3 4 5 6 7 8. ~". 9 IO. -. A - r. 8. 4 a. FIG. 4. Immunoprecipitation of endodermal cells, HeLa cells, and mouse keratinocytes with endo B and keratin anti- sera. a ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256,No. 15,Issue of August 10,pp. 8124-8133, 1981 Prrnled in U.S.A.
Identification and Immunoprecipitationof Cytoskeletal Proteinsfrom Murine Extra-embryonic EndodermalCells* (Received for publication, February 18, 1981,and in revised form, April 23, 1981)
Robert G. Oshima From theDepartment of Biology, Massachusetts Instituteof Technology, Cambridge, Massachusetts 02139
Murine extra-embryonicendodermal cells derived Embryonal carcinoma cells are the stemcells of teratocarfrom either teratocarcinomasor cultured mouse blas- cinomas (Kleinsmith and Pierce, 1964).Developmentally plutocysts contain two protein species of M, = 55,000 and ripotent EC’ cells are able to produce a variety of differenM, = 50,000 endodermal cytoskeletal proteins Aand B, tiated cell types in vivo and in vitro (Martin, 1975; Hogan, respectively) that are insoluble in nonionic detergent 1977). The differentiation in vitro of murine embryonal carand 1 M NaCl and are not found in abundance in em- cinoma cells to nonmalignant parietal and visceral extra-embryonal carcinoma cells, the stem cells of teratocarci- bryonic endodermal cells is accompanied by significant nomas.Antiserumraised against the electrophoreti- changes in cellular morphology and gene expression. EC cells cally purified endoB protein immunoprecipitated endo have little cytoplasm, grow in tight clusters with indistinct cell B from [36S]methionine-labeled cell lysates of three pa- borders, and display little or no density-dependent inhibition rietal endodermalcell lines, apresumptivevisceral endodermal cell line, and a mouse hepatoma line. Im- of growth (Pierce andBeals, 1964; Finch and Ephrussi, 1967). munoprecipitable endoB was not foundin murine em- EC cells have relatively high Ievels of alkaline phosphatase bryonal carcinoma cells, fibroblasts, myoblasts, kera- (Bemstine et al., 1973), display antigens found on early emtinocytes, erythroleukemicorneuroblastoma cells. bryonic cells (Jacob, 1977; Solter and Knowles, 1978),and do These results are consistent with the view that endoB not secrete significant amounts of plasminogen activator. Extra-embryonic endodermal cells are the fiist differenis not tubulin, vimentin, desmin, or keratin. Amino acid composition data, partial peptideanalysis of immuno- tiated cells to appear when certain murine embryonal carciprecipitated endoB, and immunoprecipitationanalysis noma cell lines are permitted to differentiate in vitro (Lehman withantikeratinserumsupportthe suggestion that et al., 1974; Martin and Evans, 1975; McBurney, 1976). Both parietal and visceral endoderm may appear in these cultures endo B is not a keratin. Indirect immunofluorescent staining of parietal endodermal cells with the endo B (Martin et al., 1977; Adamson et al., 1977),and long term cell antiserumresulted in the fluorescence of afibrillar lines of both types have been isolated from differentiated cytoskeletal network. The synthesis ofendo B was cultures of EC cells (Lehman et al., 1974; Chung et al., 1977; increaseddramaticallywhenembryonalcarcinoma Adamson et al., 1977). Cells with parietal endodermal charcells were induced to differentiate by treatment with acteristics appear when EC cell lines which normally differretinoic acid.Endo B appears to be a cytoskeletal pro- entiate infrequently are exposed to retinoic acid (Strickland tein that is synthesizedwhenmalignantembryonal and Mahdavi, 1978; Jetten et al., 1979: Strickland et al., 1980; carcinoma cells differentiate to benign extra-embry- Oshima and Linney, 1980). onic endoderm. In the present study, a cytoskeletal protein found in extraembryonic endodermal cells was isolated. Immunoprecipitation analysis indicated that the protein is found in a murine Trophectoderm is the fiist differentiated cell type to appear fetal hepatoma line as well as extra-embryonic endoderm but during the development of the mouse embryo (Snell and is not abundant in murine embryonal carcinoma cells, fibroStevens, 1966; Rossant and Papaiannou, 1977).Cavitation and blasts, myoblasts, keratinocytes, or neuroblastoma cells. The expansion of the trophectodermal outer layer of cells result in synthesis of the protein is increased greatly in embryonal the blastocyst stage embryo. Primitive extra-embryonic en- carcinoma cultures induced to differentiate by treatment with doderm then appears on the surface of the inner cell mass of retinoic acid. the blastocyst and generates visceral and parietal endoderm. EXPERIMENTAL PROCEDURES Extra-embryonic endoderm is used here in thegeneral sense, Cells-The F9.22 EC cell line is a mycoplasma-free subclone (Oshto distinguish these tissues from definitive embryonic endoderm (see Sherman, 1977). Visceralendoderm is incorporated h a , 1978) of the F9 line (Bernstine et al., 1973).FOT5 is a ouabaininto the yolk sac and is responsible for the synthesis of a- and thioguanine-resistant subclone of F9.22. The D5fibroblastic line was derived from the differentiation in oitro of PSA4 embryoid fetoprotein (Dziadek and Adamson, 1978).Parietal endoderm bodies? The PSAi EC and S T 0 fibroblast lines (Martin and Evans, migrates along the inner surface of the blastocoel and secretes 1975) were obtained from Dr. Gail Martin. Primary endodermal cells a basement membrane known as Reichart’s membrane (Snell from PSAl embryoid bodies were isolated as previously described and Stevens, 1966; Pierce et al., 1962; Pierce, 1966). * Supported by Grants CA27580 from the National Cancer Institute, United States Department of Health, Education and Welfare, and BC-314 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
’ The abbreviations used are: EC, embryonal carcinoma; EGTA, ethylene glycol bis(/3-aminoethyl ether)-N,N,N’,N”tetraacetic acid; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; PBS,phosphatebuffered saline; endo A and endo B, endodermal cytoskeletal proteins A and B; SaCI, Staphylococcus aureus Cowan Strain I, formalin-fixed as described (Kessler, 1975);SDS, sodium dodecyl sulfate. Unpublished results.
8124
Cytoskeletal
Proteins of Endodermal
(Oshima and Linney, 1980). The ST0 cell line was reselected for ouabain and thioguanine resistance and a strictly contact-inhibited subclone, designated STO-TGO, was used. The PFHRS (Chung et al., 1977), PYSL (Lehman et al., 1974), and MB2 parietal endodermal cell lines and the MB4 presumptive visceral endodermal line” (Sherman, 1975; Cantor et al., 1976; Sherman and Atienxa-Samols, 1979) were obtained from the respective investigators. The XB-2 mouse keratinocyte (Rheinwald and Green, 1975) and 3T3 (Todaro and Green, 1963) clone 52 fibroblast lines were gifts from Dr. Howard Green. The BWl-J mouse hepatoma line (Cassio and Weiss, 1979). NB-AI neuroblastoma, 984 clone 10 myoblast (Jakob et al., 19781, and 745 clone 18 erythroleukemic (Kabat el al., 1975) mouse lines were obtained from Drs. Mary Weiss, Frank Solomon, Elwood Linney, and David Kabat, respectively. HeLa cells were obtained from Dr. Jerry Schneider. Cell Culture-All cells except 3T3 and XB-2 were cultivated in Dulbecco’s modified Eagle’s medium supplemented with pyruvate (110 mg/liter), glutamine (O&i%), 10% fetal bovine serum. Calf serum was substituted for fetal bovine serum for the growth of 3T3 cells. XB-2 cells were grown in Dulbecco’s modified Eagle’s medium with 20% fetal bovine serum conditioned for 24 h by 3T3 cells or with mitomycin C-treated 3T3 feeder cells in Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum (Rheinwald and Green, 1975). PSAl embryonal carcinoma cells were propagated on feeder layers of mitomycin C-treated ST0 tibroblasts (Martin and Evans, 1975) in the presence of lo-’ M 2-mercaptoethanol (Oshima, 1978). Retinoic Acid Induction-F9.22 EC cells were plated at densities between lo” and 2 x 10” tells/60-mm dish. The densities of cells plated were chosen on the basis of preliminary experiments so that approximately the same number of cells was labeled and harvested on each day. Six hours after plating, retinoic acid was added to a final concentration of 10 ’ M. Each day, cultures were labeled for 4 h with methionine-free Dulbecco’s modified Eagle’s medium supplemented with 50 &i/ml of [““Slmethionine and 10% fetal bovine serum. Cells were harvested at room temperature by dissolving in buffer containing 0.1% SDS as described under “Immunoprecipitation.” Lysates were stored at -85 “C until use. Purification of Endo B Protein and Antiserum ProductionPFHRS parietal endodermal cells (Chung et al., 1977) were grown in roller bottles, harvested with isotonic 0.05% trypsin, 0.025% EDTA, washed with cold PBS, and broken in a Dounce homogenizer in 0.5% Nonidet P-40,0.25 M sucrose, 0.1 mM EGTA, 5 mM N-ethylmaleimide, 0.5 mM phenylmethylsulfonyl fluoride in RSB buffer (10 mM TrisHCI, pH 7.2, 10 mM NaCl, 3 mM MgCl,). The nuclei and associated cytoskeletal proteins were recovered by centrifugation through a cushion of 30% sucrose in RSB, 0.1 mM EGTA. The resulting pellet was resuspended in 0.25 M sucrose in RSB, 0.1 mM CaC12, 0.5 mM phenylmethylsulfonyl fluoride and digested with micrococcal nuclease (25 pg/ml) at 37 “C for 10 min. EDTA (pH 7.2) was added to 10 mM and NaCl was added to 1 M. After incubation for 10 min on ice with occasional vigorous mixing, the mixture was centrifuged. The pellet was washed with 1 M NaCI, and water then was dissolved in the sample buffer described by Laemmli (1970). Proteins were separated on 3-mm thick, 25-cm long slab gels of 15% acrylamide, 0.09% bisacrylamide, 0.1% SDS (Thomas and Kornberg, 1975). The protein bands of interest were excised after staining the gel briefly with 0.1% Coomassie blue R-250 in 50% methanol, 10% acetic acid or without staining by reference to dansylated derivatives of endo A, endo B, bovine serum albumin, and ovalbumin (Yphantis and Talbot, 1971). Gel bands were excised and the protein was recovered by electroelution overnight into dialysis bags. The proteins were dialyzed against 0.1% SDS (4 changes) and precipitated with 6 volumes of cold acetone. The pellets were washed with 20% trichloroacetic acid, ether twice, and acetone and then were dried under vacuum. The total yields were approximately 0.38 pg/lO” cells for endo A and 0.35 pg/lO’ cells for endo B. Approximately 2.4 x 10” cells were used for purification of the proteins. Samples for amino acid analyses were dissolved directly in 6 N HCI. Samples for immunization were dissolved in 0.1% SDS. Approximately 300 pg of endo B protein emulsified in complete Freund’s adjuvant were injected into a female New Zealand White adult rabbit. The animal was injected with a booster of approximately 125 clg of endo B and bled 10 days and 10 weeks later. Laminin antiserum was a gift of Dr. Albert Chung. It was made against the GP2 protein @hung et al., 1979). Antiserum against the 46,000-dalton human keratin was a gift from Dr. Elaine Fuchs. It has been shown to react with several molecular weight forms of both .’ M. I. Sherman, personal communication.
I
Cells
2
8125
3456 ,
130
24
18.4
Ftc. 1. kactionation of PYS parietal endodermal ceils. Twenty plates of PYS2 cells, in late log phase of growth, were labeled for 18 h in media containing %IIthe normal leucine concentration, 10% fetal bovine serum, 25 pCi/ml of [“Hlleucine. Cells were harvested after rinsing with cold PBS by scraping into buffer containing 0.5% Nonidet P-40 and protease inhibitors (lysis buffer; see “Experimental Procedures”). The nuclei and attached cytoskeletons were recovered by centrifugation, resuspended by Dounce homogenization, and centrifuged through a cushion of buffered 30% sucrose. The pellet was resuspended vigorously in 1% Tween 40, 0.5% deoxycholate in RSB and recovered by centrifugation through a sucrose cushion. The pellet was resuspended, digested with micrococcal nuclease, extracted with 1 M NaCI, washed as described under “Experimental Procedures,” and then dissolved in electrophoresis sample buffer. Approximately 33,000 cpm of radioactivity of each fraction were loaded in a 15% polyacrylamide gel containing SDS. After electrophoresis, the gel was fluorographed and exposed for 70 h at -85 “C. 2, total cell lysate; 2, cytoplasm; 3, fit pellet; 4, second pellet after homogenization and centrifugation; 5, third pellet after resuspension in double detergent buffer and centrifugation; 6, final pellet after digestion with nuclease, extraction with 1 M NaCl, and washing with 1 M NaCl and water. Band V, the protein identified as vimentin. Bands A and B, protein species of M, = 55,000 and 50,000, respectively.
Cytoskeletal Proteins of Endodermal Cells
8126
1
2
TABLE I Amino acid composition of endo B and othercytoskeletal proteins Average of two 24-h hydrolysates. No determinations of cysteine or tryptophan have been performed. Values for serine and threonine have not been corrected for hydrolytic loss. Traces of methionine oxidation products were present in the amino acid analyzer column eluants so the methionine value indicated by parentheses must be considered preliminary.
m w
68 c
c
Endo
B"
-v
A B
"
_-
-
-v
Murine CFAb
Baby hamster kidney
Porcine skeleton"
Murine tubulin'
10.3 4.8 6.5
9.2 5.2 7.6
18.7
19.5
2.9 5.7 8.4
2.9 7.75.1 9.0 4.9 2.0 3.9 10.0 2.8 2.7
9.6 6.0 5.1 12.6 4.9
I F
Human 55,000-dalton keratin'
mol %
-A -B
45"
FIG. 2. Nuclear cytoskeletal proteins of embryonal carcinoma and endodermal cells. Cells were labeled for 12-18 h with media containing 10-25 pCi/ml of ["H ]leucine.Cells were harvested and fractionated through the homogenization step described for the experiment of Fig. 1. This step was repeated and the nuclear cytoskeletal fraction was digested with micrococcal nuclease and analyzed on 151 acrylamide gels as described by Thomas andKornberg (1975). Adjacent lanes received the same amount of radioactivity and were run in the same gel. Only the middle portion of the gels is shown. 1, F9.22 EC cells; 2, PYSZ endodermal cells; 3, PSAl cells; 4, primary endodermal cells isolated from PSAl embryoid bodies; 5,F9.22 control EC cells. 6, F9.22 EC cells treated for 3 days with 10"' M retinoic acid; 7, PFHR9 parietal endodermal cells. V, the protein band identified as vimentin. A and B, endo A and B.
-bsa
Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe 1.3 His Lys Arg
10.1 6.0 8.6 15.2 2.7 9.1 7.5 6.0 5.4 (1.1) 4.7 10.4 2.1 2.6 2.3 1.7 5.6 6.7 8.1
10.2 5.6 5.9 13.1
4.9 8.4 8.2 7.1 2.0 4.8 8.4 3.2 4.0 2.3 1.3 5.7 9.7
2.0 3.8 11.2 2.3 2.3 1.5 5.5
4.7 8.6
7.6 6.3 2.2 4.8 8.3 2.8 3.8
8.6 3.9 11.9 13.8 0.4 20.0 5.0 4.1 1.5 4.4 8.7 4.3 3.5
6.1 5.2
4.9 5.5
saw
27.7 35.3 47.2 127.3 36.6 Values of the otherproteins which differ most from those of endo B are underlined. GFA, glial fibrillary acidic protein from mouse brain (Dah1 and Bignami, 1973). ' IF, intermediate fdament proteinsfrom BHK-21 cells (Starger et al., (1978). These values may represent a mixture of the intermediate filament protein, vimentin (Franke et al., 1978a), and desmin ( L a z a r ides and Hubbard, 1976; Hubbard and Lazarides, 1979). "Smalland Sobieszek, 1977. Skeleton is identical with desmin (Lazarides, 1980). e Olmstead et al., 1970. 'Fuchs and Green, 1978. The compositions of the other molecular weight forms of keratin were very similar. 'SAQ values represent the sum of the squares of the difference between each amino acid from the indicated protein and endo B. The value can beused to estimate the degree of relatedness between proteins for which the sequences are not known. Unrelated proteins have SAQ values of greater than 100 in 95% of the cases tested (Marchalonis and Weltman, 1971).
-Pk A-
>tub
B-
.ova
FIG. 3. Electrophoresis of purified endo A a n d B proteins. Purified endo A and B proteins (A and B ) were subjected to SDSpolyacrylamide gel electrophoresis. Lanes 1-5 are from a 158 acrylamide, 0.09%bisacrylamide gel as described by Thomas and Kornberg (1975). Lanes 6-10 are from a 12.5%acrylamide, 0.331 bisacrylamide gel as described by Laemmli (1970).Lanes 1,5,6, and 9 represent the detergent and 1 M NaC1-insoluble fraction of PYSZ cells as shown in Fig. 1. Lanes 2 and 7 show the migration of purified endo B protein. Lanes 3 and 8 represent endo A protein. Lane 4 shows the migration of the purified 56,000-dalton protein (vimentin) migrating slightly
human and mouse keratins (Fuchs and Green, 1978). Protein concentration was estimated by the method of Lowry et al. (1951) with bovine serum albumin in 0.1%SDS as thestandard. Immunoprecipitation-Cell cultures were labeled 4 h in methionine-free Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum and 50 pCi/ml of ["'SJmethionine (1140 Ci/ mmol, Amersham). The cultures were rinsed three times with cold PBS and dissolved by incubation in 0.1%SDS in RSB, 0.1 mMCaC12, 1 mM N-ethylmaleimide, 0.5 mM phenylmethylsulfonyl fluoride, 0.19 trypsin inhibitor units/ml of Aprotinin (Sigma) a t 8 "C. Protease-free DNase I (Otsuka and Price, 1974) was added to 10 pg/ml and the lysate was removed to a tube onice.Useof commercial DNase I, even after treatment with phenylmethylsulfonyl fluoride, resulted in considerable proteolysis. EDTA was added to 5 mM and Nonidet P40 was added to 0.5%.These lysates were stored frozen. Before use, the lysates were centrifuged at 10,000 X g for 15 min. When cell lines were compared, the same amount of acid-insoluble radioactivity was used for each cell line. slower than endo A. Lane 10 contained a mixture ofPYSZ marker proteins, porcine tubulin (tub) ( M , = 54,000), and rabbit pyruvate kinase (pk) ( M , = 57,000). The relative migration of bovine serum albumin (bsa) ( M , = 68,000) and ovalbumin (om) ( M , = 43,000) was determined from several independent gels. Gels were stained with Coomassie blue R-250.
Cytoskeletal Proteins
of Endodermal Cells
8127 I
I
2
3
4
5
6 7 8 9
2
3
4
5
IO -
~"
A - r 8
4
a
b
FIG. 4. Immunoprecipitation of endodermalcells,HeLa cells, and mouse keratinocytes withendo B and keratin antisera. a, [%]methionine-labeled whole cell lysate (2.5 X 10' cpm) of either PFHR9 endodermal or HeLa cells was incubated with 4 1-11 of nonimmune rabbit sera and S. aureus Cowan Strain I. After centrifugation, each lysate was immunoprecipitated with the equivalent of 4 pl of the indicated sera, recovered with SaCI, eluted in electrophoresis sample buffer, and analyzed on a 15%acrylamide gel containing SDS. The gel was fluorographed and exposed for 24 h a t -85 "C. I , ["Hlleucine-labeled marker proteins as for Fig. 1; 2, PFHR9, endo B antiserum previously incubated 18 h a t 4 "C with 200 ng of endo A/ pI of serum; 3, same as 2 but previously incubated with 480 ng of endo A/pl of sera; 4, PFHR9, endo B antisera;5, PFHR9, endoB antiserum previously incubated with 200 ng of endo B/pl of antiserum; 6,
PFHR9, keratin antiserum; 7, HeLa, keratin antiserum; 8, HeLa, endo B antiserum; 9, HeLa, endo B antiserum previously incubated with 200 ng of endo B/pl of antiserum; IO,same as lane 1. b, mouse XB-2 keratinocytes were cultivated, labeled with ["'SJmethionine, and harvested in the presence of mitomycin C-treated 3T3 feeder cells. The whole cell lysate (10' cpm) was incubated with 10 pl of preimmune serum and SaCI. After centrifugation, the lysates were immunoprecipitated with 10 pl of endo B antiserum, 5 pl of keratin antiserum, or a second round of10 pl of preimmune sera and SaCI. Precipitates were analyzed as in a. Exposure time was 48 h. Lane I , [:'H]leucine marker proteins as shown in Fig. 1; lane 2, not relevant to this investigation; lane 3, endo B antiserum; lane 4, preimmune serum of rabbit which produced endo B antiserum; lane 5, keratin antiserum. A and B, endo A and B.
Immunoprecipitations were performed essentially according to the method of Kessler (1975). Lysates were incubated with preimmune or nonimmune rabbit sera and the formalin fixed-Staphylococcus aureus Cowan Strain I bacteria described by Kessler (1975) (Calbiochem). Ten pl of a 10% suspension of bacteria/pl of sera were used. After centrifugation, immunoreactive proteins were recovered from these lysates by incubation with immune sera for 1 h on ice and with the formalin-fixedbacteria for an additional one-half h. The bacteria were recovered by centrifugation a t 6OOO X g and washed once with 0.1% SDS, 0.5% Nonidet P-40,lO mM Tris-HCI, pH 7.4,5 mM EDTA, three times with 0.5 M NaCI, 0.05% Nonidet P-40, 50 mM Tris-HCI, pH 7.4, 5 n" EDTA, and once with 0.15 M NaCI, 50 mM Tris-HCI, 5 mM EDTA. The radioactive proteins andrabbit immunoglobins were solubilized in sample buffer (Laemmli, 1970) with heating to 100 "C and analyzed on polyacrylamide slab gels containing SDS (Thomas and Kornberg, 1975). The gels were processed for fluorography with sulfoxide (Bonner and Laskey, either 2,5-diphenyloxazole-dimethyl 1974) or with sodium salicylate (Chamberlain, 1979) and exposed to Kodak XR-2 x-ray film a t -85 "C. Prior incubation of sera with purified endo A or B was performed by adding the solubilized proteins in 0.1% SDS, 0.5% Nonidet P-40 to aliquots of sera. Control immune sera received detergents but no protein. The final concentrations of SDS andNonidet P-40 were 0.01 and 0.05%, respectively. After incubation for 18 h a t 4OC, the sera were used for immunoprecipitation or diluted for use in immunofluorescence. Peptide Mapping-Peptide mapping of [L5S]methionine-labeled, immunoprecipitated proteins was performed according to themethod of Cleveland et al. (1977). PFHR9 endodermal cells and XB-2 kera-
tinocytes were labeled for 24 h with ["SS]methionine. Approximately 5 X lo6 cpm of acid-insoluble radioactivity from XB-2 lysate were immunoprecipitated with 5 pl of anti-46K human keratin sera (Fuchs and Green, 1978) and SaCI. PFHR9 lysate containing 5 X 10' cpm of acid-insoluble radioactivity were immunoprecipitated with 30 p1 of the endo B antiseraand SaCI. The total amount of radioactive lysate and thevolumes of immune sera were chosen to equalize the amount of precipitated radioactivity recovered. The immunoreactive proteins were recovered, washed, and eluted. Each sample was mixed with dansylated derivatives of both bovine serum albumin and ovalbumin and loaded in four lanes of a SDS slab gel. After electrophoresis, the dansylated markers were located by UV light transillumination and the region of each lane between the dansylated marker proteins was excised, equilibrated in 0.125 M Tris-HCI, pH 6.8, 0.1% SDS for 30 min a t room temperature with gentle agitation, and loaded perpendicular to the direction of electrophoresis in a 1.9-cm wide well of a second gel (1.5 mm thick). The resolving portion of the second gel was 15%acrylamide, 0.4% bisacrylamide and contained 1 mM EDTA in addition to thenormal components (Thomas and Kornberg, 1975). Spaces around the gel pieces were filled with molten 1% low temperature agarose (Bio-Rad) in 0.125 M Tris-HCI, pH 6.8,0.1% SDS, 1 mM EDTA. Each gel piece was overlaid with 20 pI of 0.125 M Tris-HCI, 0.1% SDS, 1 mM EDTA, 20% glycerol, bromphenol blue, and 6,60, or 600 ng of the S. aureus V8 protease (Miles Laboratories). Electrophoresis was carried out for 3 h a t 7.5 mA followed by 14 h at 16 mA. Gels were fixed in 50% methanol, 10%acetic acid, fluorographed, and exposed to XR-2 film a t -85 "C. Immuno~uorescence-Cells plated on glass coverslips were rinsed with PBS andfixed in a mixture of methanol and acetone (1:3). After
Cytoskeletal Proteinsof Endodermal Cells
8128
I a bc
2 a b c
3 obc
4 AB
5
6
AB
AB
W u
FIG. 5. Peptideanalysis of [3sS]methionine-labeled XB-2 keratinocyte or PFHRS endodermal proteins immunoprecipitated by keratin antiserum or endo B antiserum, respectively. XB-2 and PFHR9 cells were labeled 24 h with [%]methionine and harvested as described under “Experimental Procedures.” XB-2 lysate (5 X lo6 cpm) was reacted with 10 pI of nonimmune rabbit sera and SaCI and thenimmunoprecipitated with 5 pl of keratin antiserum. PFHR9 lysate (5 X 10’ cpm) was reacted with 30 p1 of nonimmune rabbit sera and SaCI and then immunoprecipitated with 30 pI of endo B antiserum. Each sample was eluted in sample buffer and loaded in four lanes of an acrylamide gel. After electrophoresis, the proteins of the gel lanes that contained the precipitated proteins were excised, equilibrated, and loaded, perpendicular to thedirection of electropho-
resis, into a second gel containing six large sample wells. The gel pieces were overlaid with varying amounts of V8 protease and electrophoresed in the second dimension. The insets under lanes 1 and 4 represent the areas of the gel pieces excised for protease digestion. Lanes 1, 2, and 3 represent the methionine-containing peptides of XB-2 proteins immunoprecipitated with keratin antiserum. Lanes 4, 5, and 6 represent the methionine-containing peptides of the PFHR9 proteins immunoprecipitated with endo B antiserum. Lanes 1 and 4 were overlaid with 6 ng of the V8 protease. Lanes 2 and 5 received 60 ng of protease and lanes 3 and 6 received 600 ng of protease. Arrows indicate peptides which may comigrate with endo B peptides. A and B, endo A and B; a, b, and c, three major radiolabeled protein bands precipitated by antikeratin serum as shown in Fig. 46, lane 5.
washing with PBS containing 1 mg/ml of ovalbumin, the coverslips were incubated with endo B antiserum diluted 5-fold in PBS or with the cell culture medium containing the TROMA 1 monoclonal antibody (Brulet et al., 1980)diluted IO-fold inPBS containing ovalbumin. Prior incubation of both antibody preparations with endo A or endo B was performed as described for immunoprecipitation analysis. The coverslips were incubated with the diluted antibody preparation for 30 min a t room temperature and were washed once with PBS and 4 times with either 50 mM borate buffer, pH 9.5, containing 0.15 M
NaCI, 0.5 M NaCl in phosphate buffer, pH 7.2, or 0.03%SDS in 10 mM Tris-HCI, pH 7.2. After a final wash with PBS containing ovalbumin, the antibodies were visualized with a 1:20 dilution of fluoresceinconjugated goat immunoglobin directed against rabbit IgG (MilesYeda) or a 1:30 dilution of fluorescein-conjugated rabbit immunoglobin directed against rat IgG (Miles-Yeda). The coverslips were mounted in glycine-bufferedglycerol and observed with a Leitz Dialux 20-EB microscope equipped for incident light fluorescence with a 100watt halogen lamp and H2 filter system. Photographs were taken
Cytoskeletal Proteins of Endodermal Cells
8129
identified as vimentin by immunoprecipitation analysis (data not shown) with a vimentin antiserum (Hynes and Destree, 1978). This result c o n f m s two recent studies which found vimentin in both EC cells and the PYS2 endodermal cell line (Paulin et al., 1980a and 1980b). The endo A and B proteins were purified by preparative SDS-polyacrylamide gel electrophoresis. Fig. 3 shows the migration pattern of the purified proteins. The amino acid compositions of the endo B protein and several previously isolated cytoskeletal proteins are compared in Table I. The amino acid values of the otherlisted proteins that differ most from those of endo B are underlined. An approximation of the degree of relatedness of different proteins was calculated by summing the squares of the differences between endo B and each protein for each amino acid (Marchalonis and Weltman, 1971). By amino acid composition, A+ * C endo B appears related to intermediate filament proteins B-m *D isolated from glial cells, baby hamster kidney tissue culture cells, muscle tissue, and tubulin but differs significantly from keratin. The purified endo B protein was injected into rabbits to obtain a precipitating antiserum. The serum recovered 10 days after the final injection had low but detectable activity against endodermal cells. Sera taken 10 weeks later had substantially increased activity and was used for all subsequent experiments. The specific immunoprecipitation of the endo B protein from a ["Slmethionine-labeled endodermal cell lysate is shown inFig. 4. The anti-endo B serum precipitated two FIG. 6. Immunoprecipitation of murine embryonal carci- protein bands that comigrate with marker endo A and B noma and differentiated cell lines with endo B antiserum. Total proteins. (Mixing of the precipitated proteins with marker cell lysates (5 x 10' cpm) were reacted f m t with 10 pl of preimmune serum and protein A bound to Sepharose 4B. After incubation with proteins c o n f i i e d their comigration as shown in Fig. 8,lane 1). Fig. 4 shows that the precipitation of both proteins was 10 pl of endo B antiserum, the reactive proteins were recovered with protein A-Sepharose, washed, eluted, and separated by electropho- blocked by prior incubation of the antiserum with purified resis in a 15%polyacrylamide gel containing SDS. Radioactivity was endo B. However, an equal or greater amount of purified endo visualized by fluorography. Exposure time was 4 days at -85 "C. A A protein had little effect on the subsequent precipitation of and B indicate endo A and B proteins. C and D are HeLa cell proteins the two proteins by the endo B antiserum. The endodermal recognized by the endo B antiserum. 1, F9.22 EC cells; 2, FOT5 EC cells; 3, PSAl EC cells; 4, PFHRS parietal endodermal cells; 5, PYS2 lysates used for these studieswere prepared by dissolving cells parietal endodermal cells; 6,MB4 presumptive visceral endodermal in 0.1% SDS at8 "C (see "Experimental Procedures)" before cells; 7, ST0 fibroblasts, 8, D5 presumptive fibroblasts; 9, 984 clone the addition of Nonidet P-40 to bind excessSDS (Dimitriadis, 10 myoblasts; 10, 745 clone 18 erythroleukemic cell line; 11, BWI-J 1979). If the lysate was heated to 100 "C before the addition hepatoma; 12, HeLa cells. of the Nonidet P-40, the endo B antiserum precipitated predominantly endo B. These results are shown in Fig. 8, lanes with Kodak Tri-X film with an Olympus OM2 camera and photo- 10 and 11, and may indicate a physical association between graphic tube adapter manufactured by K. A. Dawson. Exposure time endo A and B or antigenic relatedness of the two proteins. was approximately 15 s. Evidence that endo B is immunologically distinct from keratin was obtained by immunoprecipitating endodermal RESULTS cell, mousekeratinocyte, and HeLa cell lysates with either the Fig. 1 shows that two abundant protein species of M , = endo B antiserum or anti-humankeratin serum. Fig. 4a, lane 55,000 (6andA)and M , = 50,000 (band B ) are associated with 6,shows the result of precipitating an endodermal lysate with the nuclear cytoskeletal fraction of parietal endodermal cells. keratin antiserum. In comparison with the endo B antiserum These proteins remained associated with the nuclear fraction (Fig. 4a, lane 4), the keratin antiserum precipitates little through additional homogenization procedures in 0.5% Noni- presumptive endo A and B. This poor cross-reaction with det P-40 detergent or vigorous resuspension in 1%Tween 40, endo A and B is not due to the inability of the antikeratin 0.5% deoxycholate (Penman, 1966). The proteins remained serum to recognize mouseproteins because reaction of mouse insoluble after micrococcal nuclease digestion and extraction XB-2 keratinocyte lysate with the keratin antiserum resulted with 1 M NaCl. Fractionation studies of undifferentiated em- in the immunoprecipitation of large amounts of radioactive bryonal carcinoma cells or ST0 fibroblasts did not reveal presumptive keratin (Fig. 46, lune 5).The endo B antiserum similar polypeptides. Fig. 2 shows that two parietal endo- failed to recognize these presumptive keratins as shown in dermal cell lines and primary cultures of endoermal cells Figure 46, lane 3. Thus, cultured mouse keratinocytes do not derived from the spontaneous differentiation of EC cells in appear to contain signifcant amounts of radioactive endo B vitro contained proteins of similar size and solubility. F9 EC under the conditions used. cells treated with retinoic acid for 4 days synthesized similar In contrast to the resultswith mouse keratinocytes, Fig. 4a proteins (Fig. 2, lane 6).These proteins will be referred to an shows that HeLa cell lysates contained radioactive proteins endo A and B. Fig. 2 also shows that thecytoskeletal fraction that were recognizedby both keratin antiserum and the endo of EC cells and endodermal cells contain a protein slightly B antiserum. The HeLa cell proteins specifically reactive with larger than endo A of M , = 56,000. This protein band has been the endo B antiserum appear to be smaller than murine endo
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r i
t
FIG.7. Indirect immunofluorescence of PFHRS endodermal cells stained with either endo B antiserum or TROMA 1 monoclonal antibody. PFHR9 endodermal cells were fixed and incubated with either a 1:5 dilution of endo B antiserum ora 1 : l O dilution of the TROMA 1 monoclonal antibody. After washing with borate buffer, pH 9.5, antibody was visualized with either a 1:20 dilution of fluorescein-conjugated anti-rabbit IgG or a 1:30 dilution of fluores-
A and B. However, precipitation of both HeLa cell proteins was blocked byprior incubation of the endo B antiserum with purified endo B protein. The larger of the HeLa cell proteins appeared to comigrate with a major polypeptide recognized by the keratin antiserum. The smaller of the two proteins comigrated with a relatively minor component recognized by the keratin antiserum. Because of the large number of polypeptides immunoprecipitated by this keratin sera under the conditions used, it is not certain that this minor component is a keratin. However, it is clear this humancell line does contain protein(s) thatare recognized by the antiserum directed
cein-conjugated anti-rat IgG immunoglobins. Magnification was approximately X lo00 and exposure time was 15 s for all photographs. A, endo B antiserum; B , endo B antiserum previously incubated with endo B; C, endo B antiserum previously incubated with endo A D, TROMA 1 antibody; E , TROMA 1 previously incubated with endo B; F, TROMA 1 previously incubated with endo A.
against purified murine endo B (Fig. 4a, lane 8). These data are consistent with the view that HeLa cells contain both keratin-related proteins (Sun and Green, 1978; Sun et al., 1979; Franke et al., 1979)and endo B-related proteins. Partial peptide mapping (Cleveland et al., 1977) was performed to compare the possible similarity of the endo A and B proteins immunoprecipitated by the endo B antiserum. These results are shown inFig. 5 and compared to thepeptides of proteins precipitated by keratin antiserum from mouse keratinocyte lysates. Lanes 4, 5,and 6 show immunoprecipitated endo A and B proteins digested with increasing concen-
Cytoskeletal Proteins trations of the staphylococcal V8 protease. The migration of the precipitated endo A and B proteins in the fvst dimension and the approximate size of the excised gel pieces are indicated by the inset under lane 4. Endo A and B proteins share few comigrating major methionine-containing peptides. From the data shown in Fig. 5 and longer exposures of the same gel, at least 9 endo B peptides could be detected after digestion with the V8 protease. A total of 11 endo A-derived peptides was detected. Only 3 of these appeared to comigrate with endo B peptides. The endo A protein is not a higher molecular form of the endo B protein. Lanes 1, 2, and 3 show the digestion patterns of three major radiolabeled protein bands precipitated from mouse XB-2keratinocytes with anti-human keratin serum. Digestion of the c protein band precipitated by the antikeratinsera produced six detectable peptides, two of which appear to comigrate with endo B peptides. One of the peptides from the a and b protein bands precipitated by the keratin antiserum comigrated with an endo B peptide. The endo B protein is not closely related to thea, 6,or c proteins precipitated from mouse XB-2 cellsby keratin antiserum. Fig. 6 shows the results of immunoprecipitations from unheated, [%]methionine-labeled cell lysates of 11 different mousecell lines and HeLacells. The endo B antiserum precipitated both endo A and B proteins from the PFHR9, PYS2, and MB2 (data not shown) parietal endodermal cell lines. Immunoprecipitates of embryonal carcinoma cells had only traces of endo B. These levels of endo B are consistent with the observed contamination of EC cells with small numbers of spontaneously appearing endodermal cells (Oshima, 1978).The ST0 and D5 fibroblast lines, 984 clone 10 myoblast, and 745 clone 18 erythroleukemic cells did not contain significant amounts of immunologically reactive endo B protein. Other cell lines tested for the presence of endo B by immunoprecipitation and found negative were 3T3, NB-A1 neuroblastoma (data not shown), and mouse XB-2 keratinocytes (Fig. 46). In contrast, a mouse fetal hepatoma line, BWl-J (Cassio and Weiss, 1979), and the MB4 embryonic cell line contained immunoprecipitable endo A and B. As discussed earlier, HeLa cells, the only nonmurine cell line tested, contained two proteins (C and D in Fig. 6) which were reproducibly precipitated by the endo B antiserum. The two proteins just above and below protein D are barely visible in Fig. 4a (lane 8) because of differences in exposure’time. However, longer exposures of the gel shown in Fig. 4a revealed that the precipitation of these contaminating proteins was not affected by prior incubation of the endo B antiserum with purified endo B. Thus, they do not appear to be endo B-related. Evidence that the endo B protein is located in the cytoskeleton of endodermal cells and is distinct from proteins recognizedby a previously described monoclonal antibody (Brulet et al., 1980) is shown in Fig. 7. PFHR9 endodermal cells were stained with either the endo B antiserum or the TROMA 1 monoclonal antibody by the indirect immunofluorescence technique. Fig. 7A shows that thefluorescence due to the endo B antiserum was localized in a filamentous network throughout the cytoplasm of the endodermal cells and resembles the patternsobserved for intermediate filaments in other types of cells. Fig. 7Bshows that prior incubation of the antiserum with purified endo B abolishes the filamentous staining. Prior incubation with endo A has little or no affect (Fig. 7 0 . The residual fluorescence shown in Fig. 7B wasthe same as that of controls stained with nonimmune or preimmune sera. Pretreatment of the cells with Colcemid (5 p ; 18 h) did not abolish the fibrillar staining pattern. Fig. 7 0 shows a very similar fibrillar staining pattern resulting from the TROMA 1 monoclonal antibody. Prior incubation of the antibody with endo B had little effect (Fig. 7E) while prior
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incubation with endo A abolished the specific fibrillar staining. The specific fibrillar staining pattern of both antibody preparations remained when the cellswerewashedin borate buffer, pH 9.5, 0.5 M NaCl, or 0.03% SDS before visualization with the appropriate fluorescent antibody. Fig. 86 shows the results of immunoprecipitations of lysates of F9 EC cell cultures treated with retinoic acid for up to 4 days. Each day after the addition of M retinoic acid,
1 2 3 4 5 6 7 8 9
a
I
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I 2 3 4 5 6 7
“
891011
b
FIG. 8. The effect of retinoic acid on the synthesisof immunoprecipitable endo B and laminin by F9.22 embryonal carcinoma cells. F9.22 EC cells were incubated either in the presence or absence of lo-“ M retinoic acid for up to 4 days. Each day cells were labeled with [““Slmethionine, harvested, and stored frozen at -85 “C. a , lysates (10’ cpm) were cleared of nonspecific reactants with 10 p1 of nonimmune sera, immunoprecipitated with 5 pl of the CP2antisera described by Chung et al. (1979), eluted, and analyzed on a slab gel containing SDS. Only the top of the resolving gel is shown. Lanes 3, 5, 7, and 9 represent the precipitates from lysates of cells treated with retinoic acid for 12, 48, 72, and 96 h, respectively. Lanes 2, 4, 6, and 8 represent the precipitates from untreated cells for 12.48, 72, and 96 h, respectively. No sample-was placed in lane 1. Exposure time was 12 h. b, each lysate(2.3 X 10‘ cpm) was reacted with 5 pl of nonimmune rabbit sera and SaCI and then immunoprecipitated with 5 pl of endo B antiserum. Lanes 3, 5, 7, and 9 represent the immunoprecipitates of cells treated with retinoic acid for 12,48, 72, and 96 h, respectively. Lanes 2,4, 6, and 8 represent identically treated samples from control cultures. Lane 10 was loaded with the immunoprecipitate of PFHR9 cells. Lane I 1 shows the result of heating the PFHR9 lysate in SDS before the addition of Nonidet P-40 and subsequent immunoprecipitation with the endo B antiserum. Note the substantial decrease in the amount of endo A in lane 11. Lane 1 was loaded with a mixture of [RH]leucine-labeled marker proteins, as shown in Fig. 1 and the PFHR9 precipitate run in lane 10. Exposure time was 48 h. A and B, endo A and B.
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control and treated cultures were labeled for 4 h with [35S]-patterns and immunological cross-reaction (Fuchs andGreen, methionine, harvested, and assayed for the presence of the 1978). The significant differences between the amino acid endo B protein by immunoprecipitation with the endo B compositions of endo B and previously characterized keratins antiserum. The synthesis of endo B protein was detected the (Table I) and the comparison of the proteolytic fragments of secondday afterthe addition of retinoic acid. Increased immunoprecipitated endo B and mouse keratins of similar size amounts were detected 3 and 4 days after the addition of (Fig. 5) indicate that endo B is not closely related to epidermal retinoic acid. Very little endo B was found in cultures not keratin. The poor cross-reaction of an antikeratinserum with treated with retinoic acid. Fig. 8a shows the results of immu- endo B and the apparent lack of reaction of mouse keratinonoprecipitations of the lysates with an antiserum directed cytes with endo B antiserum support this conclusion. against laminin, a glycoprotein component of the basement Two recent reports have concluded that PYSB cells contain membrane secreted by extra-embryonic endodermal cells keratin-like filaments on the basis of two-dimensionalelectro(Chung et al., 1979; Timpl et al., 1979; Oshima and Linney, phoretic analysis of cytoskeletal preparations and immunoflu1980).The two laminin bands migrate slower than P-galacto- orescence of PYSB cells with a prekeratin antiserum (Paulin sidase (MI = 130,000) and have been estimated by others to et al., 1980a, and 1980b). The antibodies used in thg second have M , = -230,000 and 440,000 (Timpl et aZ.,1979). The low study were made against five to eight electrophoretically levels of laminin evident in untreated F9 cultures or cultures resolvable proteins extracted from bovine epidermis (Franke treated for only 1day are probably due to the0.1-1% contam- et al., 1978b). However, it is not clear which of the proteins ination of F9 cultures with spontaneously appearing endo- were antigenic or which of the PYSB cytoskeletal elements dermal cells (Oshima, 1978). The fraction of cells in retinoic were reactive. Further investigation is needed to determine if acid-treated cultures reacting with laminin antiserum has the endo A and B proteins are related to theelements recogbeen previously estimated by immunofluorescence and flow nized by the antibodies used by these investigators. cytometry to be approximately 2% on day 2, 10% on day 3, The first report of intermediate filament-like proteins in and 18% onday 4 (Oshima and Linney, 1980).The datashown trophectoderm utilized a monoclonal reagent made against in Fig. 8 indicate that both endo B and laminin are synthesized trophectodermal cytoskeletal preparations (Brulet et al., in increased amounts 2 days after theaddition of retinoic acid. 1980). This antibody appears to recognize endo A but not endo B (Fig. 7). This suggests that endo A is found in DISCUSSION trophectoderm as well as extra-embryonic endoderm. It is The results of this study indicate that the endo B protein possible that endo A also may be recognized by somebut not may be an excellent marker of the differentiation of EC cells all keratin antiserum. This would resolvethe apparentconflict to extra-embryonic endoderm. The immunofluorescent local- between the results reported here and those reported by ization of endo B and the solubility of the protein are consist- Paulin et al. (1980a) concerning the reaction of endodermal ent with a cytoskeletal location for the protein. The prelimi- cells with keratin antisera. The MB4 cell line, which contains endo B, may be a visceral nary amino acid composition of endo B is similar to that of intermediate filament proteins isolated by other investigators endodermal cell line3 (Cantor et al., 1976; Sherman and (Table I). However, identification of the type of cytoskeletal Atienza-Samols, 1979).If the presence of endo B is c o n f i i e d element with which the endo B protein is associated must in visceralendoderm, then endo B represents the first marker await further investigation. On the basis of its migration in of extra-embryonic endoderm present in both parietal and polyacrylamide gels containing SDS, amino acid composition, visceral types. Furthermore, the presence of endo B in the and differential distribution among different types of cultured BW1-J fetal hepatoma cell line leads to speculation concerncells, endo B does not appear to be tubulin. The apparent ing the possible presence of endo B in definitive embryonic absence of immunologically reactive proteins of similar size in endodermal tissues. The presence of proteins antigenically lysates from fibroblasts (STO, D5, and 3T3 cell lines) and a related to endo B in HeLa cells (a human tumor cell line of myoblast line indicates that the endo B is not vimentin or epithelial origin) should not be considered definitive evidence desmin. In addition, cell fractionation studies of ST0 fibro- for the presence of endo B in normal cell types of other than blasts, performed and analyzed as described for the experi- extra-embryonic or embryonic endodermal origin because of ment shown in Fig. 1, did not reveal proteins, associated with the many years that this tumorcell line has been in continuous the Nonidet P-40 and high salt-insoluble fraction, that comi- culture. Nevertheless, HeLa cells appear to contain keratingrate with either endo A or B, although vimentin was identi- related proteins and vimentin (Franke et al., 1978a). If endo B proves to be an intermediate filament protein, at least three fied as expected (data not shown). Glial fibrillary acidic protein is another intermediate fiia- different intermediate filament proteins may be found in a ment protein that has an apparent molecularweight and single cell. Embryonal carcinoma cells donot appear to contain signifamino acid composition similar to that of endo B. However, the tissue distribution of this protein is limited exclusively to icant amounts of endo B or endo A. However, EC cells do astrocytic glial cells (Bignami et al., 1980). Endodermal cells contain detectable levels of vimentin (Paulin et al., 1980a) and are apparently lacking this component (VandenBerg et al., similar amounts of actin and tubulin as PYS2 endodermal 1976). In addition, HeLa cells, which appear to contain pro- cells (Paulin et al., 1978).Treatment of EC cell cultures with teins immunologically related to endo B, are reported not to retinoic acid results in the appearance of cells that secrete react with glial fibrillary acidic protein antiserum (Bignami et plasminogen activator (Strickland and Mahdavi, 1978; Jetten et al., 1979; Oshima and Linney, 1980) and contain lamininal., 1980). The most heterogenous of the intermediate filament pro- like proteins (Jetten et al., 1979; Oshima and Linney, 1980; teins are the keratins. Epidermal bovine keratins are repre- Howe and Solter, 1980) but do not secrete a-fetoprotein sented by as many as seven polypeptides ranging in apparent (Strickland and Sawey, 1980). These results are consistent M , = 40,000-68,000. All of the polypeptides have very similar with the view that parietal endoderm is the major differenamino acid compositions and the same NHz-terminal amino tiated cell type to appear in response to retinoic acid. The acid (Steinert and Idler, 1975). Similarly, human epidermal synthesis of endo B found in EC cells treated with retinoic keratins are represented by six or seven polypeptides which acid also is consistent with this view and suggests that the appear closely related by amino acid composition, peptide regulation of the expression of endo B might be investigated
Cytoskeletal Proteins conveniently in this system. In addition, preliminary results confim that retinoic acid induces endo A synthesis as well as endo B4.Vimentin synthesis continues in both EC and endodermal cell cultures. In culture,parietal endodermalcells have a flattened appearance with distinct cell borders and usually very little overlap in confluent areas. The morphologyof these wellspread cells has previously been correlatedwith the organization of actin (Paulin et al., 1978) and is now characterized by the appearance of a new cytoskeletal protein. It seems reasonable that endo A and endo B may play roles in determining the morphology of this differentiated cell. In general, the developmental regulation of the expression of different cytoskeletal proteins may be of interest not only because of their possible involvement in cell shape and tissue morphogenesis (e.g.see Ben-ze’evet al., 1980) butalso because intracellular structural proteinsof limited tissue distribution may be excellent markers of related but divergent specialized cell types. The possible presence of endo B in both parietal and visceral endodermalcell types, which arise fromthe same area of the embryo if not from the same precursor cells, may be an example of such a marker. Acknowledgments-I am grateful to Dr. John Buchanan for laboratory space, use of equipment and his careful reading of the manuscript, Drs. Elaine Fuchs and Howard Green for antiserum to 46,000dalton human keratin, porcine tubulin, and mouse XB-2 cells, Maria Felshtinsky for technical assistance, Deborah Hagman for typing the manuscript, Dr. Rolf Kemler and Prof. F. Jacob for TROMA 1 monoclonal antibody, Dr. Paul Price for protease-free DNase I, Deborah Shackelford for advice on immunoprecipitation analysis, and Dr. Lisa Steiner for amino acid analysis. Preparative quantities of the PFHR9 endodermal cell line were grown by the Cell Culture Center, Massachusetts Institute of Technology. REFERENCES Adamson, E. D., Evans, M. J., and Magrane, G.G. (1977) Eur. J. Biochem. 79,607-615 Ben-ze’ev, A,, Farmer, S.R., and Penman, S.(1980) Cell 21, 365-372 Bernstine, E. G., Hooper, M. L., Grandchamp, S.,and Ephrussi, B. (1973) Proc. Natl. Acad. Sci.U. S. A . 70,3899-3903 Bignami, A., Dahl, D., and Rueger, D. C. (1980) Advances in Cellular Neurobiology Vol. 1, pp. 285-304, Academic Press, New York Bonner, W. M., and Laskey, R.A. (1974) Eur. J. Biochem. 46.83-88 Brulet, P., Babinet, C., Kemler, R., and Jacob, F. (1980) Proc. Natl. Acad. Sci. U. S. A . 77,4113-4117 Cantor, J., Shapiro, S. S., and Sherman, M. I. (1976) Dev. Biol. 50, 367-377 Cassio, D., and Weiss, M. (1979) Somatic Cell Genet. 5, 719-738 Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-135 Chung, A., Estes, L., Shinozuka, H., Braginski, J., Lorz, C.,and Chung, C. A. (1977) Cancer Res. 37,2072-2081 Chung, A. E., Jaffe,R., Freeman, I. L., Vergnes, J-P., Braginski, J . E., and Carlin, B. (1979) Cell 16, 277-287 Cleveland, D. W., Fischer, S.G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106 Dahl, D., and Bignami, A. (1973) Brain Res. 61,279-293 Dimitriadis, G. (1979) Anal. Biochem. 98,445-451 Dziadek, M., and Adamson, E. (1978) J . Embryol. Exp. Morphol. 43, 289-313 Finch, B., and Ephrussi, B. (1967) Proc, Natl. Acad.Sci. U. S. A . 57, 615-621 Franke, W. W., Schmid, E., Osborn, M., and Weber, K. (1978a) Proc. Natl. Acad. Sci. U. S. A . 75, 5034-5038 Franke, W. W.,Weber, K., Osborn, M., Schmid, E., and Freudenstein, C. (1978b) Exp. Cell Res. 116, 429-445 Franke, W. W., Schmiel, E., Weber, K., and Osborn, M. (1979) Ezp. Cell Res. 118, 95-109 Fuchs, E., and Green, H. (1978) Cell 15,887-897 Hogan, B. L. M. (1977) Znt. Rev. Biochem. 15, 333-376 Howe, C. C., and Solter, D. (1980) Deu. B i d . 77, 480-487 Manuscript in preparation.
of Endodermal Cells
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Hubbard, B. D., and Lazarides, E. (1979) J. Cell B i o l 8 0 , 166-182 Hynes, R. O., and Destree, A. T. (1978) Cell 13, 151-163 Jacob, F. (1977) Immunol. Rev. 33,3-32 Jakob, H., Buckingham, M. E., Cohen, A,, Dupont, L., Fiszman, M., and Jacob, F. (1978) E m . Cell Res. 114,403-408 Jetten, A. M., Jetten, M. E. R., and Sherman, M. I. (1979) Exp. Cell Res. 124,381-391 Kabat, D., Sherton, C. C., Evans, L. H., Bigley, R., and Koler, R. D. (1975) Cell 5, 331-338 Kessler, S. (1975) J. Zmmunol. 115, 1617-1624 Kleinsmith, L. J., and Pierce, G. B. (1964) Cancer Res. 24, 1544-1552 Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 Lazarides, E. (1980) Nature 283, 249-256 Lazarides, E., and Hubbard, B. D. (1976) Proc. Natl. Acad. Sci. U. S. A . 73,4344-4348 Lehman, J. M., Speers, W. C., Swartzendruber, D. E., and Pierce, G. B. (1974) J. Cell. Physiol. 84, 13-28 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 Marchalonis, J. J., and Weltman, J. K. (1971) Comp. Biochem. Physiol. B Comp. Biochem. 38,609-625 Martin, G. R.(1975) Cell 5,229-243 Martin, G. R., and Evans, M. J . (1975) Proc. Natl. Acad.Sci. U. S. A . 72, 1441-1445 Martin, G. R., Wiley, L. M., and Damjanov, I. (1977) Deu. Biol. 61, 230-244 McBurney, M. W. (1976) J. Cell. Physiol. 89,441-456 Olmstead, J . B., Carlson, K., Klebe, R., Ruddle, F., and Rosenbaum, J. (1970) Proc. Natl. Acad. Sci.U. S. A . 65, 129-136 Oshima, R. (1978) Differentiation 11, 149-155 Oshima, R., and Linney, E. (1980) Exp. Cell Res. 126,485-490 Otsuka, A. S., and Price, P. A. (1974) Anal. Biochem. 62, 180-187 Paulin, D., Nicholas, J. F., Yanit, M., Jacob, F., Weber, K., and Osborn, M. (1978) Dev. Biol. 66,488-499 Paulin, D., Babinet, C., Weber, K., and Osborn, M. (1980a) Exp. Cell Res. 130,297-304 Paulin, D., Forest, N., and Perreau, J. (1980b) J. Mol. Biol. 144, 95101 Penman, S. (1966) J . Mol. Biol. 17, 117-130 Pierce, G. B. (1966) Deu. Biol. 13, 231-249 Pierce, G. B., and Beals, J . F. (1964) Cancer Res. 24, 1553-1556 Pierce, G. B., Jr., Midgle, A. R., Sri Ram, J., and Felman, J . D. (1962) Am. J . Pathol. 41, 549-566 Rheinwald, J . G., and Green, H. (1975) Cell 6, 317-330 Rossant, J., andPapaiannou, V. E. (1977) in Concepts in Mammalian Embryogenesis (Sherman, M. I., ed) pp. 1-26, MIT Press, Cambridge, MA Sherman, M. I. (1975) Differentiation 3, 51-67 Sherman, M. I. (1977) in Concepts in Mammalian Embryogenesis (Sherman, M. I., ed) p. XV, MIT Press, Cambridge, MA Sherman, M. I., and Atienza-Samols, S . B. (1979) J. Embryol. Exp. Morphol. 52, 127-139 Small, J. V., and Sobieszek, A. (1977) J. Cell Sci. 23, 243-268 Snell, G. D., and Stevens, L. L. (1966) in Biology ofthe Laboratory Mouse (Green, E. L., ed) pp. 205-245, McGraw-Hill, New York Solter, D., and Knowles, €3. B. (1978) Proc. Natl. Acad. Sci. U . S. A . 75, 5565-5569 Starger, J. M., Brown, W. E., Goldman, A. E., and Goldman R. D. (1978) J. Cell Biol. 78, 93-109 Steinert, P., and Idler, W. W. (1975) Biochem. J. 151, 603-614 Strickland, S., and Mahdavi, V. (1978) Cell 15, 393-403 Strickland, S., and Sawey, M. (1980) Dev. Biol. 78, 76-85 Strickland, S., Smith, K. K., and Marotti, K. R. (1980) Cell 21, 347355 Sun, T-T., and Green, H. (1978) J. Biol. Chem. 253, 2053-2060 Sun, T-T., Shih, C., and Green, H. (1979) Proc. Natl. Acad.Sci. U. S. A . 76,2813-2817 Thomas, J. O., and Kornberg, R. D. (1975) Proc. Natl. Acad. Sci. U. S. A . 72,2626-2630 Timpl, R., Rohde, H., Robey, P. G., Rennard, S.I., Foidart, J-M., and Martin, G. R. (1979) J. B i d . Chem. 254,9933-9937 Todaro, G. J., and Green, H. (1963) J. Cell Biol. 17, 299-313 VandenBerg, S. R., Ludwin, S. K., Herman, M. M., and Bignami, A. (1976) Am. J . Pathol. 83, 197-206 Yphantis, D. A., and Talbot, D. N. (1971) Anal. Biochem. 44, 246253
