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Differences in Turnover Rates of Vinculin and Talin Caused by Viral Transformation and Cell Density
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
227, 352–359 (1996)
0284
Differences in Turnover Rates of Vinculin and Talin Caused by Viral Transformation and Cell Density SEUNG-WON LEE
AND
JOANN J. OTTO1
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392
Vinculin and talin are two major components of focal contacts which interact with each other. In order to understand how the relative levels of these proteins are maintained under various conditions, the synthesis rates and half-lives of vinculin and talin in chick embryonic fibroblasts were determined by autoradiography combined with immunoblotting. High cell density and transformation by Rous sarcoma virus decreased the vinculin synthesis rate by 40%. Upon viral transformation, the synthesis rate of talin decreased by 30%. In contrast to vinculin, the synthesis rate of talin was not affected by cell density. The effect of cell density on the synthesis rate of vinculin was retained after viral transformation, suggesting that cell density and viral transformation affect vinculin synthesis by two independent mechanisms. The synthesis rate of vinculin was approximately two to three times greater than that of talin under all conditions tested. The halflives of vinculin and talin remained constant at different cell densities in untransformed cells (t1/2 Å 18–21 h), but transformation slightly decreased half-lives of both proteins (t1/2 Å 16–18 h). These results suggest that the decreased expression of vinculin and talin in transformed chick fibroblasts can be attributed mainly to changes in their biosynthesis rates rather than degradation. This may contribute to a decrease in the number of focal contacts in transformed cells. q 1996 Academic Press, Inc.
INTRODUCTION
Stress fibers end at focal contacts on the ventral cell membrane. Vinculin and talin are two major elements present in focal contacts [1]. Vinculin (116 kDa) is a cytoplasmic protein widely distributed in actin-containing cell–cell junctions and cell–matrix focal adhesions [2]. In contrast, talin (225 kDa) is localized only in cell–matrix adhesions [1, 3, 4]. Vinculin and talin interact [5, 6] with moderate affinity (Kd 1008 M) [7]. Further, talin interacts with b1 integrin [8] and vin1 To whom reprint requests should be addressed at Department of Biological Sciences, Purdue University, West Lafayette, IN 479071392. Fax: (317) 494-0876. E-mail: [email protected].
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0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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culin interacts with a-actinin [6, 9, 10], tensin [11], and paxillin [12] in vitro. More recently, a direct interaction between vinculin and F-actin has been demonstrated [13]. Microinjection studies with anti-vinculin antibody [14] and anti-talin antibody [15] indicate that vinculin is a key protein in the microfilament-membrane linkage and that talin is essential for the development of focal contacts. Because vinculin and talin interact with several different focal contact components and interact with each other, they may be regulatory elements in the assembly, disassembly, and maintenance of focal contacts. Vinculin synthesis has been shown to be regulated under various circumstances and to correlate with the degree of focal contact formation. Vinculin synthesis is increased when 3T3 fibroblasts establish cell-to-cell contacts [16]. The expression of meta-vinculin, a vinculin variant restricted to muscle cells, differs in rabbit smooth muscle cell cultures seeded at different densities [17, 18]. Culture of cells on a highly adhesive matrix results in an increased synthesis of vinculin and an increased number of focal contacts in chick embryo fibroblasts [19]. Transient induction of vinculin gene expression occurs in 3T3 fibroblasts and regenerating liver cells stimulated by serum growth factors [20–22]. Vinculin expression is up-regulated in 3T3 adipose cells after treatment with growth hormone without a change in its phosphorylation level [23]. Migrating stratified squamous epithelia also express high levels of vinculin [24]. Vinculin expression is down-regulated in highly metastatic mouse-B16 melanoma cell lines [25]. A more direct correlation between vinculin expression and focal contact formation has been shown by suppression or overexpression of vinculin cDNA [26–28]. It seems clear that the expression level of vinculin is closely related to or even causal to the degree of cell motility and changes in shape resulting from changes in focal contacts. These results suggest that the formation of focal contacts may be regulated partly by the supply of their protein components. In contrast to vinculin, the expression of talin has not been well studied. Further, most of the studies on vinculin expression emphasize the synthesis rate, not the degradation rate. If intracellular protein levels of focal contact components are a determining factor for regulation of the number and
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size of focal contacts, the degradation rates of the proteins should also be examined. Transformation of chick embryo fibroblasts (CEF) with Rous sarcoma virus (RSV) results in the disruption of stress fibers and formation of filamentous actin aggregates containing vinculin, talin, a-actinin, and fimbrin on the ventral membrane [1]. Some normal focal contacts still exist but with reduced sizes and numbers compared to untransformed cells [29]. Hence, we were interested in examining the synthesis and degradation rates of vinculin and talin in untransformed and transformed CEF to elucidate whether the levels of expression of vinculin and talin are related to the transformation phenotype. In addition, because CEF at high density have fewer focal contacts, the effect of cell density on synthesis and degradation rates of both proteins was also investigated in both untransformed and transformed cells. EXPERIMENTAL PROCEDURES Materials. Unless otherwise indicated, all chemicals were reagent grade or were purchased from Sigma Chemical Co. (St. Louis, MO). The rabbit anti-chick vinculin and anti-chick talin antibodies used in this report were characterized previously [30, 31]. Cell culture. Primary CEF were prepared as previously described [32]. Viral-free chick embryo fibroblasts (from eggs obtained from SPAFAS) were infected with avian Rous sarcoma virus (RSV), type A, as previously described [33]. Both cell types (CEF and RSV-CEF) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal calf serum (Hyclone, Logan, UT), 0.125 g/liter penicillin G (1670 U/mg), and 0.5 g/liter streptomycin sulfate (736 U/mg). Transformation was confirmed by rhodamine-phalloidin staining which showed dot-like aggregations of actin filaments on the ventral side of the cells and by Northern blotting of total RNAs probed with a cDNA of v-src (a generous gift from Dr. Elizabeth J. Taparowsky, Purdue University). RSV-CEF showed typical alterations in filamentous actin as observed by others [34]. Sample preparation for biosynthesis rate determinations. Prior to labeling, cells in 5-cm plates were incubated in methionine-deficient modified Eagle’s medium (MEM, Sigma) for 15 min. To determine the relative biosynthesis rates of vinculin and talin, cells were labeled with 120 mCi [35S]methionine in 3 ml MEM/plate (sp act 1300 Ci/mM, Amersham, Arlington Heights, IL) with 5% fetal calf serum for 2 h. Labeled cells were lysed with modified RIPA containing 1.0% (w/v) Triton X-100, 0.2% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) sodium deoxycholate, 10 mM NaH2PO4 , 150 mM NaCl, 1 mM MgCl2 , 1 mM ethylene glycol-bis(b-aminoethyl ether)tetraacetic acid (EGTA), and 15 mM Tris–HCl, pH 7.4, with 1 mg/ml leupeptin, 1 mg/ml pepstatin A, and 2 mM phenylmethyl sulfonyl fluoride (PMSF). Protease inhibitors were purchased from Boehringer-Mannheim (Indianapolis, IN). Cells were lysed in 0.5 or 1.0 ml for low or high cell density plates, respectively, and scraped off the plates with a razor blade. Undissolved material was removed by a brief centrifugation (3 min at 15,000 rpm). The supernatant was quickly frozen by immersion in liquid nitrogen and kept in a freezer (0807C) until assayed. This supernatant will be called the sample. The pellet was assayed by immunoblotting to ensure that all the vinculin and talin were solubilized in the modified RIPA. In order to obtain the total biosynthesis rate, 100 ml of the samples were precipitated with trichloroacetic acid (TCA; final concentration, 10%) and the precipitates were washed two times with 10% TCA. The pellet was dissolved in 1 N NaOH in a final volume of 100 ml and its radioactivity was measured by liquid scintillation counting.
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The amount of protein in the resuspended pellet was determined with a Pierce Micro BCA assay kit (Pierce, Rockford, IL). Volumes with the same number of precipitable TCA counts were taken from each sample and quantitatively immunoprecipitated [35] with a mixture of anti-vinculin and anti-talin antibodies on ice for 1 h. The immune complex was absorbed with Protein-A bearing Staphylococcus aureus (Pansorbin, Calbiochem, San Diego, CA) on ice for 20 min. The immune complex was washed twice with modified RIPA containing 1 mM 2-mercaptoethanol and was resuspended in sample buffer and separated on 7% SDS gels [36]. The gels were blotted onto nitrocellulose and the nitrocellulose was processed as described below for autoradiography combined with immunoblotting. To compare the biosynthesis rates between vinculin and talin, cells in separate dishes were labeled for 30, 60, 90, 120, or 150 min with 120 mCi [35S]methionine. Cells were lysed after labeling as described above. The sample volumes were adjusted to contain the same amount of protein. Talin and vinculin were quantitatively immunoprecipitated and resolved on SDS–polyacrylamide gels as described above. Vinculin and talin bands visualized by Coomassie blue staining were cut out and solubilized in 0.4 ml 60% perchloric acid and 0.8 ml 30% H2O2 solution at 607C for 5 h for liquid scintillation analysis [37]. Scintillation cocktail (15 ml) (Universol, ICN, Irvine, CA) was added to each sample before counting. Sample preparation for degradation rate determinations. Cells were pulse labeled for 90 min with [35S]methionine (90 mCi/ml), washed with methionine-deficient MEM, and chased in DMEM containing 0.3 g/liter methionine and 5% fetal calf serum for 0, 4, 8, and 12 h. Cells were lysed as described above. Aliquots (20 ml) of the sample were taken to determine the protein amount. Volumes with the same total amount of protein were quantitatively immunoprecipitated with a mixture of anti-vinculin and anti-talin antibodies. The immunoprecipitates were processed as described below for autoradiography combined with immunoblotting. Autoradiography combined with immunoblotting. To determine the relative biosynthesis and degradation rates under various conditions, it was necessary to develop a method that allows for correct normalization of the degree of radiolabeling of vinculin or talin in each sample. We chose the protein amount of vinculin or talin which was detected by immunoblotting as a reference value to normalize the amount of radiolabeled vinculin or talin in each sample [38]. Proteins resolved in 7% SDS gels were semidry electroblotted onto nitrocellulose (1.14 mA/cm2 for 90 min). The transfer buffer was 48 mM Tris, 39 mM glycine, 0.1% SDS, 20% (v/v) methanol, pH 8.4. Nonspecific binding to nitrocellulose was prevented by a 30-min incubation at room temperature in blocking solution (0.25% gelatin, 3% bovine serum albumin, 150 mM NaCl, 15 mM Tris–HCl, pH 7.4). After blocking, the nitrocellulose was air-dried, and then the protein bands labeled with [35S]methionine were exposed by autoradiography without an intensifying screen at 0807C. To determine the amount of vinculin and talin, the same nitrocellulose membrane was incubated with a mixture of anti-vinculin and anti-talin antibodies in blocking solution for 4 h followed by two washes with Tris–saline (150 mM NaCl, 15 mM Tris, pH 7.4) containing 0.05% (w/v) Tween 20 and two washes with Tris–saline (15 min per wash). The nitrocellulose was incubated with 0.5 mCi/ml 125I-Protein A (sp act 54 mCi/mg, ICN, Costa Mesa, CA) in blocking solution for 2 h and extensively washed. After the nitrocellulose was dried, a blank, developed piece of film was put adjacent to the nitrocellulose in order to protect the film from b-particle emission from [35S]methionine and an autoradiogram was taken without an intensifying screen to detect the g-rays from 125I-Protein A. All the data were analyzed by laser densitometry (Pharmacia LKB, Piscataway, NJ). The values from the b-particle exposure were normalized by the relative protein amount as determined by g-ray exposure. The length of exposure was determined so that densities of autoradiograms fit in the linear range of protein amount versus the density.
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FIG. 1. Solubility and immunoprecipitation of vinculin and talin in modified RIPA. (A) A fluorogram of a SDS gel of RIPA-insoluble material. Cells were labeled with [35S]methionine and then solubilized in modified RIPA. The insoluble material was removed by centrifugation and separated by SDS–PAGE. Lane a, N-CEF; lane b, RSV-CEF. (B) Autoradiograph of an immunoblot of RIPA-insoluble material from non-radiolabeled cells. Cells were extracted as in A. The RIPA insoluble material was separated by SDS–PAGE and blotted onto nitrocellulose. The blot was incubated with a mixture of anti-vinculin and anti-talin antibodies followed by 125I-Protein A. Lane a, N-CEF; lane b, RSV-CEF. (C) Immunoblot of the supernatants after immunoprecipitation of vinculin and talin from N-CEF. The blot was incubated with a mixture of anti-vinculin and antitalin antibodies and then probed with peroxidase-conjugated Protein A. Lane a, vinculin marker; lane b, supernatant after immunoprecipitation with anti-vinculin antibody; lane c, supernatant after immunoprecipitation with anti-talin antibody; lane d, talin marker.
RESULTS
Relative Biosynthesis Rates of Vinculin and Talin The rate constant of synthesis conventionally has been defined as units or mass of a specific protein synthesized per unit time per weight of tissue [39]. For practical reasons when using cells in culture, the relative biosynthesis rate commonly has been used and is expressed as units or mass of a specific protein synthesized per unit of time per total protein. Using total biosynthesis rate or total protein synthesized for normalization can lead to underestimation of subtle changes in synthesis rates of minor cellular components. For this reason, in this study the amount of the protein of interest was used for normalization in determining the relative synthesis rates [38]. In order to determine the relative synthesis rates of vinculin and talin, cells were labeled with [35S]methionine for 2 h and then solubilized in modified RIPA. Prior to immunoprecipitation, the lysate was centrifuged to remove insoluble material (Fig. 1A). In order to be certain that all the vinculin and talin were being solubilized, the insoluble material was analyzed by immunoblotting for the presence of vinculin or talin (Fig. 1B). The pellet contained no detectable vinculin or talin. To ascertain whether we were quantitatively immunoprecipitating the vinculin and talin, the supernatants after immunoprecipitation were examined for the
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presence of vinculin and talin by immunoblotting (Fig. 1C). No detectable vinculin or talin was observed. Together, these observations demonstrate that we were examining the total population of vinculin and talin molecules rather than a subpopulation in the experiments described below. The relative biosynthesis rates were determined for vinculin and talin in CEF and RSV-CEF growing at different cell densities (Fig. 2). Cell lysates were quantitatively immunoprecipitated with a mixture of antitalin and anti-vinculin antibodies. The immune complexes were blotted onto nitrocellulose after separation by SDS gel electrophoresis. The nitrocellulose was autoradiographed and the relative amounts of [35S]methionine incorporated into vinculin and talin were determined by laser densitometry (Fig. 2A). This density value was divided by the total protein amount (nonlabeled plus labeled) of vinculin or talin as detected by immunoblotting for normalization. The total vinculin or talin protein amount was determined by probing the same piece of nitrocellulose with anti-vinculin and anti-talin antibodies followed by 125I-Protein A (Fig. 2B). Such normalized densities of [35S]methionine incorporation (Fig. 2C) represent the relative biosynthesis rate of each protein. In virally transformed cells, the synthesis rates of vinculin and talin were decreased (Fig. 2). In both normal and transformed cells grown at high cell density, the synthesis rate of vinculin was decreased, but the synthesis rate of talin was not affected (Fig. 2C). Because vinculin and talin have different transfer rates to nitrocellulose and are probed with different antibodies, the normalized values cannot be compared directly between the two proteins. We wanted to be able to compare the rates for synthesis of vinculin to that of talin under the different conditions. In order to do this, we needed to be able to convert the relative biosynthesis rates to the relative number of molecules or moles of each protein that was synthesized per unit time. We used a more conventional approach with a liquid scintillation method combined with calculations based on the relative methionine composition of the proteins as described below. Cells were continuously labeled with [35S]methionine and samples containing the same amount of total protein were quantitatively immunoprecipitated at different time points with a mixture of anti-vinculin and anti-talin antibodies. After separation on SDS gels, vinculin and talin were visualized by Coomassie blue staining, the bands containing them were cut out, and the gel pieces were subjected to liquid scintillation counting. This experiment was done only with cells at high cell density in order to obtain enough protein quantity to visualize with Coomassie blue staining. Figure 3 shows that there was a linear increase in vinculin and talin synthesized in both cell types. The slope of each line indicates the initial velocity of synthesis
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FIG. 2. Determination of the relative biosynthesis rates of vinculin and talin in different cell types by autoradiography combined with immunoblotting. Normal CEF and RSV-CEF were plated at two different cell densities (3 1 105, 3 1 106 cells/plate) 12 h prior to the experiment. Cells were labeled for 2 h with [35S]methionine and the cell lysates were immunoprecipitated with both anti-talin and antivinculin antibodies. The immunocomplexes were resolved on a 7% SDS gel and transferred to nitrocellulose (see Materials and Methods). (A) A representative autoradiogram of the nitrocellulose to detect the amount of [35S]methionine-labeled talin or vinculin. The relative density areas obtained by laser densitometry are shown below the autoradiogram. The high-molecular-weight band above talin is fibronectin, which nonspecifically binds to Pansorbin, which was used as the immunoabsorbant. (B) An autoradiogram of the same nitrocellulose which was immunoblotted with anti-talin and antivinculin antibodies and probed with 125I-Protein A. The numbers represent the relative amount of talin or vinculin determined by laser densitometry. (C) The values from Fig. 1A were divided with the corresponding values from Fig. 1B to yield the relative biosynthesis rate. The value for normal CEF at low cell density was used as the reference value for Table 1. NL, Normal, untransformed CEF at low cell density; NH, normal, untransformed CEF at high cell density; RL, RSV-CEF at low cell density; RH, RSV-CEF at high cell density; T, talin; V, vinculin.
expressed as the amount of incorporated radioactivity in vinculin or talin per unit of time per total protein amount as conventionally defined. Because vinculin and talin have different methionine compositions, the initial velocities of synthesis of the two proteins cannot be compared directly. In
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order to compare them, we calculated the ratio of methionine compositions of vinculin vs talin (0.64; 39 methionines per vinculin molecule and 61 methionines per talin molecule) based on published sequence data [40, 41]. The initial velocity of synthesis of talin (Fig. 3) in each cell type was then multiplied by 0.64 (for example, in normal CEF, 0.64 1 144.5 Å 92.5). Then, this transformed initial velocity of talin was used to determine the relative synthesis ratio between vinculin and talin in each cell type (for example, in normal CEF, 178/92.5 Å 1.9). From these data and calculations, we can estimate how many molecules of vinculin are synthesized during the synthesis of one talin molecule. By calculation, in normal CEF growing at high cell density, 1.9 vinculin molecules are synthesized per talin and, in RSV-CEF at high cell density, 1.6 vinculin molecules are synthesized per talin. With these conversion factors, the relative synthesis rates of vinculin and talin under different conditions (Fig. 2) were transformed to relative moles of vinculin and talin synthesized per unit time (Table 1). The value for vinculin in normal CEF at low cell density was set as the reference. The relative moles of talin synthesized at high cell density were obtained by dividing the relative synthesis rates of vinculin at high cell density with 1.9 for normal cells or 1.6 for transformed cells. Finally, the relative moles of talin synthesized at high cell density were used as the reference to obtain the relative moles of talin synthesized at low cell density. Such numerical transformation of five independent experiments is summarized in Table 1 which shows the relative moles of vinculin or talin synthesized per unit time.
FIG. 3. Continuous labeling of vinculin and talin to determine the initial velocities of their synthesis rates. The slope of each line is the initial velocity of synthesis. NHV (normal CEF, high cell density, vinculin; initial velocity is 178 cpm/min/mg); RHV (RSV-CEF, high cell density, vinculin; initial velocity is 97.6 cpm/min/mg); NHT (normal CEF, high cell density, talin; initial velocity is 144.5 cpm/min/ mg); RHT (RSV-CEF, high cell density, talin; initial velocity is 93.9 cpm/min/mg).
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TABLE 1 The Relative Moles of Vinculin and Talin Synthesizeda Cell densityb N-CEFe lcdg hcdh RSV-CEF f lcd hcd
Vinculin
Talin
Total biosynthesisc
2.5 1 105 3.0 1 106
1.00 (0.03)d 0.65 (0.04)
0.34 (0.04) 0.34 (0.02)
1.00 (0.00) 1.06 (0.02)
2.4 1 105 2.7 1 106
0.56 (0.01) 0.39 (0.04)
0.23 (0.03) 0.23 (0.02)
1.14 (0.08) 1.00 (0.01)
a
This table contains the average values of five independent experiments. All values are relative to the vinculin synthesis rate of normal CEF at low cell density. Therefore, the relative synthesis rates of vinculin and talin can be compared with each other, but not to the total biosynthesis rate. b The number of cells in a 5-cm plastic dish at the time of plating. c The total biosynthesis rates (cpm, counts per minute/mg protein) of each cell type were averaged and normalized to the value for normal, untransformed CEF (N-CEF) at low cell density (1.00 Å approximately 3.3 1 105 cpm/min/mg). The standard deviations (SD) of the total biosynthesis rates are noted in parentheses. d The synthesis rate was determined by the ratio of density areas from b-particle exposure to those from g-ray exposure to detect total vinculin or talin. The synthesis rates of each cell type were normalized to the value of normal CEF at low cell density for both vinculin and talin. SD of the synthesis rates are noted in parentheses. The unit is b-particle density area/g-ray density area/2 h which reflects the amount of newly synthesized vinculin or talin per time per total vinculin or talin. Since the transfer efficiency during the electroblotting varies between proteins of different sizes and since different antibodies were used to detect each protein, it was not possible to compare the density of vinculin with that of talin until we determined the synthesis rates of vinculin or talin by using the liquid scintillation method (Fig. 2). As described in the text, the initial velocity of synthesis (the slope of each line) for NHV, NHT, RHV, and RHT was determined, and the relative ratio between vinculin and talin was calculated (N-CEF Å 1.9, RSV-CEF Å 1.6; the numbers reflect the number of vinculin molecules synthesized per one talin molecule per unit time). These values were used to convert the relative synthesis rates of talin obtained by direct autoradiography combined with immunoblotting and made it possible to compare the relative synthesis rate of vinculin with talin. e Normal, untransformed chicken embryonic fibroblasts. f Rous sarcoma virus transformed-chicken embryonic fibroblasts. g Low cell density. h High cell density.
Half-Lives of Vinculin and Talin To determine the half-lives of vinculin and talin in both normal and RSV-transformed cells at different cell densities, pulse–chase experiments were done and the results were analyzed by the same method as is described for the determination of relative biosynthesis rate (autoradiography combined with immunoblotting). Figure 4 is a representative autoradiogram of a degradation experiment of CEF grown at high cell density. Vinculin and talin have similar half-lives at the two cell densities (t1/2 Å 18–21 h) while viral transformation slightly decreases their half-lives (t1/2 Å 16– 18 h) as shown in Table 2, which contains data from four independent experiments. DISCUSSION
The regulation of vinculin expression under various conditions has been well studied in a variety of cells [16–25]. However, a detailed understanding of talin expression has been lacking until now. Further, considering that the level of a particular protein in cells is controlled by both synthesis and degradation, information about vinculin and talin degradation is necessary to determine to what degree an altered synthesis rate will contribute to the pool of protein. Finally, because
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vinculin and talin interact and function in focal contacts, it is important to understand how cells coordinate the synthesis and degradation of both proteins. Experiments addressing these issues have not been feasible partly because of difficulties in normalization of biosynthesis rates and degradation rates of proteins of interest under different conditions. We believe that autoradiography, combined with immunoblotting [38], allows us to normalize protein synthesis and degradation rates with more precision and to compare the relative biosynthesis rates between vinculin and talin when combined with conventional methods. The synthesis rate of vinculin was decreased by viral transformation by 40% at both cell densities (Table 1). The synthesis rate of talin was also decreased by 30% in transformed cells but was not affected by cell density. Since the total biosynthesis rates of transformed cells were the same as normal cells, we suggest that these decreases are caused specifically by viral transformation. Chick fibroblasts and their RSV transformants at high cell density showed an approximately 40% decrease in their vinculin synthesis rates relative to low cell density. It is notable that cell density-dependent type regulation of the vinculin synthesis rate is preserved in RSV-transformed CEF even though RSV
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TABLE 2 The Half-Lives of Vinculin and Talina Cell densityb N-CEFd lcd f hcdg RSV-CEFe lcd hcd
Vinculin t1/2 (hr)
Talin t1/2 (hr)
3.1 1 105 3.8 1 106
19 (1)c 20 (2)
18 (2) 21 (1)
2.4 1 105 2.8 1 106
16 (2) 18 (2)
16 (1) 17 (2)
a
This table contains the average values of four independent experiments. The half-lives were obtained as described previously [39]. b The number of cells in a 5-cm plastic dish at the time of plating. c SD is in parentheses. d Normal chick embryonic fibroblasts. e Rous sarcoma virus-transformed chick embryonic fibroblasts. f Low cell density. g High cell density.
FIG. 4. Determination of half-lives of vinculin and talin in normal CEF grown at high cell density by autoradiography combined with immunoblotting. Cells (3.4 1 106 cells/plate) were pulse labeled for 90 min with [35S]methionine and then chased for 0, 4, 8, or 12 h. Each sample was immunoprecipitated with both anti-talin and antivinculin antibodies. The immunocomplex was resolved on a 7% SDS gel and transferred onto nitrocellulose (see Materials and Methods). (A) A representative autoradiogram of the nitrocellulose after the proteins were transferred shows the relative amount of [35S]methionine-labeled talin or vinculin. The numbers below the autoradiogram are the densitometric determinations of the amount of label incorporated into vinculin or talin. The high-molecular-weight band above talin is fibronectin, which nonspecifically binds to Pansorbin, which was used as the immunoabsorbant. (B) An autoradiogram of
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transformation decreases the vinculin synthesis rate by 40%. This suggests that there may be two independent mechanisms to regulate vinculin synthesis. At high cell density, both normal and transformed cells formed fewer cell-to-substrate contacts than at low cell density (data not shown). Thus, we suspect that the vinculin synthesis rate is regulated by the level of cellto-substrate interaction; that is, the availability of substrate which allows the formation of focal contacts may affect the supply of vinculin. Such a relationship between vinculin synthesis and substrate availability is well documented by Bendori et al. [19], who show that CEF grown on a highly adhesive matrix have increased vinculin expression and increased focal contact formation. In contrast to our results, increased vinculin synthesis is observed when 3T3 fibroblasts are grown at high cell density [16]. However, unlike CEF, 3T3 fibroblasts in culture grow as a monolayer and establish definitive cell-to-cell contacts at high cell density, and vinculin is localized in the cell-to-cell contact areas as well as focal contacts. Chick fibroblasts do not show any indication of vinculin localization in cell-to-cell contact areas at high cell density (data not shown). This difference in the localization of vinculin in the two cell types may account for the observed differences in synthesis rates relative to cell density. It is unlikely that the decrease in vinculin synthesis in cells at high cell den-
the same nitrocellulose which was immunoblotted with anti-talin and anti-vinculin antibodies and probed with 125I-Protein A. The numbers below the autoradiogram represent the relative total amount of talin or vinculin. (C) The values from A were normalized with the corresponding values from B. The graph is a plot of the logarithm of the normalized values (the amount of radioactivity remaining in the protein) vs time according to Doyle and Tweto (1975) [39]. Kd (rate constant of degradation; time01) of talin Å 03.31 1 1002 (t1/2 Å 21 h), r (linear regression) Å 0.95. Kd of vinculin Å 03.88 1 1002 (t1/2 Å 18 h), r Å 0.99.
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sity is due to a diminished general metabolic rate imposed by contact inhibition since the total biosynthesis rates and talin synthesis rates remain the same at high cell density in both normal and transformed cells. In contrast to vinculin, the talin synthesis rate did not show any change at different cell densities in either normal or RSV-transformed CEF. The biological implication of this remains unclear. Perhaps talin may participate in other functions in addition to cell–substrate adhesion or its protein level is not regulated by the availability of substrate. Even though talin is usually found in cell-to-matrix contacts where microfilaments are attached to membranes [4], talin is also recruited to the cell-to-cell contacts of effector cells upon activation of T-lymphocytes [42, 43]. This coincides with the reorganization of the microtubule organizing center and Golgi complex and eventually results in unidirectional killing of the target cells [42, 43]. This suggests that cell–matrix adhesion may not be the sole role of talin in vivo and raises the possibility that talin expression may not be regulated by the availability of the substrate such as appears to be the case for vinculin. The half-lives of both proteins are long and similar at different cell densities (the half lives of vinculin and talin are about 18–21 h in normal cells and 16–18 h in transformed cells). We conclude that the levels of vinculin and talin at steady state in a cell are largely dependent on their biosynthesis rates not on degradation rates. Transformation of CEF by Rous sarcoma virus type A causes an altered phenotype [34]. A disruption of stress fibers occurs with viral transformation, and the actin is reorganized into dot-like aggregates on the ventral membrane. These actin aggregates also contain vinculin and talin. Normal focal contacts were also observed along the cell edge in transformed cells, but they were fewer in number and small in size (data not shown). The decreased number of focal contacts could be attributed to the decreased amounts of vinculin and talin caused by transformation. This idea is supported by the observation that when vinculin expression is inhibited in cells transfected with antisense vinculin constructs, decreased numbers of focal contacts are present [28]. Prolonged treatment of cells with cycloheximide prior to or even after cell harvesting does not inhibit cell attachment, spreading, or organization of focal contacts [44], which suggests that synthesis of vinculin or talin is not crucial for the formation of focal contacts; rather, decreased synthesis may affect the number and the size of the focal contacts by limiting the supply of vinculin and/or talin. Our data predict that talin expression may have similar effects on focal contact formation as vinculin. Based on our observations and those of others, we suggest that the level of expression of both vinculin and talin is one of the mechanisms that controls focal contact formation both in terms of their numbers and size and is determined
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largely by changes in the synthesis rates not by degradation rates. In addition, viral transformation and high cell density cause decreases in biosynthesis of vinculin via independent mechanisms. The biosynthesis of talin, in contrast, is only affected by transformation. We thank Drs. David Asai, John Anderson, Claudia Kent, and Jim Forney for their helpful comments. This work was supported by grants from the American Cancer Society (CD-108) and an Elks graduate fellowship from the Purdue Cancer Center.
REFERENCES 1. Burridge, K., Fath, K., Kelly, T., Nuckolls, G., and Turner, C. (1988) Annu. Rev. Cell Biol. 4, 487–525. 2. Otto, J. J. (1990) Cell Motil. Cytoskeleton 16, 1–6. 3. Geiger, B., Volk, T., and Bendori, R. (1987) J. Cell Sci. (Suppl.) 8, 251–272. 4. Beckerle, M., and Yeh, R. (1990) Cell Motil. Cytoskeleton 16, 7–13. 5. Otto, J. J. (1983) J. Cell Biol. 97, 1283–1287. 6. Wilkins, J. A., Chen, K. Y., and Lin, S. (1983) Biochem. Biophys. Res. Commun. 116, 1026–1032. 7. Burridge, K., and Mangeat, P. (1984) Nature 308, 744–746. 8. Horwitz, A., Duggan, K., Buck, C., Beckerle, M., and Burridge, K. (1986) Nature 320, 531–533. 9. Belkin, A. M., and Koteliansky, V. E. (1987) FEBS Lett. 220, 291–294. 10. Wachsstock, D. H., Wilkins, J. A., and Lin, S. (1987) Biochem. Biophys. Res. Commun. 146, 554–560. 11. Wilkins, J. A., Risinger, M. A., Coffey, E., and Lin, S. (1987) J. Cell Biol. 104, 130a. 12. Turner, C., Glenney, J. R., and Burridge, K. (1990) J. Cell Biol. 111, 1059–1068. 13. Johnson, R. P., and Craig, S. W. (1995) Nature 373, 261–264. 14. Westmeyer, A., Ruhnau, K., Wegner, A., and Jockusch, B. (1990) EMBO J. 9, 2071–2078. 15. Nuckolls, G. H., Romer, L. H., and Burridge, K. (1992) J. Cell Sci. 102, 753–762. 16. Ungar, F., Geiger, B., and Ben-Ze’ev, A. (1986) Nature 319, 787–791. 17. Shirinsky, V. P., Birukov, K. G., Koteliansky, V. E., Glukhova, M. A., Spanidis, E., Rogers, J. D., Campbell, J. H., and Campbell, G. R. (1991) Exp. Cell Res. 194, 186–189. 18. Birukov, K. G., Frid, M. G., Rogers, J. D., Shirinsky, V. P., Koteliansky, V. E., Campbell, J. H., and Campbell, G. R. (1993) Exp. Cell Res. 204, 46–53. 19. Bendori, R., Salomon, D., and Geiger, B. (1987) EMBO J. 6, 2897–2905. 20. Ben-Ze’ev, A., Reiss, R., Bendori, R., and Gorodecki, B. (1990) Cell Regul. 1, 621–636. 21. Bellas, R. E., Bendori, R., and Farmer, S. R. (1991) J. Biol. Chem. 266, 12008–12014. 22. Gluck, U., Rodriguez-Fernandez, J. L., Pankov, R., and BenZe’ev, A. (1992) Exp. Cell Res. 202, 477–486. 23. Guller, S., Corin, R. E., Yuan-wu, K., and Sonenberg, M. (1991) Endocrinology 129, 527–533. 24. Zieske, J. D., Bukusoglu, G., and Gipson, I. K. (1989) J. Cell Biol. 109, 571–576. 25. Sadano, H., Inoue, M., and Taniguchi, S. (1992) Jpn. J. Cancer Res. 83, 625–630.
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SYNTHESIS AND TURNOVER RATES OF VINCULIN AND TALIN 26. Samuels, M., Ezzell, R. M., Cardozo, T. J., Critchley, D. R., Coll, J.-L., and Adamson, E. D. (1993) J. Cell Biol. 121, 909–921. 27. Rodriguez-Fernandez, J. L., Geiger, B., Salomon, D., and BenZe’ev, A. (1992) Cell Motil. Cytoskeleton 22, 127–134. 28. Rodriguez-Fernandez, J. L., Geiger, B., Salomon, D., and BenZe’ev, A. (1993) J. Cell Biol. 122, 1285–1294. 29. Rohrschneider, L. R. (1980) Proc. Natl. Acad. Sci. USA 77, 3514–3518. 30. Groesch, M. E., and Otto, J. J. (1990) Cell Motil. Cytoskeleton 15, 41–50. 31. Lee, S.-w., Wulfkuhle, J. D., and Otto, J. J. (1992) J. Biol. Chem. 267, 16355–16358. 32. Abercrombie, M., and Dunn, G. A. (1975) Exptl. Cell Res. 92, 57–62. 33. Olden, K., and Yamada, K. M. (1977) Cell 11, 957–969. 34. David-Pfeuty, T., and Singer, S. J. (1980) Proc. Natl. Acad. Sci. USA 77, 6687–6691.
35. Otto, J. J., and Lee, S.-w. (1993) Meth. Cell Biol. 37, 119–127. 36. Laemmli, U. K. (1970) Nature 227, 680–685. 37. Sefton, B. M., Patschinsky, T., Berdot, C., Hunter, T., and Elliot, T. (1982) J. Virol. 41, 813–820. 38. Oron, L., and Bar-Nun, S. (1989) Anal. Biochem. 179, 248–287. 39. Doyle, D., and Tweto, J. (1975) Methods Cell Biol. 10, 235–260. 40. Price, G. J., Jones, P., Davison, M. D., Patel, B., Bendori, R., Geiger, B., and Critchley, D. R. (1988) Biochem. J. 259, 453– 461. 41. Rees, D. J. G., Ades, S. E., Singer, S. J., and Hynes, R. O. (1990) Nature 347, 685–689. 42. Kupfer, A., Singer, S. J., and Dennert, G. (1986) J. Exp. Med. 163, 489–498. 43. Kupfer, A., Swain, S. L., and Singer, S. J. (1987) J. Exp. Med. 165, 1565–1580. 44. Grenz, H., Carbonetto, S., and Goodman, S. L. (1993) J. Cell Sci. 105, 739–751.
Received March 4, 1996 Revised version received June 10, 1996
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0284
Differences in Turnover Rates of Vinculin and Talin Caused by Viral Transformation and Cell Density SEUNG-WON LEE
AND
JOANN J. OTTO1
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392
Vinculin and talin are two major components of focal contacts which interact with each other. In order to understand how the relative levels of these proteins are maintained under various conditions, the synthesis rates and half-lives of vinculin and talin in chick embryonic fibroblasts were determined by autoradiography combined with immunoblotting. High cell density and transformation by Rous sarcoma virus decreased the vinculin synthesis rate by 40%. Upon viral transformation, the synthesis rate of talin decreased by 30%. In contrast to vinculin, the synthesis rate of talin was not affected by cell density. The effect of cell density on the synthesis rate of vinculin was retained after viral transformation, suggesting that cell density and viral transformation affect vinculin synthesis by two independent mechanisms. The synthesis rate of vinculin was approximately two to three times greater than that of talin under all conditions tested. The halflives of vinculin and talin remained constant at different cell densities in untransformed cells (t1/2 Å 18–21 h), but transformation slightly decreased half-lives of both proteins (t1/2 Å 16–18 h). These results suggest that the decreased expression of vinculin and talin in transformed chick fibroblasts can be attributed mainly to changes in their biosynthesis rates rather than degradation. This may contribute to a decrease in the number of focal contacts in transformed cells. q 1996 Academic Press, Inc.
INTRODUCTION
Stress fibers end at focal contacts on the ventral cell membrane. Vinculin and talin are two major elements present in focal contacts [1]. Vinculin (116 kDa) is a cytoplasmic protein widely distributed in actin-containing cell–cell junctions and cell–matrix focal adhesions [2]. In contrast, talin (225 kDa) is localized only in cell–matrix adhesions [1, 3, 4]. Vinculin and talin interact [5, 6] with moderate affinity (Kd 1008 M) [7]. Further, talin interacts with b1 integrin [8] and vin1 To whom reprint requests should be addressed at Department of Biological Sciences, Purdue University, West Lafayette, IN 479071392. Fax: (317) 494-0876. E-mail: [email protected].
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0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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culin interacts with a-actinin [6, 9, 10], tensin [11], and paxillin [12] in vitro. More recently, a direct interaction between vinculin and F-actin has been demonstrated [13]. Microinjection studies with anti-vinculin antibody [14] and anti-talin antibody [15] indicate that vinculin is a key protein in the microfilament-membrane linkage and that talin is essential for the development of focal contacts. Because vinculin and talin interact with several different focal contact components and interact with each other, they may be regulatory elements in the assembly, disassembly, and maintenance of focal contacts. Vinculin synthesis has been shown to be regulated under various circumstances and to correlate with the degree of focal contact formation. Vinculin synthesis is increased when 3T3 fibroblasts establish cell-to-cell contacts [16]. The expression of meta-vinculin, a vinculin variant restricted to muscle cells, differs in rabbit smooth muscle cell cultures seeded at different densities [17, 18]. Culture of cells on a highly adhesive matrix results in an increased synthesis of vinculin and an increased number of focal contacts in chick embryo fibroblasts [19]. Transient induction of vinculin gene expression occurs in 3T3 fibroblasts and regenerating liver cells stimulated by serum growth factors [20–22]. Vinculin expression is up-regulated in 3T3 adipose cells after treatment with growth hormone without a change in its phosphorylation level [23]. Migrating stratified squamous epithelia also express high levels of vinculin [24]. Vinculin expression is down-regulated in highly metastatic mouse-B16 melanoma cell lines [25]. A more direct correlation between vinculin expression and focal contact formation has been shown by suppression or overexpression of vinculin cDNA [26–28]. It seems clear that the expression level of vinculin is closely related to or even causal to the degree of cell motility and changes in shape resulting from changes in focal contacts. These results suggest that the formation of focal contacts may be regulated partly by the supply of their protein components. In contrast to vinculin, the expression of talin has not been well studied. Further, most of the studies on vinculin expression emphasize the synthesis rate, not the degradation rate. If intracellular protein levels of focal contact components are a determining factor for regulation of the number and
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size of focal contacts, the degradation rates of the proteins should also be examined. Transformation of chick embryo fibroblasts (CEF) with Rous sarcoma virus (RSV) results in the disruption of stress fibers and formation of filamentous actin aggregates containing vinculin, talin, a-actinin, and fimbrin on the ventral membrane [1]. Some normal focal contacts still exist but with reduced sizes and numbers compared to untransformed cells [29]. Hence, we were interested in examining the synthesis and degradation rates of vinculin and talin in untransformed and transformed CEF to elucidate whether the levels of expression of vinculin and talin are related to the transformation phenotype. In addition, because CEF at high density have fewer focal contacts, the effect of cell density on synthesis and degradation rates of both proteins was also investigated in both untransformed and transformed cells. EXPERIMENTAL PROCEDURES Materials. Unless otherwise indicated, all chemicals were reagent grade or were purchased from Sigma Chemical Co. (St. Louis, MO). The rabbit anti-chick vinculin and anti-chick talin antibodies used in this report were characterized previously [30, 31]. Cell culture. Primary CEF were prepared as previously described [32]. Viral-free chick embryo fibroblasts (from eggs obtained from SPAFAS) were infected with avian Rous sarcoma virus (RSV), type A, as previously described [33]. Both cell types (CEF and RSV-CEF) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal calf serum (Hyclone, Logan, UT), 0.125 g/liter penicillin G (1670 U/mg), and 0.5 g/liter streptomycin sulfate (736 U/mg). Transformation was confirmed by rhodamine-phalloidin staining which showed dot-like aggregations of actin filaments on the ventral side of the cells and by Northern blotting of total RNAs probed with a cDNA of v-src (a generous gift from Dr. Elizabeth J. Taparowsky, Purdue University). RSV-CEF showed typical alterations in filamentous actin as observed by others [34]. Sample preparation for biosynthesis rate determinations. Prior to labeling, cells in 5-cm plates were incubated in methionine-deficient modified Eagle’s medium (MEM, Sigma) for 15 min. To determine the relative biosynthesis rates of vinculin and talin, cells were labeled with 120 mCi [35S]methionine in 3 ml MEM/plate (sp act 1300 Ci/mM, Amersham, Arlington Heights, IL) with 5% fetal calf serum for 2 h. Labeled cells were lysed with modified RIPA containing 1.0% (w/v) Triton X-100, 0.2% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) sodium deoxycholate, 10 mM NaH2PO4 , 150 mM NaCl, 1 mM MgCl2 , 1 mM ethylene glycol-bis(b-aminoethyl ether)tetraacetic acid (EGTA), and 15 mM Tris–HCl, pH 7.4, with 1 mg/ml leupeptin, 1 mg/ml pepstatin A, and 2 mM phenylmethyl sulfonyl fluoride (PMSF). Protease inhibitors were purchased from Boehringer-Mannheim (Indianapolis, IN). Cells were lysed in 0.5 or 1.0 ml for low or high cell density plates, respectively, and scraped off the plates with a razor blade. Undissolved material was removed by a brief centrifugation (3 min at 15,000 rpm). The supernatant was quickly frozen by immersion in liquid nitrogen and kept in a freezer (0807C) until assayed. This supernatant will be called the sample. The pellet was assayed by immunoblotting to ensure that all the vinculin and talin were solubilized in the modified RIPA. In order to obtain the total biosynthesis rate, 100 ml of the samples were precipitated with trichloroacetic acid (TCA; final concentration, 10%) and the precipitates were washed two times with 10% TCA. The pellet was dissolved in 1 N NaOH in a final volume of 100 ml and its radioactivity was measured by liquid scintillation counting.
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The amount of protein in the resuspended pellet was determined with a Pierce Micro BCA assay kit (Pierce, Rockford, IL). Volumes with the same number of precipitable TCA counts were taken from each sample and quantitatively immunoprecipitated [35] with a mixture of anti-vinculin and anti-talin antibodies on ice for 1 h. The immune complex was absorbed with Protein-A bearing Staphylococcus aureus (Pansorbin, Calbiochem, San Diego, CA) on ice for 20 min. The immune complex was washed twice with modified RIPA containing 1 mM 2-mercaptoethanol and was resuspended in sample buffer and separated on 7% SDS gels [36]. The gels were blotted onto nitrocellulose and the nitrocellulose was processed as described below for autoradiography combined with immunoblotting. To compare the biosynthesis rates between vinculin and talin, cells in separate dishes were labeled for 30, 60, 90, 120, or 150 min with 120 mCi [35S]methionine. Cells were lysed after labeling as described above. The sample volumes were adjusted to contain the same amount of protein. Talin and vinculin were quantitatively immunoprecipitated and resolved on SDS–polyacrylamide gels as described above. Vinculin and talin bands visualized by Coomassie blue staining were cut out and solubilized in 0.4 ml 60% perchloric acid and 0.8 ml 30% H2O2 solution at 607C for 5 h for liquid scintillation analysis [37]. Scintillation cocktail (15 ml) (Universol, ICN, Irvine, CA) was added to each sample before counting. Sample preparation for degradation rate determinations. Cells were pulse labeled for 90 min with [35S]methionine (90 mCi/ml), washed with methionine-deficient MEM, and chased in DMEM containing 0.3 g/liter methionine and 5% fetal calf serum for 0, 4, 8, and 12 h. Cells were lysed as described above. Aliquots (20 ml) of the sample were taken to determine the protein amount. Volumes with the same total amount of protein were quantitatively immunoprecipitated with a mixture of anti-vinculin and anti-talin antibodies. The immunoprecipitates were processed as described below for autoradiography combined with immunoblotting. Autoradiography combined with immunoblotting. To determine the relative biosynthesis and degradation rates under various conditions, it was necessary to develop a method that allows for correct normalization of the degree of radiolabeling of vinculin or talin in each sample. We chose the protein amount of vinculin or talin which was detected by immunoblotting as a reference value to normalize the amount of radiolabeled vinculin or talin in each sample [38]. Proteins resolved in 7% SDS gels were semidry electroblotted onto nitrocellulose (1.14 mA/cm2 for 90 min). The transfer buffer was 48 mM Tris, 39 mM glycine, 0.1% SDS, 20% (v/v) methanol, pH 8.4. Nonspecific binding to nitrocellulose was prevented by a 30-min incubation at room temperature in blocking solution (0.25% gelatin, 3% bovine serum albumin, 150 mM NaCl, 15 mM Tris–HCl, pH 7.4). After blocking, the nitrocellulose was air-dried, and then the protein bands labeled with [35S]methionine were exposed by autoradiography without an intensifying screen at 0807C. To determine the amount of vinculin and talin, the same nitrocellulose membrane was incubated with a mixture of anti-vinculin and anti-talin antibodies in blocking solution for 4 h followed by two washes with Tris–saline (150 mM NaCl, 15 mM Tris, pH 7.4) containing 0.05% (w/v) Tween 20 and two washes with Tris–saline (15 min per wash). The nitrocellulose was incubated with 0.5 mCi/ml 125I-Protein A (sp act 54 mCi/mg, ICN, Costa Mesa, CA) in blocking solution for 2 h and extensively washed. After the nitrocellulose was dried, a blank, developed piece of film was put adjacent to the nitrocellulose in order to protect the film from b-particle emission from [35S]methionine and an autoradiogram was taken without an intensifying screen to detect the g-rays from 125I-Protein A. All the data were analyzed by laser densitometry (Pharmacia LKB, Piscataway, NJ). The values from the b-particle exposure were normalized by the relative protein amount as determined by g-ray exposure. The length of exposure was determined so that densities of autoradiograms fit in the linear range of protein amount versus the density.
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FIG. 1. Solubility and immunoprecipitation of vinculin and talin in modified RIPA. (A) A fluorogram of a SDS gel of RIPA-insoluble material. Cells were labeled with [35S]methionine and then solubilized in modified RIPA. The insoluble material was removed by centrifugation and separated by SDS–PAGE. Lane a, N-CEF; lane b, RSV-CEF. (B) Autoradiograph of an immunoblot of RIPA-insoluble material from non-radiolabeled cells. Cells were extracted as in A. The RIPA insoluble material was separated by SDS–PAGE and blotted onto nitrocellulose. The blot was incubated with a mixture of anti-vinculin and anti-talin antibodies followed by 125I-Protein A. Lane a, N-CEF; lane b, RSV-CEF. (C) Immunoblot of the supernatants after immunoprecipitation of vinculin and talin from N-CEF. The blot was incubated with a mixture of anti-vinculin and antitalin antibodies and then probed with peroxidase-conjugated Protein A. Lane a, vinculin marker; lane b, supernatant after immunoprecipitation with anti-vinculin antibody; lane c, supernatant after immunoprecipitation with anti-talin antibody; lane d, talin marker.
RESULTS
Relative Biosynthesis Rates of Vinculin and Talin The rate constant of synthesis conventionally has been defined as units or mass of a specific protein synthesized per unit time per weight of tissue [39]. For practical reasons when using cells in culture, the relative biosynthesis rate commonly has been used and is expressed as units or mass of a specific protein synthesized per unit of time per total protein. Using total biosynthesis rate or total protein synthesized for normalization can lead to underestimation of subtle changes in synthesis rates of minor cellular components. For this reason, in this study the amount of the protein of interest was used for normalization in determining the relative synthesis rates [38]. In order to determine the relative synthesis rates of vinculin and talin, cells were labeled with [35S]methionine for 2 h and then solubilized in modified RIPA. Prior to immunoprecipitation, the lysate was centrifuged to remove insoluble material (Fig. 1A). In order to be certain that all the vinculin and talin were being solubilized, the insoluble material was analyzed by immunoblotting for the presence of vinculin or talin (Fig. 1B). The pellet contained no detectable vinculin or talin. To ascertain whether we were quantitatively immunoprecipitating the vinculin and talin, the supernatants after immunoprecipitation were examined for the
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presence of vinculin and talin by immunoblotting (Fig. 1C). No detectable vinculin or talin was observed. Together, these observations demonstrate that we were examining the total population of vinculin and talin molecules rather than a subpopulation in the experiments described below. The relative biosynthesis rates were determined for vinculin and talin in CEF and RSV-CEF growing at different cell densities (Fig. 2). Cell lysates were quantitatively immunoprecipitated with a mixture of antitalin and anti-vinculin antibodies. The immune complexes were blotted onto nitrocellulose after separation by SDS gel electrophoresis. The nitrocellulose was autoradiographed and the relative amounts of [35S]methionine incorporated into vinculin and talin were determined by laser densitometry (Fig. 2A). This density value was divided by the total protein amount (nonlabeled plus labeled) of vinculin or talin as detected by immunoblotting for normalization. The total vinculin or talin protein amount was determined by probing the same piece of nitrocellulose with anti-vinculin and anti-talin antibodies followed by 125I-Protein A (Fig. 2B). Such normalized densities of [35S]methionine incorporation (Fig. 2C) represent the relative biosynthesis rate of each protein. In virally transformed cells, the synthesis rates of vinculin and talin were decreased (Fig. 2). In both normal and transformed cells grown at high cell density, the synthesis rate of vinculin was decreased, but the synthesis rate of talin was not affected (Fig. 2C). Because vinculin and talin have different transfer rates to nitrocellulose and are probed with different antibodies, the normalized values cannot be compared directly between the two proteins. We wanted to be able to compare the rates for synthesis of vinculin to that of talin under the different conditions. In order to do this, we needed to be able to convert the relative biosynthesis rates to the relative number of molecules or moles of each protein that was synthesized per unit time. We used a more conventional approach with a liquid scintillation method combined with calculations based on the relative methionine composition of the proteins as described below. Cells were continuously labeled with [35S]methionine and samples containing the same amount of total protein were quantitatively immunoprecipitated at different time points with a mixture of anti-vinculin and anti-talin antibodies. After separation on SDS gels, vinculin and talin were visualized by Coomassie blue staining, the bands containing them were cut out, and the gel pieces were subjected to liquid scintillation counting. This experiment was done only with cells at high cell density in order to obtain enough protein quantity to visualize with Coomassie blue staining. Figure 3 shows that there was a linear increase in vinculin and talin synthesized in both cell types. The slope of each line indicates the initial velocity of synthesis
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FIG. 2. Determination of the relative biosynthesis rates of vinculin and talin in different cell types by autoradiography combined with immunoblotting. Normal CEF and RSV-CEF were plated at two different cell densities (3 1 105, 3 1 106 cells/plate) 12 h prior to the experiment. Cells were labeled for 2 h with [35S]methionine and the cell lysates were immunoprecipitated with both anti-talin and antivinculin antibodies. The immunocomplexes were resolved on a 7% SDS gel and transferred to nitrocellulose (see Materials and Methods). (A) A representative autoradiogram of the nitrocellulose to detect the amount of [35S]methionine-labeled talin or vinculin. The relative density areas obtained by laser densitometry are shown below the autoradiogram. The high-molecular-weight band above talin is fibronectin, which nonspecifically binds to Pansorbin, which was used as the immunoabsorbant. (B) An autoradiogram of the same nitrocellulose which was immunoblotted with anti-talin and antivinculin antibodies and probed with 125I-Protein A. The numbers represent the relative amount of talin or vinculin determined by laser densitometry. (C) The values from Fig. 1A were divided with the corresponding values from Fig. 1B to yield the relative biosynthesis rate. The value for normal CEF at low cell density was used as the reference value for Table 1. NL, Normal, untransformed CEF at low cell density; NH, normal, untransformed CEF at high cell density; RL, RSV-CEF at low cell density; RH, RSV-CEF at high cell density; T, talin; V, vinculin.
expressed as the amount of incorporated radioactivity in vinculin or talin per unit of time per total protein amount as conventionally defined. Because vinculin and talin have different methionine compositions, the initial velocities of synthesis of the two proteins cannot be compared directly. In
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order to compare them, we calculated the ratio of methionine compositions of vinculin vs talin (0.64; 39 methionines per vinculin molecule and 61 methionines per talin molecule) based on published sequence data [40, 41]. The initial velocity of synthesis of talin (Fig. 3) in each cell type was then multiplied by 0.64 (for example, in normal CEF, 0.64 1 144.5 Å 92.5). Then, this transformed initial velocity of talin was used to determine the relative synthesis ratio between vinculin and talin in each cell type (for example, in normal CEF, 178/92.5 Å 1.9). From these data and calculations, we can estimate how many molecules of vinculin are synthesized during the synthesis of one talin molecule. By calculation, in normal CEF growing at high cell density, 1.9 vinculin molecules are synthesized per talin and, in RSV-CEF at high cell density, 1.6 vinculin molecules are synthesized per talin. With these conversion factors, the relative synthesis rates of vinculin and talin under different conditions (Fig. 2) were transformed to relative moles of vinculin and talin synthesized per unit time (Table 1). The value for vinculin in normal CEF at low cell density was set as the reference. The relative moles of talin synthesized at high cell density were obtained by dividing the relative synthesis rates of vinculin at high cell density with 1.9 for normal cells or 1.6 for transformed cells. Finally, the relative moles of talin synthesized at high cell density were used as the reference to obtain the relative moles of talin synthesized at low cell density. Such numerical transformation of five independent experiments is summarized in Table 1 which shows the relative moles of vinculin or talin synthesized per unit time.
FIG. 3. Continuous labeling of vinculin and talin to determine the initial velocities of their synthesis rates. The slope of each line is the initial velocity of synthesis. NHV (normal CEF, high cell density, vinculin; initial velocity is 178 cpm/min/mg); RHV (RSV-CEF, high cell density, vinculin; initial velocity is 97.6 cpm/min/mg); NHT (normal CEF, high cell density, talin; initial velocity is 144.5 cpm/min/ mg); RHT (RSV-CEF, high cell density, talin; initial velocity is 93.9 cpm/min/mg).
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TABLE 1 The Relative Moles of Vinculin and Talin Synthesizeda Cell densityb N-CEFe lcdg hcdh RSV-CEF f lcd hcd
Vinculin
Talin
Total biosynthesisc
2.5 1 105 3.0 1 106
1.00 (0.03)d 0.65 (0.04)
0.34 (0.04) 0.34 (0.02)
1.00 (0.00) 1.06 (0.02)
2.4 1 105 2.7 1 106
0.56 (0.01) 0.39 (0.04)
0.23 (0.03) 0.23 (0.02)
1.14 (0.08) 1.00 (0.01)
a
This table contains the average values of five independent experiments. All values are relative to the vinculin synthesis rate of normal CEF at low cell density. Therefore, the relative synthesis rates of vinculin and talin can be compared with each other, but not to the total biosynthesis rate. b The number of cells in a 5-cm plastic dish at the time of plating. c The total biosynthesis rates (cpm, counts per minute/mg protein) of each cell type were averaged and normalized to the value for normal, untransformed CEF (N-CEF) at low cell density (1.00 Å approximately 3.3 1 105 cpm/min/mg). The standard deviations (SD) of the total biosynthesis rates are noted in parentheses. d The synthesis rate was determined by the ratio of density areas from b-particle exposure to those from g-ray exposure to detect total vinculin or talin. The synthesis rates of each cell type were normalized to the value of normal CEF at low cell density for both vinculin and talin. SD of the synthesis rates are noted in parentheses. The unit is b-particle density area/g-ray density area/2 h which reflects the amount of newly synthesized vinculin or talin per time per total vinculin or talin. Since the transfer efficiency during the electroblotting varies between proteins of different sizes and since different antibodies were used to detect each protein, it was not possible to compare the density of vinculin with that of talin until we determined the synthesis rates of vinculin or talin by using the liquid scintillation method (Fig. 2). As described in the text, the initial velocity of synthesis (the slope of each line) for NHV, NHT, RHV, and RHT was determined, and the relative ratio between vinculin and talin was calculated (N-CEF Å 1.9, RSV-CEF Å 1.6; the numbers reflect the number of vinculin molecules synthesized per one talin molecule per unit time). These values were used to convert the relative synthesis rates of talin obtained by direct autoradiography combined with immunoblotting and made it possible to compare the relative synthesis rate of vinculin with talin. e Normal, untransformed chicken embryonic fibroblasts. f Rous sarcoma virus transformed-chicken embryonic fibroblasts. g Low cell density. h High cell density.
Half-Lives of Vinculin and Talin To determine the half-lives of vinculin and talin in both normal and RSV-transformed cells at different cell densities, pulse–chase experiments were done and the results were analyzed by the same method as is described for the determination of relative biosynthesis rate (autoradiography combined with immunoblotting). Figure 4 is a representative autoradiogram of a degradation experiment of CEF grown at high cell density. Vinculin and talin have similar half-lives at the two cell densities (t1/2 Å 18–21 h) while viral transformation slightly decreases their half-lives (t1/2 Å 16– 18 h) as shown in Table 2, which contains data from four independent experiments. DISCUSSION
The regulation of vinculin expression under various conditions has been well studied in a variety of cells [16–25]. However, a detailed understanding of talin expression has been lacking until now. Further, considering that the level of a particular protein in cells is controlled by both synthesis and degradation, information about vinculin and talin degradation is necessary to determine to what degree an altered synthesis rate will contribute to the pool of protein. Finally, because
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vinculin and talin interact and function in focal contacts, it is important to understand how cells coordinate the synthesis and degradation of both proteins. Experiments addressing these issues have not been feasible partly because of difficulties in normalization of biosynthesis rates and degradation rates of proteins of interest under different conditions. We believe that autoradiography, combined with immunoblotting [38], allows us to normalize protein synthesis and degradation rates with more precision and to compare the relative biosynthesis rates between vinculin and talin when combined with conventional methods. The synthesis rate of vinculin was decreased by viral transformation by 40% at both cell densities (Table 1). The synthesis rate of talin was also decreased by 30% in transformed cells but was not affected by cell density. Since the total biosynthesis rates of transformed cells were the same as normal cells, we suggest that these decreases are caused specifically by viral transformation. Chick fibroblasts and their RSV transformants at high cell density showed an approximately 40% decrease in their vinculin synthesis rates relative to low cell density. It is notable that cell density-dependent type regulation of the vinculin synthesis rate is preserved in RSV-transformed CEF even though RSV
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TABLE 2 The Half-Lives of Vinculin and Talina Cell densityb N-CEFd lcd f hcdg RSV-CEFe lcd hcd
Vinculin t1/2 (hr)
Talin t1/2 (hr)
3.1 1 105 3.8 1 106
19 (1)c 20 (2)
18 (2) 21 (1)
2.4 1 105 2.8 1 106
16 (2) 18 (2)
16 (1) 17 (2)
a
This table contains the average values of four independent experiments. The half-lives were obtained as described previously [39]. b The number of cells in a 5-cm plastic dish at the time of plating. c SD is in parentheses. d Normal chick embryonic fibroblasts. e Rous sarcoma virus-transformed chick embryonic fibroblasts. f Low cell density. g High cell density.
FIG. 4. Determination of half-lives of vinculin and talin in normal CEF grown at high cell density by autoradiography combined with immunoblotting. Cells (3.4 1 106 cells/plate) were pulse labeled for 90 min with [35S]methionine and then chased for 0, 4, 8, or 12 h. Each sample was immunoprecipitated with both anti-talin and antivinculin antibodies. The immunocomplex was resolved on a 7% SDS gel and transferred onto nitrocellulose (see Materials and Methods). (A) A representative autoradiogram of the nitrocellulose after the proteins were transferred shows the relative amount of [35S]methionine-labeled talin or vinculin. The numbers below the autoradiogram are the densitometric determinations of the amount of label incorporated into vinculin or talin. The high-molecular-weight band above talin is fibronectin, which nonspecifically binds to Pansorbin, which was used as the immunoabsorbant. (B) An autoradiogram of
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transformation decreases the vinculin synthesis rate by 40%. This suggests that there may be two independent mechanisms to regulate vinculin synthesis. At high cell density, both normal and transformed cells formed fewer cell-to-substrate contacts than at low cell density (data not shown). Thus, we suspect that the vinculin synthesis rate is regulated by the level of cellto-substrate interaction; that is, the availability of substrate which allows the formation of focal contacts may affect the supply of vinculin. Such a relationship between vinculin synthesis and substrate availability is well documented by Bendori et al. [19], who show that CEF grown on a highly adhesive matrix have increased vinculin expression and increased focal contact formation. In contrast to our results, increased vinculin synthesis is observed when 3T3 fibroblasts are grown at high cell density [16]. However, unlike CEF, 3T3 fibroblasts in culture grow as a monolayer and establish definitive cell-to-cell contacts at high cell density, and vinculin is localized in the cell-to-cell contact areas as well as focal contacts. Chick fibroblasts do not show any indication of vinculin localization in cell-to-cell contact areas at high cell density (data not shown). This difference in the localization of vinculin in the two cell types may account for the observed differences in synthesis rates relative to cell density. It is unlikely that the decrease in vinculin synthesis in cells at high cell den-
the same nitrocellulose which was immunoblotted with anti-talin and anti-vinculin antibodies and probed with 125I-Protein A. The numbers below the autoradiogram represent the relative total amount of talin or vinculin. (C) The values from A were normalized with the corresponding values from B. The graph is a plot of the logarithm of the normalized values (the amount of radioactivity remaining in the protein) vs time according to Doyle and Tweto (1975) [39]. Kd (rate constant of degradation; time01) of talin Å 03.31 1 1002 (t1/2 Å 21 h), r (linear regression) Å 0.95. Kd of vinculin Å 03.88 1 1002 (t1/2 Å 18 h), r Å 0.99.
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sity is due to a diminished general metabolic rate imposed by contact inhibition since the total biosynthesis rates and talin synthesis rates remain the same at high cell density in both normal and transformed cells. In contrast to vinculin, the talin synthesis rate did not show any change at different cell densities in either normal or RSV-transformed CEF. The biological implication of this remains unclear. Perhaps talin may participate in other functions in addition to cell–substrate adhesion or its protein level is not regulated by the availability of substrate. Even though talin is usually found in cell-to-matrix contacts where microfilaments are attached to membranes [4], talin is also recruited to the cell-to-cell contacts of effector cells upon activation of T-lymphocytes [42, 43]. This coincides with the reorganization of the microtubule organizing center and Golgi complex and eventually results in unidirectional killing of the target cells [42, 43]. This suggests that cell–matrix adhesion may not be the sole role of talin in vivo and raises the possibility that talin expression may not be regulated by the availability of the substrate such as appears to be the case for vinculin. The half-lives of both proteins are long and similar at different cell densities (the half lives of vinculin and talin are about 18–21 h in normal cells and 16–18 h in transformed cells). We conclude that the levels of vinculin and talin at steady state in a cell are largely dependent on their biosynthesis rates not on degradation rates. Transformation of CEF by Rous sarcoma virus type A causes an altered phenotype [34]. A disruption of stress fibers occurs with viral transformation, and the actin is reorganized into dot-like aggregates on the ventral membrane. These actin aggregates also contain vinculin and talin. Normal focal contacts were also observed along the cell edge in transformed cells, but they were fewer in number and small in size (data not shown). The decreased number of focal contacts could be attributed to the decreased amounts of vinculin and talin caused by transformation. This idea is supported by the observation that when vinculin expression is inhibited in cells transfected with antisense vinculin constructs, decreased numbers of focal contacts are present [28]. Prolonged treatment of cells with cycloheximide prior to or even after cell harvesting does not inhibit cell attachment, spreading, or organization of focal contacts [44], which suggests that synthesis of vinculin or talin is not crucial for the formation of focal contacts; rather, decreased synthesis may affect the number and the size of the focal contacts by limiting the supply of vinculin and/or talin. Our data predict that talin expression may have similar effects on focal contact formation as vinculin. Based on our observations and those of others, we suggest that the level of expression of both vinculin and talin is one of the mechanisms that controls focal contact formation both in terms of their numbers and size and is determined
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largely by changes in the synthesis rates not by degradation rates. In addition, viral transformation and high cell density cause decreases in biosynthesis of vinculin via independent mechanisms. The biosynthesis of talin, in contrast, is only affected by transformation. We thank Drs. David Asai, John Anderson, Claudia Kent, and Jim Forney for their helpful comments. This work was supported by grants from the American Cancer Society (CD-108) and an Elks graduate fellowship from the Purdue Cancer Center.
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SYNTHESIS AND TURNOVER RATES OF VINCULIN AND TALIN 26. Samuels, M., Ezzell, R. M., Cardozo, T. J., Critchley, D. R., Coll, J.-L., and Adamson, E. D. (1993) J. Cell Biol. 121, 909–921. 27. Rodriguez-Fernandez, J. L., Geiger, B., Salomon, D., and BenZe’ev, A. (1992) Cell Motil. Cytoskeleton 22, 127–134. 28. Rodriguez-Fernandez, J. L., Geiger, B., Salomon, D., and BenZe’ev, A. (1993) J. Cell Biol. 122, 1285–1294. 29. Rohrschneider, L. R. (1980) Proc. Natl. Acad. Sci. USA 77, 3514–3518. 30. Groesch, M. E., and Otto, J. J. (1990) Cell Motil. Cytoskeleton 15, 41–50. 31. Lee, S.-w., Wulfkuhle, J. D., and Otto, J. J. (1992) J. Biol. Chem. 267, 16355–16358. 32. Abercrombie, M., and Dunn, G. A. (1975) Exptl. Cell Res. 92, 57–62. 33. Olden, K., and Yamada, K. M. (1977) Cell 11, 957–969. 34. David-Pfeuty, T., and Singer, S. J. (1980) Proc. Natl. Acad. Sci. USA 77, 6687–6691.
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Received March 4, 1996 Revised version received June 10, 1996
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