ANALlTICAL
95, 236-244
BIOCHEMISTRY
An Isoelectric
(1979)
Focusing
Study of Plasma Membrane
Actin’
JANETTANNENBAUMANDALEXANDERRICH Massachusetts
Institute
of Technology,
Cambridge,
Massachusetts
02139
Received November 8, 1978 Plasma membranes were purified from secondary chick embryo fibroblasts labeled with [%]methionine for 1 or 18 h. The total cell homogenate, postnuclear supernatant and plasma membrane fractions were analyzed by two-dimensional electrophoresis (isoelectric focusing followed by SDS-slab gel electrophoresis). The (Y, p, and y isoelectric variants of actin were present in similar proportion in membranes, supernatant, and cell homogenate as determined by incorporation of Y5’ into each species of actin. These results indicate that the plasma membrane actin of chick fibroblasts is heterogeneous and that no isoelectric variant of actin is unique to the plasma membrane.
Analysis of various muscle and nonmuscle cells by isoelectric focusing has demonstrated the existence of multiple species of actin with identical molecular weights but different isoelectric points [reviewed in reference (l)]. The major species of actin (a, /3, y) appear to be products of different genes (2,3). The (Y species of actin constitutes the actin of the thin filaments in striated muscle (4-6). It has been suggested (7) that the y species in chick gizzard has predominantly a structural role, while the p species participates in smooth muscle contraction. In nonmuscle cells, however, the structural or functional specificity of the /3 and y actins is unknown. Attempts have been made to elucidate the significance of this heterogeneity of actin by examining whether each actin is localized to a distinct cell fraction. Cytoskeletal preparations of chick embryo fibroblasts were reported to contain LY,p, and y actin in the same proportions as intact cells (6). The nuclear pellet, 150,OOOg pellet, and soluble fractions of glial and nerve cell lines also exhibited no selectivity in type or relative amounts of p and y actin present (5). Sim-
ilarly, p and y actin were present in a constant ratio in various cytoplasmic fractions of human platelets (8). Analysis of unpolymerized actin from chick brain or of detergent-soluble and insoluble material from neuronal cells also failed to demonstrate a specific location for either F or y actin (9). Both p and y actin were present in erythrocyte ghosts (5). Since the actin associated with the plasma membrane is thought to be involved,in a variety of cell functions [reviewed in reference (IO)], we chose to analyze purified plasma membrane by isoelectric focusing in order to determine which actins were present. We report here that the plasma membrane actin of secondary chick embryo fibroblasts is heterogeneous and that the (Y, p, and y species occur in the same proportions in plasma membrane, total cell homogenate, and postnuclear supernatant. METHODS
Primary chick embryo fibroblasts (CEF)2 were obtained by differential plating of trypsinized thigh-muscle tissue from 1 l-day chick embryos in the course of preparing pri-
r This paper is dedicated to the memory of Dr. Alvin Nason. 0003-2697/79/070236-09$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
2 Abbreviations used: CEF, chick embryo fibroEBSS, Earle’s balanced salt solution.
blasts; 236
PLASMA
MEMBRANE
mary muscle cultures. The cells were dispersed in 0.25% trypsin in calcium-magnesium-free Earle’s balanced salt solution (EBSS), centrifuged, and suspended in growth medium (72% Dulbecco’s Modified Eagle’s Medium, 18% Medium 199, 10% horse serum, 1% chick embryo extract, 100 units penicillin, 100 pg streptomycin, 0.25 pg Fungizone, and 0.25 g glucose per 100 ml). Cells which attached to IOO-mm plastic tissue culture dishes (Falcon) in 15-20 min at 37°C were used to initiate the primary CEF cultures. CEF were cultured in growth medium at 37°C in a humidified atmosphere of 5% CO,-95% air and subcultured weekly. Secondary CEF were used for experiments after 2 to 6 weeks in culture. For experiments, CEF were plated in loo-mm plastic dishes at a density of 1 x lo6 cells/dish and used when confluent (3 days of growth). Cells were labeled with [35S]methionine (Amersham, 700-1200 Ci/mmol) in Dulbecco’s modified Eagle’s medium without methionine, plus 10% horse serum, chick embryo extract, antibiotics, and glucose as detailed above (labeling medium). Growth medium was removed from the dishes and replaced with 5 ml of labeling medium containing 20 pCi/ml of [35S]methionine (1 h labeling period), or with 7 ml of labeling medium containing 5 &i/ml of [35S]methionine (18-h labeling period). At the end of the labeling period, the dishes were drained, rinsed with EBSS, and then with 4.3 mM EDTA in calcium-magnesium-free EBSS (5 ml/dish). After incubation at 37°C for 5 min, cells were easily detached by pipetting EBSS onto the monolayer. The cell suspension was washed once in EBSS and the cell pellet obtained after centrifugation was used for the isolation of plasma membranes by the method of Atkinson and Summers (11). A sample of the homogenized cell pellet was taken for assay of total cellular actin. Postnuclear supematant was obtained by centrifugation of the cell homogenate ( 1000~ , 30 s) to remove nuclei. All samples were stored at -70°C.
ACTINS
237
Samples for isoelectric focusing were generally lyophilized to dryness and then dissolved in SDS sample buffer 0 (12) which contained 10% glycerol, 2.3% SDS, 0.0625 M Tris-Cl, pH 6.8, and 0.1 M dithiothreitol instead of the mercaptoethanol listed in reference (12). Samples containing about lOO,OOO-200,000 cpm of 35S were taken up in 10 ~1 of SDS buffer 0 and mixed with 5 mg of urea plus 50 ~1 of O’Farrell’s (12) lysis buffer (9.5 M urea, 2% NP-40, 2% ampholytes, and 0.1 M dithiothreitol), essentially as described by Alton and Lodish (13). The SDS buffer was used to ensure that all of the membrane components would be completely solubilized. In two experiments (Table 1, Nos. 1 and 8), the lyophilized samples were dissolved directly in lo-20 ~1 of lysis buffer A without addition of buffer 0 and extra urea; this procedure gave comparable results. Isoelectric focusing in cylindrical polyacrylamide gels was performed as described by O’Farrell (12) with modifications as noted below. Bio-Bad amphoiytes of pH range 5-7 (1.6%) and pH range 3-10 (0.4%) were used to form the pH gradient. The focusing gels (2 x 230 mm) were prerun for 30 min each at 500 and 1000 V. After application of the samples, the gels were run at 1000 V for 18-20 h. Gels were then extruded from the glass tubing. A 30-mm length of gel from the acidic end and 80 mm of the alkaline end were cut off, yielding a 120-mm section which contained the actin spots approximately at its midpoint. The 120-mm segment of the gel was then equilibrated with 5 ml of SDS sample buffer 0, frozen in a dry-ice ethanol bath, and stored at -70°C. For the second dimension, we used a Laemmli slab gel (14) with 10% or 12%‘acrylamide in the running gel. The stacking gel (3% acrylamide) contained 2 M urea to facilitate overlaying of the stacker with 0.1% SDS during polymerization ( 15). Gels were run at 100 V (ca. 50 mA) until the tracking dye entered the running gel and
238
TANNENBAIJM
AND RICH
TABLE QUANTITATION
Experiment No. A
1 2 3 4
Average 2 SD B
5 6 7 8
Average ? SD
OF ACTINS
1
IN CELLS
AND
FRACTIONS”
0
YIP
Cells
Membrane
Supernatant
Cells
Membrane
0.15 0.26 0.38 0.21
0.21 0.19, 0.21” 0.39 0.29
ND’ 0.27 0.39 0.39
0.55 0.53 0.75 0.59
0.38 0.45, 0.526 0.60 0.52
ND’ 0.52 0.59 0.74
0.25 F 0.10
0.26 2 0.08
0.35 + 0.07
0.61 + 0.10
0.49 2 0.08
0.62 4 0.11
0.14 ND’ ND’ 0.20
NDC 0.28 0.07 0.40
0.12 0.16 0.26 0.46
0.39 NDC ND’ 0.42
0.40 0.44 0.48 0.54
0.41 0.47 0.47 0.49
0.17 r 0.04
0.25 2 0.18
0.25 + 0.15
0.41 + 0.02
0.47 t 0.06
0.46 -r- 0.03
Supernatant
U Samples of total cell homogenate, postnuclear supernatant, and plasma membrane from [%]methioninelabeled CEF were subjected to two-dimensional electrophoresis. Spots corresponding to the (Y,p, and y species of actin (shown in Figs. l-3) were cut from the gels; radioactivity was determined by scintillation counting. The ratio of cpm in (Yor y actin relative to p actin was calculated after correcting values for background cpm. (A) Cells labeled 1 h. (B).CeIls labeled 18 h. * Duplicate determinations. c Not done.
then 200 V (ca. 50 mA) until the dye reached the front. The gels were stained with 0.2% Coomassie blue in 50% methanol-IO% acetic acid for 1-2 h, destained overnight in 50% methanol-IO% acetic acid, rinsed 30-60 min in 10% acetic acid (to remove methanol), dried onto Whatman 3 MM paper, and exposed to Kodak XR-5 X-ray film. Developed films were aligned with the dried gels in order to locate the actin spots, which were then excised from the gels. Each spot was placed in a scintillation vial to which 1 ml of 90% NCS (Amersham)-10% distilled water was added. The tightly capped vials were shaken overnight at 37°C and cooled to room temperature. Ten milliliters of Liquifluor-toluene scintillation cocktail (New England Nuclear) were added to each and the capped vials were incubated at room temperature for 3 days to allow maximal elution of radioactivity from the gels. The samples were then counted at room temperature. Background counts in
the gels were determined by cutting out pieces of gel devoid of spots on the X-ray film and processing them in the same way. To test the trypsin sensitivity of membrane actin, isolated plasma membranes in 10 mM Tris-Cl, pH 8.0, were incubated with 0.025% trypsin (Trypsin-TPCK, Worthington Biochemical Corporation) for 2.5 h at room temperature. A replicate sample of membrane was dissolved in 5% Tritop X- 100 (New England Nuclear) by incubation at 37°C for 30 min and then similarly treated with trypsin. The trypsinized samples and an untreated sample of membrane were mixed with equal volumes of boiling 2x concentrated SDS sample buffer 0 and boiled 2 min. The samples were stored at -70°C and subsequently run on a 10% acrylamide slab gel as described above for the second dimension of the isoelectric focusing gels. Actin bands were cut out and counted as described above. All chemicals were of reagent grade. The
PLASMA
MEMBRANE
ACTINS
239
FIG. 1. Autoradiogram of [35S]methionine-labeled CEF cell homogenate (A) and plasma membrane (B) analyzed by two-dimensional electrophoresis. CEF monolayers were labeled for 1 h. The direction of migration in the first dimension was left (alkaline) to right (acidic); only the central 120.mm segment of the isoelectric focusing gel was used for the separation in the second dimension on 12% acrylamide SDS-slab gels (migration from top to bottom). Arrow indicates actin.
urea used was ultrapure grade from Schwa& Mann. NP-40 was from Shell or Bethesda research Labs. Other chemicals for isoelectric focusing and acrylamide slab gel
electrophoresis were purchased from BioRad. Media components and serum were from Grand Island Biological Company. Chick striated muscle actin was purified
240
TANNENBAUM
AND
RICH
FIG. 2. Autoradiogram showing two-dimensional analysis of [35S]methionine-labeled CEF cell homogenate (A) and plasma membrane (B) from cells labeled for 18 h. The acidic end of the pH gradient is towards the right (see legend to Fig. 1). Separation in the second dimension was on 10% acrylamide slab gels. Arrow indicates actin.
PLASMA
MEMBRANE
from an acetone powder by the method of Spudich and Watt (16); the actin was used after one repolymerization.
241
ACTINS
ilar patterns were obtained for samples of postnuclear supernatant (not shown). Quantitation
of Actin Species
RESULTS
Heterogeneity Actin
of Plasma
Membrane
Monolayers of CEF were labeled for 1 h with [35S]methionine, and plasma membranes were purified as described under Methods. In each experiment, samples of cell homogenate, postnuclear supernatant, and plasma membranes were analyzed by two-dimensional electrophoresis: isoelectric focusing followed by SDS-slab gel electrophoresis. As reported by others (6) and shown in Figs. IA (arrow) and 3A, actin from CEF was resolved into three major ((Y, /3, y) and two minor (6, E) species. When we examined the actin associated with the purified plasma membrane from CEF (Figs. 1B and 3B), we found these same five species of actin. The postnuclear supernatant gave a similar pattern (not shown). We verified that these spots corresponded to actin by submitting a mixture of labeled CEF plasma membranes and unlabeled purified chick striated muscle actin (a) to twodimensional electrophoresis. Figure 3E demonstrates that the labeled membrane actin migrated to the same position as the unlabeled actin marker. The CEF were also labeled for about 18 h in order to measure the equilibrium distribution of the several species of actin. Comparison of Figs. 2A and B or 3C and D again indicates that membrane actin exhibits the same heterogeneity as total cellular actin in CEF. The minor species of actin (6 and E), which may be precursors, respectively, of p and y actins (3), do not accumulate during long-term labeling and therefore are barely visible in these autoradiograms. A slight smearing of material in the region of the a-actin spot (Figs. 3C. D) may reflect some autooxidation of the 35S-label during long-term labeling. Sim-
To determine the relative distribution of the major species of actin in CEF cells and fractions, we cut the actin spots from the dried slab gels and measured the amount of labeled material in them by scintillation counting. As shown in Table 1, for both l- and 18-h labeling experiments, the distribution of 01, p, and y actin was not significantly different for CEF cells, supernatant, and plasma membrane. The greater variability in the OJp values for some samples labeled 18 h compared to 1 h was probably caused by the smearing of actin spots seen in the (Y region (see above and Figs. 3C, D). Although the membrane purification procedure we used preferentially yields unsealed “ghosts” of plasma membrane, we considered the possibility that the apparent heterogeneity of membrane actin might result from the presence of some sealed vesicles of plasma membrane which contained trapped cytoplasmic actin. Actin trapped inside of a sealed vesicle would be resistant to trypsin on the outside unless the vesicle was broken or dissolved, e.g., by detergent. To measure the amount of trypsin-resistant actin, we incubated [35S] methionine-labeled CEF plasma membrane with dilute trypsin in the absence or presence of 5% Triton X-100 and then subjected these samples and untreated plasma membranes to slab gel electrophoresis. The actin band of each sample was cut from the gel and counted. As shown in Table 2, no more than 4% of the membrane-associated actin was trypsin resistant and therefore possibly inside sealed vesicles. All of the actin was digested when Triton X-100 was present to dissolve the membrane. Since no more than 4% of the membrane actin might correspond to trapped cytoplasmic actin, it is apparent that the sim-
242
TANNENBAUM
AND
RICH
PLASMA
MEMBRANE
ilarity in the distribution of actins between plasma membrane and total cell homogenate cannot be attributed to trapping of actin within membrane vesicles. Thus, compared to total cellular actin, the plasma membrane actins of CEF do not appear to be unique either in species or relative amount. DISCUSSION
Purified plasma membranes from CEF contain cy, p, and y actins in the same ratio as whole CEF cells. The sensitivity of the membrane actin to proteolysis when trypsin was added indicates that this heterogeneous actin was membrane associated and not merely trapped inside sealed vesicles. Therefore, it appears that no individual species of actin in CEF may be considered membrane specific. The similar distribution of actins in postnuclear supernatant and total cell homogenate also indicates that none of the actins is unique to the nucleus, in agreement with results using other cell types (5). The inability to demonstrate structural or functional specificity for isoelectric variants of actin in nonmuscle cells by cell fractionation suggests that these variants lack specific roles and instead are completely interchangeable. Alternatively, specificity may be found only in highly specialized substructures of the cell. In that case, the presence of multiple species of actin attached to the plasma membrane might reflect its multiple functions (e.g., membrane motility, cell adhesion, endocytosis). Isolation of the
243
ACTINS TABLE TRYPSIN
RESISTANCE
OF MEMBRANE Actin (cpm “jS1
Sample Plasma membrane, Plasma membrane, Plasma membrane. + Triton X-100
2
untreated +trypsin +trypsin
ACTIN” (%) Trypsin resistant
2436 98
4
8
0
U Replicate samples of plasma membrane isolated from [:?5]methionine-labeled CEF were analyzed on a 10% acrylamide slab gel after no further treatment (untreated), digestion with 0.025% trypsin (+trypsin). or incubation with 5% Triton X-100 followed by digestion with trypsin (+trypsin + Triton X-100). Actin bands were cut from the gels and radioactivity was determined by scintillation counting. Values shown have been corrected for background counts.
retricted portion of plasma membrane involved in a particular function, such as formation of a microvillus or phagocytic vacuole, might then be expected to produce a fraction with a distinct composition of actins compared to the total membrane actin. A recent publication (17) reporting the threefold enrichment of two minor species of rat brain actin in the specialized brain synapse fraction tends to support this latter alternative. However it is clear that further work will be required before we understand the biological significance of the different forms of actin. ACKNOWLEDGMENTS Janet Tannenbaum Dystrophy Society
is a Fellow of the Muscular of America at Massachusetts In-
FIG. 3. (A-D). Region of autoradiograms containing actin in Figs. 1 and 2. Positions of three major ((Y. p, and y) and two minor (6, E) species of actin are indicated; acidic end of gradient is towards the right. Cell homogenate (A) and plasma membrane (B) of cells labeled 1 h show 6 and t species; these are barely visible in cell homogenate (C) and plasma membrane (D) labeled 18 h. (E) Comigration of labeled actin in CEF plasma membrane with purified n-actin marker. A mixture of unlabeled (Y actin and “5S-labeled plasma membrane (one third the amount used in (D) was analyzed by twodimensional electrophoresis. Circle (arrow) indicates position of Coomassie blue-stained actin marker; only the actin region of the autoradiogram is shown.
244
TANNENBAUM
stitute of Technology, Cambridge, Massachusetts. We thank E. Helitzer for help in obtaining Figures l-3 and J. Simpson for secretarial assistance. This work was supported by grants from the NIH, NSF, NASA, and the American Cancer Society.
REFERENCES 1. Korn, E. D. (1978) Proc. Nat. Acad. Sci. U. S. A. 75, 588-599. 2. Storti, R. V., and Rich, A. (1976) Proc. Nat. Acad. Sci. U. S. A. 73, 2346-2350. 3. Hunter, T., and Garrels, J. I. (1977) Cell 12, 767781. 4. Whalen, R. G., Butler-Browne, G. S., and Gros, F. (1976) Proc. Nat. Acad. Sci. U. S. A. 73, 2018-2022. 5. Carrels, J. I., and Gibson, W. (1976) Cell 9, 793805. 6. Rubenstein, P. A., and Spudich, J. A. (1977) Proc. Nat. Acad. Sci. U. S. A. 74, 120-123. 7. Izant, J. G., and Lazarides, E. (1977) Proc. Naf. Acad. Sci. U. S. A. 74, 1450-1454.
AND RICH 8. Landon, F., Hut, C., Thomt, F., Oriol, C., and Olomucki, A., (1977) Eur. J. Biochem. 81,571577. 9. Choo, Q. L., and Bray, D. (1978) .I. Neurochem. 31, 217-224. 10. Pollard, T. D. (1975) in Molecules and Cell Movement (Inoue, S., and Stephens, R. E., eds.), pp. 259-286, Raven Press, New York. 11. Atkinson, P. H., and Summers, D. F. (1971) J. Biol. Chem. 246, 5162-5175. 12. O’Farrell, P. H. (1975) 1. Biol. Chem. 250, 40074021. 13. Alton, T. H. and Lodish, H. F. (1977) Develop. Biol. 60, 180-206. 14. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 15. Storti, R. V., Horovitch, S. J., Scott, M. P., Rich, A., and Pardue, M. L. (1978) Cell 13, 589-598. 16. Spudich, J. A., and Watt, S. (197l)J. Biol. Chem. 246, 4866-4871. 17. Marotta, C. A., Strocchi, P., and Gilbert, J. M. (1978) J. Neurochem. 30, 1441-1451.
95, 236-244
BIOCHEMISTRY
An Isoelectric
(1979)
Focusing
Study of Plasma Membrane
Actin’
JANETTANNENBAUMANDALEXANDERRICH Massachusetts
Institute
of Technology,
Cambridge,
Massachusetts
02139
Received November 8, 1978 Plasma membranes were purified from secondary chick embryo fibroblasts labeled with [%]methionine for 1 or 18 h. The total cell homogenate, postnuclear supernatant and plasma membrane fractions were analyzed by two-dimensional electrophoresis (isoelectric focusing followed by SDS-slab gel electrophoresis). The (Y, p, and y isoelectric variants of actin were present in similar proportion in membranes, supernatant, and cell homogenate as determined by incorporation of Y5’ into each species of actin. These results indicate that the plasma membrane actin of chick fibroblasts is heterogeneous and that no isoelectric variant of actin is unique to the plasma membrane.
Analysis of various muscle and nonmuscle cells by isoelectric focusing has demonstrated the existence of multiple species of actin with identical molecular weights but different isoelectric points [reviewed in reference (l)]. The major species of actin (a, /3, y) appear to be products of different genes (2,3). The (Y species of actin constitutes the actin of the thin filaments in striated muscle (4-6). It has been suggested (7) that the y species in chick gizzard has predominantly a structural role, while the p species participates in smooth muscle contraction. In nonmuscle cells, however, the structural or functional specificity of the /3 and y actins is unknown. Attempts have been made to elucidate the significance of this heterogeneity of actin by examining whether each actin is localized to a distinct cell fraction. Cytoskeletal preparations of chick embryo fibroblasts were reported to contain LY,p, and y actin in the same proportions as intact cells (6). The nuclear pellet, 150,OOOg pellet, and soluble fractions of glial and nerve cell lines also exhibited no selectivity in type or relative amounts of p and y actin present (5). Sim-
ilarly, p and y actin were present in a constant ratio in various cytoplasmic fractions of human platelets (8). Analysis of unpolymerized actin from chick brain or of detergent-soluble and insoluble material from neuronal cells also failed to demonstrate a specific location for either F or y actin (9). Both p and y actin were present in erythrocyte ghosts (5). Since the actin associated with the plasma membrane is thought to be involved,in a variety of cell functions [reviewed in reference (IO)], we chose to analyze purified plasma membrane by isoelectric focusing in order to determine which actins were present. We report here that the plasma membrane actin of secondary chick embryo fibroblasts is heterogeneous and that the (Y, p, and y species occur in the same proportions in plasma membrane, total cell homogenate, and postnuclear supernatant. METHODS
Primary chick embryo fibroblasts (CEF)2 were obtained by differential plating of trypsinized thigh-muscle tissue from 1 l-day chick embryos in the course of preparing pri-
r This paper is dedicated to the memory of Dr. Alvin Nason. 0003-2697/79/070236-09$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
2 Abbreviations used: CEF, chick embryo fibroEBSS, Earle’s balanced salt solution.
blasts; 236
PLASMA
MEMBRANE
mary muscle cultures. The cells were dispersed in 0.25% trypsin in calcium-magnesium-free Earle’s balanced salt solution (EBSS), centrifuged, and suspended in growth medium (72% Dulbecco’s Modified Eagle’s Medium, 18% Medium 199, 10% horse serum, 1% chick embryo extract, 100 units penicillin, 100 pg streptomycin, 0.25 pg Fungizone, and 0.25 g glucose per 100 ml). Cells which attached to IOO-mm plastic tissue culture dishes (Falcon) in 15-20 min at 37°C were used to initiate the primary CEF cultures. CEF were cultured in growth medium at 37°C in a humidified atmosphere of 5% CO,-95% air and subcultured weekly. Secondary CEF were used for experiments after 2 to 6 weeks in culture. For experiments, CEF were plated in loo-mm plastic dishes at a density of 1 x lo6 cells/dish and used when confluent (3 days of growth). Cells were labeled with [35S]methionine (Amersham, 700-1200 Ci/mmol) in Dulbecco’s modified Eagle’s medium without methionine, plus 10% horse serum, chick embryo extract, antibiotics, and glucose as detailed above (labeling medium). Growth medium was removed from the dishes and replaced with 5 ml of labeling medium containing 20 pCi/ml of [35S]methionine (1 h labeling period), or with 7 ml of labeling medium containing 5 &i/ml of [35S]methionine (18-h labeling period). At the end of the labeling period, the dishes were drained, rinsed with EBSS, and then with 4.3 mM EDTA in calcium-magnesium-free EBSS (5 ml/dish). After incubation at 37°C for 5 min, cells were easily detached by pipetting EBSS onto the monolayer. The cell suspension was washed once in EBSS and the cell pellet obtained after centrifugation was used for the isolation of plasma membranes by the method of Atkinson and Summers (11). A sample of the homogenized cell pellet was taken for assay of total cellular actin. Postnuclear supematant was obtained by centrifugation of the cell homogenate ( 1000~ , 30 s) to remove nuclei. All samples were stored at -70°C.
ACTINS
237
Samples for isoelectric focusing were generally lyophilized to dryness and then dissolved in SDS sample buffer 0 (12) which contained 10% glycerol, 2.3% SDS, 0.0625 M Tris-Cl, pH 6.8, and 0.1 M dithiothreitol instead of the mercaptoethanol listed in reference (12). Samples containing about lOO,OOO-200,000 cpm of 35S were taken up in 10 ~1 of SDS buffer 0 and mixed with 5 mg of urea plus 50 ~1 of O’Farrell’s (12) lysis buffer (9.5 M urea, 2% NP-40, 2% ampholytes, and 0.1 M dithiothreitol), essentially as described by Alton and Lodish (13). The SDS buffer was used to ensure that all of the membrane components would be completely solubilized. In two experiments (Table 1, Nos. 1 and 8), the lyophilized samples were dissolved directly in lo-20 ~1 of lysis buffer A without addition of buffer 0 and extra urea; this procedure gave comparable results. Isoelectric focusing in cylindrical polyacrylamide gels was performed as described by O’Farrell (12) with modifications as noted below. Bio-Bad amphoiytes of pH range 5-7 (1.6%) and pH range 3-10 (0.4%) were used to form the pH gradient. The focusing gels (2 x 230 mm) were prerun for 30 min each at 500 and 1000 V. After application of the samples, the gels were run at 1000 V for 18-20 h. Gels were then extruded from the glass tubing. A 30-mm length of gel from the acidic end and 80 mm of the alkaline end were cut off, yielding a 120-mm section which contained the actin spots approximately at its midpoint. The 120-mm segment of the gel was then equilibrated with 5 ml of SDS sample buffer 0, frozen in a dry-ice ethanol bath, and stored at -70°C. For the second dimension, we used a Laemmli slab gel (14) with 10% or 12%‘acrylamide in the running gel. The stacking gel (3% acrylamide) contained 2 M urea to facilitate overlaying of the stacker with 0.1% SDS during polymerization ( 15). Gels were run at 100 V (ca. 50 mA) until the tracking dye entered the running gel and
238
TANNENBAIJM
AND RICH
TABLE QUANTITATION
Experiment No. A
1 2 3 4
Average 2 SD B
5 6 7 8
Average ? SD
OF ACTINS
1
IN CELLS
AND
FRACTIONS”
0
YIP
Cells
Membrane
Supernatant
Cells
Membrane
0.15 0.26 0.38 0.21
0.21 0.19, 0.21” 0.39 0.29
ND’ 0.27 0.39 0.39
0.55 0.53 0.75 0.59
0.38 0.45, 0.526 0.60 0.52
ND’ 0.52 0.59 0.74
0.25 F 0.10
0.26 2 0.08
0.35 + 0.07
0.61 + 0.10
0.49 2 0.08
0.62 4 0.11
0.14 ND’ ND’ 0.20
NDC 0.28 0.07 0.40
0.12 0.16 0.26 0.46
0.39 NDC ND’ 0.42
0.40 0.44 0.48 0.54
0.41 0.47 0.47 0.49
0.17 r 0.04
0.25 2 0.18
0.25 + 0.15
0.41 + 0.02
0.47 t 0.06
0.46 -r- 0.03
Supernatant
U Samples of total cell homogenate, postnuclear supernatant, and plasma membrane from [%]methioninelabeled CEF were subjected to two-dimensional electrophoresis. Spots corresponding to the (Y,p, and y species of actin (shown in Figs. l-3) were cut from the gels; radioactivity was determined by scintillation counting. The ratio of cpm in (Yor y actin relative to p actin was calculated after correcting values for background cpm. (A) Cells labeled 1 h. (B).CeIls labeled 18 h. * Duplicate determinations. c Not done.
then 200 V (ca. 50 mA) until the dye reached the front. The gels were stained with 0.2% Coomassie blue in 50% methanol-IO% acetic acid for 1-2 h, destained overnight in 50% methanol-IO% acetic acid, rinsed 30-60 min in 10% acetic acid (to remove methanol), dried onto Whatman 3 MM paper, and exposed to Kodak XR-5 X-ray film. Developed films were aligned with the dried gels in order to locate the actin spots, which were then excised from the gels. Each spot was placed in a scintillation vial to which 1 ml of 90% NCS (Amersham)-10% distilled water was added. The tightly capped vials were shaken overnight at 37°C and cooled to room temperature. Ten milliliters of Liquifluor-toluene scintillation cocktail (New England Nuclear) were added to each and the capped vials were incubated at room temperature for 3 days to allow maximal elution of radioactivity from the gels. The samples were then counted at room temperature. Background counts in
the gels were determined by cutting out pieces of gel devoid of spots on the X-ray film and processing them in the same way. To test the trypsin sensitivity of membrane actin, isolated plasma membranes in 10 mM Tris-Cl, pH 8.0, were incubated with 0.025% trypsin (Trypsin-TPCK, Worthington Biochemical Corporation) for 2.5 h at room temperature. A replicate sample of membrane was dissolved in 5% Tritop X- 100 (New England Nuclear) by incubation at 37°C for 30 min and then similarly treated with trypsin. The trypsinized samples and an untreated sample of membrane were mixed with equal volumes of boiling 2x concentrated SDS sample buffer 0 and boiled 2 min. The samples were stored at -70°C and subsequently run on a 10% acrylamide slab gel as described above for the second dimension of the isoelectric focusing gels. Actin bands were cut out and counted as described above. All chemicals were of reagent grade. The
PLASMA
MEMBRANE
ACTINS
239
FIG. 1. Autoradiogram of [35S]methionine-labeled CEF cell homogenate (A) and plasma membrane (B) analyzed by two-dimensional electrophoresis. CEF monolayers were labeled for 1 h. The direction of migration in the first dimension was left (alkaline) to right (acidic); only the central 120.mm segment of the isoelectric focusing gel was used for the separation in the second dimension on 12% acrylamide SDS-slab gels (migration from top to bottom). Arrow indicates actin.
urea used was ultrapure grade from Schwa& Mann. NP-40 was from Shell or Bethesda research Labs. Other chemicals for isoelectric focusing and acrylamide slab gel
electrophoresis were purchased from BioRad. Media components and serum were from Grand Island Biological Company. Chick striated muscle actin was purified
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FIG. 2. Autoradiogram showing two-dimensional analysis of [35S]methionine-labeled CEF cell homogenate (A) and plasma membrane (B) from cells labeled for 18 h. The acidic end of the pH gradient is towards the right (see legend to Fig. 1). Separation in the second dimension was on 10% acrylamide slab gels. Arrow indicates actin.
PLASMA
MEMBRANE
from an acetone powder by the method of Spudich and Watt (16); the actin was used after one repolymerization.
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ilar patterns were obtained for samples of postnuclear supernatant (not shown). Quantitation
of Actin Species
RESULTS
Heterogeneity Actin
of Plasma
Membrane
Monolayers of CEF were labeled for 1 h with [35S]methionine, and plasma membranes were purified as described under Methods. In each experiment, samples of cell homogenate, postnuclear supernatant, and plasma membranes were analyzed by two-dimensional electrophoresis: isoelectric focusing followed by SDS-slab gel electrophoresis. As reported by others (6) and shown in Figs. IA (arrow) and 3A, actin from CEF was resolved into three major ((Y, /3, y) and two minor (6, E) species. When we examined the actin associated with the purified plasma membrane from CEF (Figs. 1B and 3B), we found these same five species of actin. The postnuclear supernatant gave a similar pattern (not shown). We verified that these spots corresponded to actin by submitting a mixture of labeled CEF plasma membranes and unlabeled purified chick striated muscle actin (a) to twodimensional electrophoresis. Figure 3E demonstrates that the labeled membrane actin migrated to the same position as the unlabeled actin marker. The CEF were also labeled for about 18 h in order to measure the equilibrium distribution of the several species of actin. Comparison of Figs. 2A and B or 3C and D again indicates that membrane actin exhibits the same heterogeneity as total cellular actin in CEF. The minor species of actin (6 and E), which may be precursors, respectively, of p and y actins (3), do not accumulate during long-term labeling and therefore are barely visible in these autoradiograms. A slight smearing of material in the region of the a-actin spot (Figs. 3C. D) may reflect some autooxidation of the 35S-label during long-term labeling. Sim-
To determine the relative distribution of the major species of actin in CEF cells and fractions, we cut the actin spots from the dried slab gels and measured the amount of labeled material in them by scintillation counting. As shown in Table 1, for both l- and 18-h labeling experiments, the distribution of 01, p, and y actin was not significantly different for CEF cells, supernatant, and plasma membrane. The greater variability in the OJp values for some samples labeled 18 h compared to 1 h was probably caused by the smearing of actin spots seen in the (Y region (see above and Figs. 3C, D). Although the membrane purification procedure we used preferentially yields unsealed “ghosts” of plasma membrane, we considered the possibility that the apparent heterogeneity of membrane actin might result from the presence of some sealed vesicles of plasma membrane which contained trapped cytoplasmic actin. Actin trapped inside of a sealed vesicle would be resistant to trypsin on the outside unless the vesicle was broken or dissolved, e.g., by detergent. To measure the amount of trypsin-resistant actin, we incubated [35S] methionine-labeled CEF plasma membrane with dilute trypsin in the absence or presence of 5% Triton X-100 and then subjected these samples and untreated plasma membranes to slab gel electrophoresis. The actin band of each sample was cut from the gel and counted. As shown in Table 2, no more than 4% of the membrane-associated actin was trypsin resistant and therefore possibly inside sealed vesicles. All of the actin was digested when Triton X-100 was present to dissolve the membrane. Since no more than 4% of the membrane actin might correspond to trapped cytoplasmic actin, it is apparent that the sim-
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MEMBRANE
ilarity in the distribution of actins between plasma membrane and total cell homogenate cannot be attributed to trapping of actin within membrane vesicles. Thus, compared to total cellular actin, the plasma membrane actins of CEF do not appear to be unique either in species or relative amount. DISCUSSION
Purified plasma membranes from CEF contain cy, p, and y actins in the same ratio as whole CEF cells. The sensitivity of the membrane actin to proteolysis when trypsin was added indicates that this heterogeneous actin was membrane associated and not merely trapped inside sealed vesicles. Therefore, it appears that no individual species of actin in CEF may be considered membrane specific. The similar distribution of actins in postnuclear supernatant and total cell homogenate also indicates that none of the actins is unique to the nucleus, in agreement with results using other cell types (5). The inability to demonstrate structural or functional specificity for isoelectric variants of actin in nonmuscle cells by cell fractionation suggests that these variants lack specific roles and instead are completely interchangeable. Alternatively, specificity may be found only in highly specialized substructures of the cell. In that case, the presence of multiple species of actin attached to the plasma membrane might reflect its multiple functions (e.g., membrane motility, cell adhesion, endocytosis). Isolation of the
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ACTINS TABLE TRYPSIN
RESISTANCE
OF MEMBRANE Actin (cpm “jS1
Sample Plasma membrane, Plasma membrane, Plasma membrane. + Triton X-100
2
untreated +trypsin +trypsin
ACTIN” (%) Trypsin resistant
2436 98
4
8
0
U Replicate samples of plasma membrane isolated from [:?5]methionine-labeled CEF were analyzed on a 10% acrylamide slab gel after no further treatment (untreated), digestion with 0.025% trypsin (+trypsin). or incubation with 5% Triton X-100 followed by digestion with trypsin (+trypsin + Triton X-100). Actin bands were cut from the gels and radioactivity was determined by scintillation counting. Values shown have been corrected for background counts.
retricted portion of plasma membrane involved in a particular function, such as formation of a microvillus or phagocytic vacuole, might then be expected to produce a fraction with a distinct composition of actins compared to the total membrane actin. A recent publication (17) reporting the threefold enrichment of two minor species of rat brain actin in the specialized brain synapse fraction tends to support this latter alternative. However it is clear that further work will be required before we understand the biological significance of the different forms of actin. ACKNOWLEDGMENTS Janet Tannenbaum Dystrophy Society
is a Fellow of the Muscular of America at Massachusetts In-
FIG. 3. (A-D). Region of autoradiograms containing actin in Figs. 1 and 2. Positions of three major ((Y. p, and y) and two minor (6, E) species of actin are indicated; acidic end of gradient is towards the right. Cell homogenate (A) and plasma membrane (B) of cells labeled 1 h show 6 and t species; these are barely visible in cell homogenate (C) and plasma membrane (D) labeled 18 h. (E) Comigration of labeled actin in CEF plasma membrane with purified n-actin marker. A mixture of unlabeled (Y actin and “5S-labeled plasma membrane (one third the amount used in (D) was analyzed by twodimensional electrophoresis. Circle (arrow) indicates position of Coomassie blue-stained actin marker; only the actin region of the autoradiogram is shown.
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stitute of Technology, Cambridge, Massachusetts. We thank E. Helitzer for help in obtaining Figures l-3 and J. Simpson for secretarial assistance. This work was supported by grants from the NIH, NSF, NASA, and the American Cancer Society.
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