoplasm, nuclei, and nuclear membrane in many areas of control cells (Fig. 7A). In DFMO-treated cells (Fig. 7B), cytoplasmic staining increased in some cells, but nuclear staining still predominated. Thirty minutes after EGF (Fig. 7, column 3), Tbeta 4 staining had disappeared from nearly all areas in control cells (Fig. 7A). In DFMO-treated cells (Fig. 7B), Tbeta 4 remained unchanged. With or without EGF, Tbeta 4 in DFMO-putrescine-treated cells (Fig. 7C) resembled the controls.


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Fig. 7.   Staining for thymosin beta 4 (Tbeta 4). Cells were untreated (control; A) or treated with 5 mM DFMO (B) or with 5 mM DFMO and 10 µM putrescine (C). Serum was removed on day 3, and cells were wounded 24 h later and allowed to migrate for 3 h. Column 1, no EGF; column 2, 2 min of EGF; column 3, 30 min of EGF. A representative composite from 3 experiments. Scale bar, 20 µm.

Autoradiographs of RNA blots were quantitated by densitometric scanning and normalized with respect to the density of actin. The amounts of Tbeta 4 mRNA in control, DFMO, and DFMO-putrescine-treated cells were not different (Fig. 8).


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Fig. 8.   Tbeta 4 mRNA. Cells were untreated (control) or treated with 5 mM DFMO or with 5 mM DFMO-10 µM putrescine and serum starved on day 3. Total RNA was extracted 24 h later, and Northern blots were prepared as described in MATERIALS AND METHODS. Autoradiographs of RNA blots were quantitated by densitometric scanning and monitored by hybridization to labeled actin probe. Experiment was repeated twice with similar results.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Structural and functional changes occur in IEC-6 cells in which polyamine biosynthesis has been interrupted. Cell shape changes and striking rearrangements of filamentous actin follow DFMO treatment, and cell migration into a denuded area is reduced by 70-80% (18). We have previously shown that polyamine-depleted cells also have an abnormal distribution of the EGF receptor and fail to respond to an EGF stimulus for growth and migration (17). All of these changes can be prevented by providing the DFMO-treated cells with a polyamine supplement. To gain an understanding of the mechanisms involved in the disruption of migration, we investigated the distribution and quantity of F-actin and G-actin and the localization of Tbeta 4, a major actin-monomer binding protein, in migrating polyamine-deficient cells after stimulation with EGF. We also investigated the effect of DFMO on the mRNA of Tbeta 4.

Confocal images of the confluent cell monolayer 3 h after wounding show that both F- and G-actin were concentrated on the migrating margin of control cells. Tbeta 4 was distributed at low levels around the nucleus. In DFMO-treated cells, interior actin stress fibers were greatly reduced, whereas F-actin in the cell cortex was increased. This pattern of F-actin distribution has been noted by us previously and earlier by Pohjanpelto et al. (26) in a polyamine-deficient CHO cell variant. Others have made the same observation under other experimental conditions. Several possibilities have been proposed to explain the abnormally dense cortex, namely, that peripheral actin filaments are more resistant to depolymerization because they are composed primarily of beta -actin, whereas inner stress fibers consist of more easily depolymerized beta - and gamma -actin (14, 24). Yu and co-workers (40) have suggested that there is less Tbeta 4 in the cell cortex to sequester actin monomers (although this has not been demonstrated) and also that cortical actin is protected by other actin binding proteins. In transfected cells that overexpress Tbeta 4, central F-actin disappears and peripheral actin is retained (35, 40). Adhesion plaques and cytoskeletal proteins increase (9). We found nuclear staining of Tbeta 4 in DFMO-treated cells but not in control cells unless they were treated with EGF. Nuclear staining of Tbeta 4 has been attributed to diffusion from the cytoplasm (39) or to increased cell thickness over the nucleus (40). Because Northern blots did not show an increase in total Tbeta 4 mRNA after DFMO, we believe the increased nuclear staining of Tbeta 4 is more likely to be due to its translocation from the cytoplasm than to an actual increase in quantity.

After 2 min of EGF treatment, control and DFMO-putrescine-supplemented cells showed a striking increase in the staining of F-actin and Tbeta 4 throughout the cell. In DFMO-treated cells, any increase in F-actin occurred mainly in the cortex. Similar examples of rapid actin polymerization have been reported in polymorphonuclear leukocytes exposed to a chemoattractant (3) and in A431 cells after EGF treatment (29). In A431 cells, newly assembled actin filaments localize and bind selectively to the tyrosine-phosphorylated EGF receptor in the plasma membrane (30), where they provide force to advance the lamellipodia (21). In view of the fact that DFMO reduces tyrosine phosphorylation of the EGF receptor in IEC-6 cells (17), the association of newly polymerized actin filaments with the EGF receptor at the membrane may be reduced as well. Decreased phosphotyrosine content in several cellular substrates has been reported in DFMO-treated thymoma cells also (23). Thirty minutes after EGF, the early changes in actin and Tbeta 4 in control and DFMO-putrescine-supplemented cells were returning to their pretreatment condition. The DFMO-treated cells, however, showed little evidence of change.

Changes in F- and G-actin quantity 2 min after EGF were suggestive but not statistically significant in spite of the visual evidence that suggested an increase in F-actin and a decrease in G-actin. Although it is possible for F-actin to be underestimated due to depolymerization in actin assays that depend on centrifugation alone (35), the Heacock and Bamburg (12) DNase assay avoids depolymerization by complexing F-actin with activated myosin at lysis. Visibly, the perceived density of actin filaments may have been enhanced by reinforcement, bundling, and cross-linking of preexisting very fine filaments by actin binding proteins that made the filaments increasingly visible. Decreased F-actin in the cell interior may interfere with the normal transport of monomers and mRNA and contribute to increased cortical actin and changes in the cellular distribution of Tbeta 4. Western blot analysis showed no changes in total actin caused by either DFMO or EGF, and the assayed amount of total actin per cell was within the reported range of other cell lines (1, 12, 34).

Tbeta 4 binds and sequesters free actin monomers, simultaneously preventing premature actin polymerization and providing a reserve of monomers for new polymerization in areas of F-actin remodeling (22). In control and DFMO-putrescine-supplemented cells, Tbeta 4 responded rapidly to EGF by increasing from few stainable areas over the majority of the monolayer to many areas showing bright fluorescence in the nucleus and cytoplasm. Rapid induction of Tbeta 4 has been observed by others as well, namely, in thymocytes stimulated by conconavalin A (33) and in NIH/3T3 serum-starved cells stimulated by serum (41). Both investigators attributed the rapid increase in Tbeta 4 to translational control (33, 41). Zalvide et al. (41) found that Tbeta 4 mRNA had pronounced stability and that protein biosynthesis was not necessary for Tbeta 4 elevation in response to a stimulus. In the present study, we show that Tbeta 4 staining is primarily located in the nucleus in DFMO-treated cells and does not change significantly with EGF.

In summary, novel findings in our study are as follows. 1) In migrating control cells, stimulation by EGF caused the redistribution of F-actin to favor strong stress fibers, attachment sites at the migrating edge, and attenuation of G-actin without changes in the quantity of total actin. If the migrating cells were polyamine depleted, F-actin was redistributed primarily to the cell cortex, and G-actin became scattered and disorganized. 2) In migrating control cells, the extent and intensity of Tbeta 4 immunostaining increased markedly within 2 min of exposure to EGF and then returned to the original condition within 30 min. In migrating polyamine-depleted cells, immunostaining of Tbeta 4 was primarily located in the nucleus and was not significantly changed by EGF. The quantity of Tbeta 4 mRNA was not affected by DFMO. 3) Supplementation of polyamine-depleted cells with putrescine maintained both actin forms and Tbeta 4 in their normal patterns of localization and did not affect Tbeta 4 mRNA.

We hypothesize that two consequences of polyamine deficiency contribute to the reduced ability of polyamine-deficient cells to migrate. These are, first, aberrant actin monomer sequestration due to changes in Tbeta 4 intracellular distribution and, second, increased cortical actin polymerization impeding the localization of newly polymerized actin filaments on activated EGF receptors in the plasma membrane.

The link between polyamines and Tbeta 4 remains unclear. It is known that Tbeta 4 binds to monomeric actin through its amino-terminal residues in an alpha -helical conformation (7, 31, 37). It is tempting to speculate that the structure of the polyamines, namely a flexible carbon chain with positive charges distributed at fixed lengths, may assist Tbeta 4 in maintaining the alpha -helical conformation required for binding to G-actin. To our knowledge, this study is the first report that attempts to relate F-actin, G-actin, and Tbeta 4 to an underlying role for the polyamines in intestinal cell migration.


    ACKNOWLEDGEMENTS

We thank Danny Morse for graphic assistance and Easter Jenkins for final manuscript preparation. For the confocal images, we used the Confocal Laser Scanning Microscope Facility, University of Tennessee (Memphis, TN; National Institutes of Health Grant CLSM 1 S10-RR-08385, Dr. Andrea Elberger, principal investigator). We gratefully acknowledge the gifts of DFMO from Marion Merrell Dow (Cincinnati, OH), Tbeta 4 antibody from Dr. Gregory Evangelatos (Radioimmunochemistry Laboratory, National Centre for Scientific Research "Demokritos," Athens, Greece), and Tbeta 4 murine beta -4 cDNA from Dr. Grazyna Bozek (Dept. of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL).


    FOOTNOTES

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52784 (to L. R. Johnson).

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. §1734 solely to indicate this fact.

Address for reprint requests: S. A. McCormack, Dept. of Physiology and Biophysics, University of Tennessee, Memphis, 894 Union Ave., Memphis, TN 38163.

Received 20 July 1998; accepted in final form 12 November 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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