Cell Biology
International
Reports,
75
Vol. 15, No. I, 7997
SOME OF EUKARYOTIC ELONGATION FACTOR 2 IS COLOCALIZED WITH ACTIN MICROFILAMENT
BUNDLES
IN MOUSE EMBRYO FIBROBLASTS
E.A.Shestakova,
L.P.Motuz,
A.A.Minin,
V.I.Gelfand,
and
L.P.Gavrilova'
Institute the
Academy of Sciences of Protein Research, USSR, Pushchino, Moscow Region, 142292, USSR
of
ABSTRACT Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts.The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables with actin coincided bundles of microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with EF-2 the antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.
1
To whom correspondence
0309-l
651/91/010075-1
O/$03.00/0
should
be addressed
0 1991 Academic
Press Ltd
76
Cell Biology
International
Reports,
Vol. 75, No. 1, 1991
INTRODUCTION There are some data that in eukaryotic cells components involved in protein synthesis could be bound to the cytoskeletal structures. Biochemical results showed that polysomes, mRNA and several initiation factors bound to the Triton-insoluble material of the cells where all main cytoskeletal structures were preserved (Lenk et al., 1977; Bonneau et al., 1985; Howe and Hershey, 1984). Using immunogold microscopy Singer et al. (1989) showed, that actin, tubulin and vimentin mRNAs bound to actin filaments . Zumbe et al. (1982) claimed that the antibody to the 50 kDa mRNA cap-binding protein stained the intermediate filaments in BHK-21 cells . In our earlier work we showed that EF-2, the eukaryotic initiation factor 2 (eIF-2) and ribosomes in mouse embryo fibroblasts were located mainly in the perinuclear region of these cells (Gavrilova et al., 1987). However, in many cells we observed also staining of the straight cables similar in the distribution to that of the actin microfilament bundles. Here we studied this phenomenon in greater detail and found that EF-2 is many (but not located along all) of the actin microfilament bundles in the embryo cultured mouse fibroblasts. MATERIALS
AND METHODS
Immunization of the animals was done Antibodies. with EF-2 purified from rat liver according to Motuz et the purification of rat liver EF-2 al. (1984). In brief, A crude supernatant fraction from rat was as follows. livers was subsequently purified by chromatography on phosphocellulose, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex A-50 and Ultrogel AcA 44. EF-2 preparation pure judged SDS-polyacrylamide was by gel assayed by its ADP-ribosylation electrophoresis and catalyzed by the A-fragment of diphtheria toxin and by system of poly-Phe activity in poly(U)-dependent its synthesis. Rabbits were immunized with EF-2 denatured with 5% native the with the acid and goats trichloroacetic in phosphate buffered saline (PBS). protein solution After denaturation the protein pellets were washed with acetone, dried and resuspended in PBS.The suspension or protein solution were emulsified with complete Freund's adjuvant at a ratio of I:1 (v/v) and injected into the popliteal lymph nodes (0.5 mg per node). Rabbits were boosted in an ear vein with a solution of EF-2 in PBS 40
Cell Biology International
Reports, Vol. 15, No. 7, 7997
77
and 70 days later (0.5 mg per injection) and bled 7-14 Immunoglobulins were injection. days after the last chromatography on isolated from the serum by DEAE-cellulose. Goats were boosted by injections of the mixture of the EF-2 solution (0.5 mg per injection) with incomplete Freund's adjuvant into the popliteal lymph nodes 10, 20, 34 and 37 days after first immunization and bled 3 days were obtained using the last boost. after 1t.S DEAE-cellulose chromatography. The monospecific rabbit or goat EF-2 antibodies fractions using an were purified from the I&G EF-Z-Sepharose column (1 mg of EF-2 per ml of Sepharose Antibodies were eluted with 3M KSCN and the salt gel). was removed by gel-filtration on a Sephadex G-50 column. SDS-polyacrylamide Biochemical methods. gel according to electrophoresis was performed Laemmli (1970). For immunoblotting the proteins were transferred to nitrocellulose according to Kyhse-Andersen (1984). (the EF-2 antibodies) Binding of the first antibodies were second antibodies (rabbit visualized using the antibodies) conjugated anti-goat or goat anti-rabbit Chemical Co.). with peroxidase (Sigma Peroxidase-conjugated antibodies were used at a dilution of 1:lOO. Cells. Mouse embryo fibroblasts were obtained by trypsinization of 15-17 day embryos and cultured in L-15 medium (Flow Laboratories) supplemented with 10% fetal streptomycin. calf serum, penicillin and Secondary cultures were plated o?to coverslips at a density of about 7000 cells per cm and were grown for 24-96 hrs. To obtain a detergent-insoluble material where all main cellular cytoskeletal structures were preserv?$ the cells grown on the coverslips were washed with Ca -free PBS and extracted before fixation for 3 min at room temperature with 1% Triton X-100 in buffer M (50 mM imidazole-HCl pH 6.8, 50 mM KCl, 2 mM MgC12, 1 mM 2-mercaptoethanol, 1 mM EGTA, 0.1 mM EDTA) supplemented with 4% polyethylene glycol 40,000 (Bershadsky et al., 1978). After extraction the Triton-insoluble material was washed with buffer M. The Triton-extracted and non-extracted cells were fixed with 4% formaldehyde in PBS for at least IO-15 min at room temperature and then washed with PBS. Immunofluorescence staining and microscopy. Fixed and washed cells were treated with 1% Triton X-100 in PBS for 'IO rllin at room temperature. This procedure was done to make membrane permeable to antibodies and was used for two cell samples, one of which was extracted with Triton X-100 prior to fixation and the other was fixed without preliminary Triton-treatment. Sometimes instead of 1% Triton X-100 fixed cells were treated at
78
Cell Biology
International
Reports,
Vol. 75, No. 7, 1997
4OC with a 1:l mixture of acetone and water for 3 min. then with pure acetone for 5 min and again with the acetone-water mixture for 3 min. After those treatments the cells were washed with PBS and stained for 50-60 min at room temperature with primary antibodies. The concentrations of antibodies were 3 rig/ml for the rabbit antibodies and 4-30 pg/ml for the goat antibodies to EF-2. After extensive washing the cells were incubated with secondary fluorescein-conjugated antibodies (Sigma Chemical Co.), diluted I:100 (anti-rabbit) and 1:300 (anti-goat). If simultaneous visualization of actin was required, rhodaminyl-phalloidin (0.03 pg/ml) was added to the solution of secondary antibodies. Controls of staining specificity included incubation with non-immune neutralization of antibodies with the excess of I&, EF-2 or omission of the primary antibodies. All the controls were negative. Cells were observed with a Photomicroscope III (Opton) equipped with phase-contrast and epifluorescent optics and filter sets 487714 and 487710 (selective excitation) for rhodamine and fluorescein, respectively. RESULTS The distribution of EF-2 in formaldehyde fixed fibroblasts was studied by indirect mouse embryo Results of staining with immunofluorescence technique. the goat antibodies to EF-2 are presented in Fig. IA,C. These antibodies stained the area around the nucleus. were stained also with the The straight threads and the distribution of the antibodies. The shape filaments stained with the EF-2 antibodies resembled that of the actin microfilament bundles. For a direct comparison of the two sets of filaments we stained the antibodies and cell with the EF-2 same 1 A-D). It is seen that the rhodaminyl-phalloidin (Fig. bind also filaments stained by the EF-2 antibodies EF-2 containing Consequently, rhodaminyl-phalloidin. filaments coincided with actin microfilament bundles. actin all the that not should be noted It EF-2 were stained with the bundles microfilament Most often the actin microfilament bundles antibodies. located at the cell periphery contained no EF-2 (at least no EF-2 accessible to the antibodies). EF-2 was microfilament actin from some of the also absent junctions (Fig. 1). It is interesting to note that EF-2 was also found in the polygonal net of microfilaments above the nucleus (Fig. 1 C-E) (Lazarides, 1976 A) antibodies fibroblasts EF-2 embryo In mouse showed a distribution similar to developed in rabbit
Cell Biology
International
Reports,
Vol. 15, No. 1, 1997
staining Fig. 1. Double embryo mouse of with goat fibroblasts antibodies to EF-2 (A,C), and tetramethylrhodaminyl actin (for phalloidin microfilament detection) Phase-contrast (B,D). the same cell image of (E). Scale bar, 20 pm.
80
Cell Biology International
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that of the EF-2 goat antibodies. Again the actin microfilament bundles crossing the cells could bind the EF-2 rabbit antibodies while some of the thick bundles were almost not stained (Fig. 2). To study the strength of retaining of EF-2 along actin microfilament bundles, native cells were extracted X-100 in buffer M (see before fixation with 1% Triton Materials and Methods). This treatment had no effect on the distribution of actin microfilament bundles in the cultured fibroblasts (Fig. but practically 3B) completely removed EF-2 from the cells (Fig. 3A).These results were confirmed by immunoblotting experiments which also showed that EF-2 was absent from the Triton -extracted residue of the non-fixed cells rather than being just masked from staining by protein any molecules after such Triton X-100 treatment (Fig. 5 B,C, lanes 2). These data do not agree with those published previously which indicated that native cells treated
Fig.
2. Double staining of mouse embryo fibroblasts to rabbit antibodies EF-2 (A), with tetramethylrhodaminyl-phalloidin (B). bar, 20 m.
with and Scale
with Triton X-100 before fixation sometimes showed the bundles with the microfilament actin staining of 1987). We (Gavrilova et al., to EF-2 antibodies consider it was the result of a noncomplete extraction of Triton-soluble material from the cells. staining the that some evidences We obtained reported here is not the result of nonspecific binding of the EF-2 antibodies to actin microfilament bundles. mouse embryo homogenates of intact First, in the fibroblasts the EF-2 antibodies recognized practically mobility electrophoretic with the polypeptide one identical to that of EF-2 (Fig. 5). Thus, the antibodies themselves have a high specificity. Second, the staining two different batches of using obtained patterns
CeliGiology
International
Reports,
Vol. 75, No. 7, 1991
81
antibodies (goat and rabbit) were similar, minimizing the probability of a minor impurity in the antibody 1 and 2). Third, the ten times excess preparation (Fig. of rat liver EF-2 prevented staining of the cells with antibodies both preparations of EF-2 (Fig.4 A), suggesting binding to the that the antibody microfilament bundles was via the antigen-binding site IgG from preimmune sera of of the antibodies. Fourth, both the rabbit and the goat used to raise antibodies at the concentrations of at least gave no staining two-fold greater than those required to give a bright fluorescence with the antibodies. Fifth, not all the were stained by the actin microfilament bundles
Fig.
3.
X-100 extraction on mouse Effect of Triton fibroblasts embryo staining with rabbit antibodies to EF-2. Native cells were treated with Triton X-100 before fixation. EF-2 (A) antibody staining; staining with (B) actin tetramethylrhodaminyl-phalloidin. Scale bar, 20
Fig.
4.
pm.
Effect of preincubation of EF-2 rabbit antibodies with ten-fold EF-2 excess. Cells were fixed without preliminary Triton treatment. (A) EF-2 antibody staining; (B) actin staining with tetramethylrhodaminyl-phalloidin. Scale bar, 20 w.
82
Cell Biology
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Reports,
Vol. 15, No. 1, 1991
antibodies, suggesting that the non-specific staining of fibers is the stress highly 1,2). unlikely (Fig. Finally, when cells were extracted with Triton X-100 EF-2 antibody binding was not observed before fixation by immunofluoresience microscopy while staining of actin microfilament bundles with rhodaminyl-phalloidin was not changed (Fig. 3). These results were confirmed by immunoblotting experiments which also indicated that Triton treatment of native cells removed EF-2 from the cytoplasm (Fig. 5 B,C, lanes 2). DISCUSSION We have prepared antibodies to rat liver EF-2 and used them to localize the corresponding antigen in fixed mouse embryo fibroblasts. Previous studies of EF-2 localization by immunocytochemistry have demonstrated a high concentration of EF-2 in the area near the nucleus 1987). Here we show that together (Gavrilova et al., with the distribution in the perinuclear region EF-2 actin antibodies also stains O’f the bundles microfilaments. shown that EF-2 is localized the Having along A
C
II-
94
),
y
67 43
30
( M,J
Fig.
5.
23
1
2
1
2
3
Immunoblotting of mouse embryo fibroblast antibodies to lysate with rabbit and goat of molecular mass EF-2. Lane Mw, the migrat.on of intact kDa standards; lane 1, proteins remained that cells; lane 2, proteins X-100 extraction; lane insoluble after Triton liver EF-2. (A) Coomassie staining of 3, rat (B) immunoblots of the proteins the gel; labeled with rabbit antibodies to EF-2; (C) immunoblots of the proteins labeled with goat antibodies to EF-2.
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83
bundles of actin microfilaments it is interesting to ask what function it might serve there. One possibility is that microfilament-bound EF-2 is involved in protein synthesis on mRNAs for proteins without signal peptides. Kecent studies have indicated that EF-2 in animal phosphorylated and that cells may be reversibly phosphorylated EF-2 is inactive in protein synthesis 1987; Ryazanov, 1987; Ryazanov et al., (Palfrey et al., 1988). Another possibility is that one of these forms of EF-2 (either phosphorylated or non-phosphorylated) has an affinity to stress fibers. fact is that not all actin The interesting with EF-2 microfilament bundles were stained the EF-2 Probably selective localization of antibodies. with a bundles correlates along some microfilament similar distribution of some actin-binding proteins. For example, it is known that tropomyosin is absent. from some of the stress-fibers (Lazarides, 1976 B). Therefore suggestion is that EF-2 binding to the a reasonable stress fibers could be mediated by actin-binding protein rather than by a direct interaction between actin and EF-2. At least it is likely that EF-2 is not an integral the stress-fibers as it was completely protein of extracted from the cytoskeleton by 1% Triton X-100. Further studies are required to determine whether microfilament-bound EF-2 is active in protein synthesis and which proteins are synthesized using this fraction of EF-2. ACKNOWLEDGEMENTS We are grateful to Professor A.S.Spirin for permanent interest in our work, to Professor Th. Wieland (Max Plank Institute for Medical Research, Heidelberg, FRG) for the gift of rhodaminyl-phalloidin, to Mrs. V.P.Burmistrova for expert technical assistance. REFERENCES Bershadsky, A.D., Gelfand, V.G., Svitkina, T.M., and Tint, 1-S. (1978) Microtubules in mouse embryo fibroblasts extracted with Triton X-100. Cell Viol. lnt. Rep. 2, 425-432. Bonneau, A.-M., Darveau, A., and Sonenberg, N. (1985) Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure. J. Cell Biol. 100, 1209-1218. Gavrilova, L.P., Rutkevitch, N.M., Gelfand, V.I., Motuz, L.P., Stahl, J., Bommer, U.-A., and Bielka, H. (1937)
84
Cell Biology
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Reports,
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Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts. Cell Biol. Int. Rep. 11, 745-753. Howe, J.G., Hershey, J.W.B. (1984) Translation initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells. Cell 37, 85-93. Kyhse-Andersen, J. (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. 10, Biophys. Meth. 203-209.
Laemmli, during
U.K. the
assembly
Nature
227,
680-685.
(1970)
Cleavage of of the head
structural proteins of bacteriophage T4.
Lazarides, E. (1976 A) Actin, alpha-actinin and tropomyosin interaction ' the structural organization of actin filamenltns in nonmuscle cells. J. Cell Biol. 68, 202-219. Lazarides, E. (1976 B) Two general classes of actin filaments in tissue culture cells: the role of tropomyosin. J. Supramol. Struct. 5, 531-563. Lenk, R.,Ransom, L., Kaufmann, Y., and Penman Sh. (1977) A cytoskeletal structure associated with polyribosomes obtained from HeLa cells. Cell 10, 67-78. Motuz,
L.P., Tsyuryupa, G.P., Aprikyan, A.G., and Ovodov, Isolation S.Yu. (1984) comparative and physicochemical characteristics of elongation factor EF-2 from rat liver and cyst Artemia Sali.na. 16 th Meeting FEBS (Moscow, 25-30, June 1984): Palfrey, H.C., Nairn, A.C., Muldoon, L.L., and activation Villereal, M.L. (1987) Rapid of calmodulin-dependent protein III kinase in mitogen-stimulated human fibroblasts. J. Biol. Chem. 262,
9785-9792.
Ryazanov, A.G. phosphorylation 214,
(1987)
of
elongation
Ca'+/calmodulin-dependent factor 2. FEBS Lett.
331-334.
Ryazanov,
A.G., Shestakova, Phosphorylation of affects rate of
(1988)
kinase
and Natapov, P.G. E.A., elongation factor 2 by EF-2 Nature 334, translation.
170-173.
R.H.,Langevin, G.L., and Lawrence, J.B. Singer, Ultrastructural visualization of cytoskeletal and their associated proteins using double-label situ hybridization. J. Cell Biol. 108, 2343-2353. and Trachsel, H. Zumbe, A., Staehli, C., Association of a Mr 50000 cap-binding protein the cytoskeleton in baby hamster kidney cells. Natl. Acad. Sci. USA 79, 2927-2931. Paper
received
29.06.90.
Revised
paper
accepted
09.11.90.
(1989)
mRNAs in (1982)
with Proc.
International
Reports,
75
Vol. 15, No. I, 7997
SOME OF EUKARYOTIC ELONGATION FACTOR 2 IS COLOCALIZED WITH ACTIN MICROFILAMENT
BUNDLES
IN MOUSE EMBRYO FIBROBLASTS
E.A.Shestakova,
L.P.Motuz,
A.A.Minin,
V.I.Gelfand,
and
L.P.Gavrilova'
Institute the
Academy of Sciences of Protein Research, USSR, Pushchino, Moscow Region, 142292, USSR
of
ABSTRACT Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts.The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables with actin coincided bundles of microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with EF-2 the antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.
1
To whom correspondence
0309-l
651/91/010075-1
O/$03.00/0
should
be addressed
0 1991 Academic
Press Ltd
76
Cell Biology
International
Reports,
Vol. 75, No. 1, 1991
INTRODUCTION There are some data that in eukaryotic cells components involved in protein synthesis could be bound to the cytoskeletal structures. Biochemical results showed that polysomes, mRNA and several initiation factors bound to the Triton-insoluble material of the cells where all main cytoskeletal structures were preserved (Lenk et al., 1977; Bonneau et al., 1985; Howe and Hershey, 1984). Using immunogold microscopy Singer et al. (1989) showed, that actin, tubulin and vimentin mRNAs bound to actin filaments . Zumbe et al. (1982) claimed that the antibody to the 50 kDa mRNA cap-binding protein stained the intermediate filaments in BHK-21 cells . In our earlier work we showed that EF-2, the eukaryotic initiation factor 2 (eIF-2) and ribosomes in mouse embryo fibroblasts were located mainly in the perinuclear region of these cells (Gavrilova et al., 1987). However, in many cells we observed also staining of the straight cables similar in the distribution to that of the actin microfilament bundles. Here we studied this phenomenon in greater detail and found that EF-2 is many (but not located along all) of the actin microfilament bundles in the embryo cultured mouse fibroblasts. MATERIALS
AND METHODS
Immunization of the animals was done Antibodies. with EF-2 purified from rat liver according to Motuz et the purification of rat liver EF-2 al. (1984). In brief, A crude supernatant fraction from rat was as follows. livers was subsequently purified by chromatography on phosphocellulose, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex A-50 and Ultrogel AcA 44. EF-2 preparation pure judged SDS-polyacrylamide was by gel assayed by its ADP-ribosylation electrophoresis and catalyzed by the A-fragment of diphtheria toxin and by system of poly-Phe activity in poly(U)-dependent its synthesis. Rabbits were immunized with EF-2 denatured with 5% native the with the acid and goats trichloroacetic in phosphate buffered saline (PBS). protein solution After denaturation the protein pellets were washed with acetone, dried and resuspended in PBS.The suspension or protein solution were emulsified with complete Freund's adjuvant at a ratio of I:1 (v/v) and injected into the popliteal lymph nodes (0.5 mg per node). Rabbits were boosted in an ear vein with a solution of EF-2 in PBS 40
Cell Biology International
Reports, Vol. 15, No. 7, 7997
77
and 70 days later (0.5 mg per injection) and bled 7-14 Immunoglobulins were injection. days after the last chromatography on isolated from the serum by DEAE-cellulose. Goats were boosted by injections of the mixture of the EF-2 solution (0.5 mg per injection) with incomplete Freund's adjuvant into the popliteal lymph nodes 10, 20, 34 and 37 days after first immunization and bled 3 days were obtained using the last boost. after 1t.S DEAE-cellulose chromatography. The monospecific rabbit or goat EF-2 antibodies fractions using an were purified from the I&G EF-Z-Sepharose column (1 mg of EF-2 per ml of Sepharose Antibodies were eluted with 3M KSCN and the salt gel). was removed by gel-filtration on a Sephadex G-50 column. SDS-polyacrylamide Biochemical methods. gel according to electrophoresis was performed Laemmli (1970). For immunoblotting the proteins were transferred to nitrocellulose according to Kyhse-Andersen (1984). (the EF-2 antibodies) Binding of the first antibodies were second antibodies (rabbit visualized using the antibodies) conjugated anti-goat or goat anti-rabbit Chemical Co.). with peroxidase (Sigma Peroxidase-conjugated antibodies were used at a dilution of 1:lOO. Cells. Mouse embryo fibroblasts were obtained by trypsinization of 15-17 day embryos and cultured in L-15 medium (Flow Laboratories) supplemented with 10% fetal streptomycin. calf serum, penicillin and Secondary cultures were plated o?to coverslips at a density of about 7000 cells per cm and were grown for 24-96 hrs. To obtain a detergent-insoluble material where all main cellular cytoskeletal structures were preserv?$ the cells grown on the coverslips were washed with Ca -free PBS and extracted before fixation for 3 min at room temperature with 1% Triton X-100 in buffer M (50 mM imidazole-HCl pH 6.8, 50 mM KCl, 2 mM MgC12, 1 mM 2-mercaptoethanol, 1 mM EGTA, 0.1 mM EDTA) supplemented with 4% polyethylene glycol 40,000 (Bershadsky et al., 1978). After extraction the Triton-insoluble material was washed with buffer M. The Triton-extracted and non-extracted cells were fixed with 4% formaldehyde in PBS for at least IO-15 min at room temperature and then washed with PBS. Immunofluorescence staining and microscopy. Fixed and washed cells were treated with 1% Triton X-100 in PBS for 'IO rllin at room temperature. This procedure was done to make membrane permeable to antibodies and was used for two cell samples, one of which was extracted with Triton X-100 prior to fixation and the other was fixed without preliminary Triton-treatment. Sometimes instead of 1% Triton X-100 fixed cells were treated at
78
Cell Biology
International
Reports,
Vol. 75, No. 7, 1997
4OC with a 1:l mixture of acetone and water for 3 min. then with pure acetone for 5 min and again with the acetone-water mixture for 3 min. After those treatments the cells were washed with PBS and stained for 50-60 min at room temperature with primary antibodies. The concentrations of antibodies were 3 rig/ml for the rabbit antibodies and 4-30 pg/ml for the goat antibodies to EF-2. After extensive washing the cells were incubated with secondary fluorescein-conjugated antibodies (Sigma Chemical Co.), diluted I:100 (anti-rabbit) and 1:300 (anti-goat). If simultaneous visualization of actin was required, rhodaminyl-phalloidin (0.03 pg/ml) was added to the solution of secondary antibodies. Controls of staining specificity included incubation with non-immune neutralization of antibodies with the excess of I&, EF-2 or omission of the primary antibodies. All the controls were negative. Cells were observed with a Photomicroscope III (Opton) equipped with phase-contrast and epifluorescent optics and filter sets 487714 and 487710 (selective excitation) for rhodamine and fluorescein, respectively. RESULTS The distribution of EF-2 in formaldehyde fixed fibroblasts was studied by indirect mouse embryo Results of staining with immunofluorescence technique. the goat antibodies to EF-2 are presented in Fig. IA,C. These antibodies stained the area around the nucleus. were stained also with the The straight threads and the distribution of the antibodies. The shape filaments stained with the EF-2 antibodies resembled that of the actin microfilament bundles. For a direct comparison of the two sets of filaments we stained the antibodies and cell with the EF-2 same 1 A-D). It is seen that the rhodaminyl-phalloidin (Fig. bind also filaments stained by the EF-2 antibodies EF-2 containing Consequently, rhodaminyl-phalloidin. filaments coincided with actin microfilament bundles. actin all the that not should be noted It EF-2 were stained with the bundles microfilament Most often the actin microfilament bundles antibodies. located at the cell periphery contained no EF-2 (at least no EF-2 accessible to the antibodies). EF-2 was microfilament actin from some of the also absent junctions (Fig. 1). It is interesting to note that EF-2 was also found in the polygonal net of microfilaments above the nucleus (Fig. 1 C-E) (Lazarides, 1976 A) antibodies fibroblasts EF-2 embryo In mouse showed a distribution similar to developed in rabbit
Cell Biology
International
Reports,
Vol. 15, No. 1, 1997
staining Fig. 1. Double embryo mouse of with goat fibroblasts antibodies to EF-2 (A,C), and tetramethylrhodaminyl actin (for phalloidin microfilament detection) Phase-contrast (B,D). the same cell image of (E). Scale bar, 20 pm.
80
Cell Biology International
Reports, Vol. 15, No. 1, 1991
that of the EF-2 goat antibodies. Again the actin microfilament bundles crossing the cells could bind the EF-2 rabbit antibodies while some of the thick bundles were almost not stained (Fig. 2). To study the strength of retaining of EF-2 along actin microfilament bundles, native cells were extracted X-100 in buffer M (see before fixation with 1% Triton Materials and Methods). This treatment had no effect on the distribution of actin microfilament bundles in the cultured fibroblasts (Fig. but practically 3B) completely removed EF-2 from the cells (Fig. 3A).These results were confirmed by immunoblotting experiments which also showed that EF-2 was absent from the Triton -extracted residue of the non-fixed cells rather than being just masked from staining by protein any molecules after such Triton X-100 treatment (Fig. 5 B,C, lanes 2). These data do not agree with those published previously which indicated that native cells treated
Fig.
2. Double staining of mouse embryo fibroblasts to rabbit antibodies EF-2 (A), with tetramethylrhodaminyl-phalloidin (B). bar, 20 m.
with and Scale
with Triton X-100 before fixation sometimes showed the bundles with the microfilament actin staining of 1987). We (Gavrilova et al., to EF-2 antibodies consider it was the result of a noncomplete extraction of Triton-soluble material from the cells. staining the that some evidences We obtained reported here is not the result of nonspecific binding of the EF-2 antibodies to actin microfilament bundles. mouse embryo homogenates of intact First, in the fibroblasts the EF-2 antibodies recognized practically mobility electrophoretic with the polypeptide one identical to that of EF-2 (Fig. 5). Thus, the antibodies themselves have a high specificity. Second, the staining two different batches of using obtained patterns
CeliGiology
International
Reports,
Vol. 75, No. 7, 1991
81
antibodies (goat and rabbit) were similar, minimizing the probability of a minor impurity in the antibody 1 and 2). Third, the ten times excess preparation (Fig. of rat liver EF-2 prevented staining of the cells with antibodies both preparations of EF-2 (Fig.4 A), suggesting binding to the that the antibody microfilament bundles was via the antigen-binding site IgG from preimmune sera of of the antibodies. Fourth, both the rabbit and the goat used to raise antibodies at the concentrations of at least gave no staining two-fold greater than those required to give a bright fluorescence with the antibodies. Fifth, not all the were stained by the actin microfilament bundles
Fig.
3.
X-100 extraction on mouse Effect of Triton fibroblasts embryo staining with rabbit antibodies to EF-2. Native cells were treated with Triton X-100 before fixation. EF-2 (A) antibody staining; staining with (B) actin tetramethylrhodaminyl-phalloidin. Scale bar, 20
Fig.
4.
pm.
Effect of preincubation of EF-2 rabbit antibodies with ten-fold EF-2 excess. Cells were fixed without preliminary Triton treatment. (A) EF-2 antibody staining; (B) actin staining with tetramethylrhodaminyl-phalloidin. Scale bar, 20 w.
82
Cell Biology
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Reports,
Vol. 15, No. 1, 1991
antibodies, suggesting that the non-specific staining of fibers is the stress highly 1,2). unlikely (Fig. Finally, when cells were extracted with Triton X-100 EF-2 antibody binding was not observed before fixation by immunofluoresience microscopy while staining of actin microfilament bundles with rhodaminyl-phalloidin was not changed (Fig. 3). These results were confirmed by immunoblotting experiments which also indicated that Triton treatment of native cells removed EF-2 from the cytoplasm (Fig. 5 B,C, lanes 2). DISCUSSION We have prepared antibodies to rat liver EF-2 and used them to localize the corresponding antigen in fixed mouse embryo fibroblasts. Previous studies of EF-2 localization by immunocytochemistry have demonstrated a high concentration of EF-2 in the area near the nucleus 1987). Here we show that together (Gavrilova et al., with the distribution in the perinuclear region EF-2 actin antibodies also stains O’f the bundles microfilaments. shown that EF-2 is localized the Having along A
C
II-
94
),
y
67 43
30
( M,J
Fig.
5.
23
1
2
1
2
3
Immunoblotting of mouse embryo fibroblast antibodies to lysate with rabbit and goat of molecular mass EF-2. Lane Mw, the migrat.on of intact kDa standards; lane 1, proteins remained that cells; lane 2, proteins X-100 extraction; lane insoluble after Triton liver EF-2. (A) Coomassie staining of 3, rat (B) immunoblots of the proteins the gel; labeled with rabbit antibodies to EF-2; (C) immunoblots of the proteins labeled with goat antibodies to EF-2.
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bundles of actin microfilaments it is interesting to ask what function it might serve there. One possibility is that microfilament-bound EF-2 is involved in protein synthesis on mRNAs for proteins without signal peptides. Kecent studies have indicated that EF-2 in animal phosphorylated and that cells may be reversibly phosphorylated EF-2 is inactive in protein synthesis 1987; Ryazanov, 1987; Ryazanov et al., (Palfrey et al., 1988). Another possibility is that one of these forms of EF-2 (either phosphorylated or non-phosphorylated) has an affinity to stress fibers. fact is that not all actin The interesting with EF-2 microfilament bundles were stained the EF-2 Probably selective localization of antibodies. with a bundles correlates along some microfilament similar distribution of some actin-binding proteins. For example, it is known that tropomyosin is absent. from some of the stress-fibers (Lazarides, 1976 B). Therefore suggestion is that EF-2 binding to the a reasonable stress fibers could be mediated by actin-binding protein rather than by a direct interaction between actin and EF-2. At least it is likely that EF-2 is not an integral the stress-fibers as it was completely protein of extracted from the cytoskeleton by 1% Triton X-100. Further studies are required to determine whether microfilament-bound EF-2 is active in protein synthesis and which proteins are synthesized using this fraction of EF-2. ACKNOWLEDGEMENTS We are grateful to Professor A.S.Spirin for permanent interest in our work, to Professor Th. Wieland (Max Plank Institute for Medical Research, Heidelberg, FRG) for the gift of rhodaminyl-phalloidin, to Mrs. V.P.Burmistrova for expert technical assistance. REFERENCES Bershadsky, A.D., Gelfand, V.G., Svitkina, T.M., and Tint, 1-S. (1978) Microtubules in mouse embryo fibroblasts extracted with Triton X-100. Cell Viol. lnt. Rep. 2, 425-432. Bonneau, A.-M., Darveau, A., and Sonenberg, N. (1985) Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure. J. Cell Biol. 100, 1209-1218. Gavrilova, L.P., Rutkevitch, N.M., Gelfand, V.I., Motuz, L.P., Stahl, J., Bommer, U.-A., and Bielka, H. (1937)
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