01Neiiimheiiiisir!.. Val 32. pp. 133 143 Pergamon Press Ltd. 1979 Printed in Crear B ~ ~ r a i n 0 International Society for Neurochemistry Ltd.
0022-3042 79/010 1-0I33502 00,O
JIJI1I’nil~
PROTEIN PROFILES O F RAT EMBRYO CEREBRAL CELLS DUR NG DIFFERENTIATION IN C U .TURE C. RICHTER-LANDSBERG’ and E. YAWN’ Department of Neurobiology, Thc Weizmann Institute of Science, Rehovot, Israel (Received 6 January 1978 R m w d 22 May 1978. Accepted 19 Jurie 1978)
Abstract--The protein composition of thc particulate fraction of dissociated foctal rat cerebral cells during maturation in culture was investigated. SDS polyacrylamide gel electrophoresis showed a general decrease in the histonal components and significant changes in composition of a group of polypeptides with molecular weights ranging from 42 to 6 0 K . Two of these polypeptides coelectrophoresed with tubulin and actin whereas a 48 K polypeptide comigrated with the major component of the Wolfgram myelin protein. Its relative quantity appeared to approach a plateau after 8 days in culture. The myelin basic and proteolipid proteins were below detection levels in cultured cells at any time point investigated. A group of polypeptides with estimated molecular weights of 47, 51 and 52 K possibly representing synaptic proteins increased with time in culture. The appearance of a prominent band (60K) in brain cultures and in other cells of divergent origin was demonstrated. This protein may be related to thc process of cell adaptation to culture conditions. The developmental changes in the protein profile are discussed in the context of an in tiitro myelinogenesis and synaptogenesis and compared with whole brain particulate and subcellnlar fractions.
DURING the first few weeks of postnatal life, the devel- teins which may be of relevance to the synaptic strucoping r a t brain undergoes distinct constitutional ture (FEITet a/., 1977; KELLY& COTMAN, 1977; MORchanges which are marked by an increase in the brain GAN et a/., 1973). Neither are we aware of systematic solids, i.e. nucleic acids, proteins and lipids, and by studies designed to follow-up the appearance of myea decrease in the water content (BENJAMINS & lin-related proteins in myelinating explants or disMCKHANN, 1976; DAVISON& DOBRING, 1968; sociated cell cultures (BORNSTEIN & MURRAY,1958; WELLS& DITTMER, 1967; MATTHEWet a/., 1973). The LODJNet al., 1973; SHAHARel a/., 1975). The present most characteristic cellular events accompanying report, which is an extension of our previous work these macroniolecular changes are maturation of on the metabolic aspects of in uitro brain cell differenneuronal processes, and extensive synaptogenesis and tiation (YAWN & ZIEGLER,1977), documents the myelinogenesis (EAYRS& GOODHEAD, 1959; GONATAS chronological changes in the protein composition of the particulate fraction of embryonic cell culture and e f a/., 1971 ; NORTON, 1976: BLOOM,1972). We have recently shown that dissociated cells from discusses its possible relevance to normal brain develthe fetal rat forebrain express in culture several dis- opment. tinct features of the developing nervous tissue such as a n abundant neuritic aborization. establishment of MATERIALS AND METHODS synaptic contacts and appearance of myelin (YAWN Cell culture. Cerebral hemispheres of 16-day-old rat & YAVIN, 1977). While considerable information embryos were dissociated and cultured on plastic Petri exists on the protein pattern during brain ontogenesis dishes precoated with poly L-lysine under the same growth (GROSSFELD & SHOOTER,197 1 ; WAEHNELDT & NEU- conditions as previously described (YAVIN& YAWN, 1974). HOFF, 1974; SCHMITT rt a / . , 1977) no systematic studies Prrpuratiori OJ the particulate protein fraction. Prior to are available on the protein composition in relation- harvesting, cells were washed three times with 2 ml each ship t o brain cell maturation in cultwe. To our of 0.01 M-Tris -CI buffer, p H 7.5, scraped off and centrifuged knowledge, no attempts havc been made to correlate for 1 min in an Eppendorf 3200 microcentrifuge. The cell the ample ultrastructural evidence for synapses in cul- pellct was stored at -20°C and re-homogenized in the tured cells and the presence of membrane specific pro- above buffer for subsequent biochemical analysis.
’
Present address: Department of Neuroscience, The Children’s Hospital Medical Center. Boston, MA, U.S.A. * T o whom all correspondence should be addressed. Abhretiiatioris used: SDS. sodium dodecyl sulphate; CNP, 2‘3’-cyclic nucleotide phosphohydrolase; K, Kilodalton. N.C 32.1
I
133
Rut bruin Jructioriarion. Rat forebrains were taken out after decapitation, homogenized in 0.01 M-Tris-C1 buffer, p H 7.5 (10% homogenate w/v) and centrifuged as above. The resulting pellet was re-homogenized and centrifuged under the same conditions, yielding the particulate fraction of the total homogenate. Synaptosomes and synaptic membranes were prepared from adult rat brain according to MORGANet al. (1971). Rat brain myelin was prepared as
134
C. RICHTER-LANDSBERG and E. YAVIN
described by WAEHNELDT & MANDEL (1972) with somc by seeding cells at a relatively high density on polymodifications. described elsewhere (WAEHNELDT, 1975). All lysine-coated surfaces (YAVIN & YAWN, 1977). As a protein fractions were stored at -20°C until used for result of establishment of early contacts between further biochemical studies. homogeneously distributed cells at close proximities, SDS-polyacrylarnide gel electrophorrsis. Protein samplcs the appearance of typical fibroblastic elements was ( I mg proteinjml) were mixed with concentrated sample buffer (2:1, v/v) to yield a final concentration of 10% gly- greatly reduced for extended periods of growth thus cerol, 5% 2-mercaptoethanol, 3% SDS, 0.001 bromophenol enabling cells of neural origin to express some of their blue, 60 mM-Tris-CI (pH 6.8) and then hcated for 5 min characteristic properties in oitro. Figure 1 depicts the changes in the D N A content at 100°C. Aliquots of about 13pg protein in 20pI volume were routinely subjected to electrophoresis. Electrophoretic and the rate of C3H]thymidine incorporation under separation was carried out in 1G20% gradient slab gels these experimental conditions. It is evident that the unless otherwise stated as described by LAEMMLI ( I 970) daily D N A increment reaches a value of 8% by the with 0.025 ~-Tris-Cilycine,pH 8.3, containing 0.1% SDS as second day, and is subsequently reduced to a value electrophoresis buffer, at constant voltage of 120 V at 18°C of 2.5% per day by the end of the second week in for 5 h. Gels were stained with Coomassie brilliant blue culture. The daily increment, cxpressed as percent of (0.2% in methanolLacetic acid-water, 5 :1 :4, by vol) and the amount of DNA/plate on day 1, probably sugdestained with 10% acetic acid containing 20% methanol. Scanning of the photographic transparencies was carried gests that a stable population of relatively long-lived cells is obtained. This is further corroborated by the out in a Gilford 24003 spectrophotometer at 560 nm: the densitometric tracings were analysed with a Keuffler and finding that L3H]thyrnidine incorporation into cellular DNA is markcdly reduced after 1 week in culture, Esser planimeter. Metabolic studies. About 20 pCi ~-[~~S]methionine.indicating that there is little or no cell turnover, which (400 mCi/mmol, New England Nuclear) in 2 ml meth- would be measured by synthesis of new DNA. Thereionine-free medium was added per dish and cells incubated fore, changes in the protein profiles to be subsefor 5 h at 37'C. For glycoprotein labeling either quently described are presumably related to a popula25 pCi r-[6-'H]fucose (5.3Ci/mrnol New England Nuclear) tion of cells that is preponderantly non-dividing. or 4 pci AcetyI-o-[l-'4C]mannosamine (54.5 mCi/mmol, New England Nuclear) was added per plate. Cells were Alterations in protein prqfiles with culture rnaturatioii harvested after 6 days incubation. DNA labeling was The electrophoretic pattern of the particulate fracaccomplished by pulsing cells with 3 pCiC3H]thymidinc tion of the fetal rat brain is shown in Fig. 2a. Apart (6.58 Ci/mmol, Kamag, Dimona) for 1 h. Procedural details for DNA quantitation and radioactivity determination are from a fast migrating group of polypeptides (I 1-15 K) described elsewhere (YAVIN,1977). which presumably represent the cellular histones, a Derermiizarion of toral proteirt-bound radioactivity. Ali- 56 K and a 42 K band, coinciding with tubulin and quots of the cell homogenate was precipitated at 4°C o n actin markers, respectively, were the predominant Whatman 3 M M paper discs with 7.5% trichloracetic acid components of the complex protein pattern. During containing 2 mg/ml of Casamino acids (Difco). The filters subsequent culture, characteristic changes in the prowere heated at 90°C in 7.5% TCA for 20 min, washed three times in cold TCA, rinsed with ethanol and diethyl-ether tein composition of the fetal brain cells were seen. and dried. Radioactivity was measured in a Packard Tri- In contrast to the fairly steady level of the 42 K component and other minor proteins, a slight decrease carb liquid scintillator spectrometer. Autoradiography For [35S]methionine autoradiography in the concentration of the 56K component and a the stained gels were dried and exposed to an X-ray film marked decrease of the nuclear histones is evident. (Kodak XRP 54) for 48 h. In order to enhance the effi- With progressive maturation in culture, a significant ciency of the autoradiography of [3H]fucose or [14C]N- increasc in a polypeptide (band I), migrating slightly acetyl-mannosamine-labeled proteins, the gels were im- slower than the 56 K component, was apparent. The pregnated with a scintillation cocktail (BONNEK & LASKEY, protein components (bands 3 and 4) migrating with 1974) and aRer drying exposed to the film for 5-6 weeks a relative mobility between the 56 K and 42 K bands at - 70°C. Developed films were scanned and evaluated were also more pronounced at 15 days than at 2 days as described above. Miscellaneous analytical methods. Protein was deter- in culture. These findings are consistent in different mined according to LOWRYet al. (1951) using bovine gel systems as clearly demonstrated by the use of 7.5% serum albumin as standard. 2',3'-cycIic nucleotide 3'-phos- homogeneous (Fig. 2b) and 5-150/, polyacrylamide phohydrolase (CNP, EC 3.1.4.16) activity was measured by gradient gels (Fig. 2c). et al. (1973). the method of PROHASKA The relative pcrcentage of the various proteins was assessed by densitometric measurements of the Coomassie blue stained gels. As shown in Fig. 3, a deRESULTS crease of the histonal-like components from 35% at The phenomenon of fibroblast proliferation is one day one t o 20% after three weeks in culturc was of the major problems encountered in adapting pri- apparent. A decline of the 5 6 K band from 15% to mary cultures originating from highly specialized tis- a constant level of approximately 10% is seen after sues to grow and express their specific properties in the first week in oitro. As already noticed, the relative oitro. In our attempts to grow dissociated fetal rat amount of the 42 K component was practically uncerebral cells, this problem has been largely curtailed changed whereas the proteins migrating between the
Protein profiles in cultured brain cells
135 10
0 A TI L
a
a
s +
5
=
E
2
c a
z n
0
6
12
18
24
Days in culture
FIG.I . Incorporation of C3H]thymidine and changes in the DNA content during cell.growth in culturc. Experimental details were described under Materials and Methods. [3H]Thymidine incorporation (-@) is expressed as c.p.m./pg DNA. DNA daily increment (o----
0022-3042 79/010 1-0I33502 00,O
JIJI1I’nil~
PROTEIN PROFILES O F RAT EMBRYO CEREBRAL CELLS DUR NG DIFFERENTIATION IN C U .TURE C. RICHTER-LANDSBERG’ and E. YAWN’ Department of Neurobiology, Thc Weizmann Institute of Science, Rehovot, Israel (Received 6 January 1978 R m w d 22 May 1978. Accepted 19 Jurie 1978)
Abstract--The protein composition of thc particulate fraction of dissociated foctal rat cerebral cells during maturation in culture was investigated. SDS polyacrylamide gel electrophoresis showed a general decrease in the histonal components and significant changes in composition of a group of polypeptides with molecular weights ranging from 42 to 6 0 K . Two of these polypeptides coelectrophoresed with tubulin and actin whereas a 48 K polypeptide comigrated with the major component of the Wolfgram myelin protein. Its relative quantity appeared to approach a plateau after 8 days in culture. The myelin basic and proteolipid proteins were below detection levels in cultured cells at any time point investigated. A group of polypeptides with estimated molecular weights of 47, 51 and 52 K possibly representing synaptic proteins increased with time in culture. The appearance of a prominent band (60K) in brain cultures and in other cells of divergent origin was demonstrated. This protein may be related to thc process of cell adaptation to culture conditions. The developmental changes in the protein profile are discussed in the context of an in tiitro myelinogenesis and synaptogenesis and compared with whole brain particulate and subcellnlar fractions.
DURING the first few weeks of postnatal life, the devel- teins which may be of relevance to the synaptic strucoping r a t brain undergoes distinct constitutional ture (FEITet a/., 1977; KELLY& COTMAN, 1977; MORchanges which are marked by an increase in the brain GAN et a/., 1973). Neither are we aware of systematic solids, i.e. nucleic acids, proteins and lipids, and by studies designed to follow-up the appearance of myea decrease in the water content (BENJAMINS & lin-related proteins in myelinating explants or disMCKHANN, 1976; DAVISON& DOBRING, 1968; sociated cell cultures (BORNSTEIN & MURRAY,1958; WELLS& DITTMER, 1967; MATTHEWet a/., 1973). The LODJNet al., 1973; SHAHARel a/., 1975). The present most characteristic cellular events accompanying report, which is an extension of our previous work these macroniolecular changes are maturation of on the metabolic aspects of in uitro brain cell differenneuronal processes, and extensive synaptogenesis and tiation (YAWN & ZIEGLER,1977), documents the myelinogenesis (EAYRS& GOODHEAD, 1959; GONATAS chronological changes in the protein composition of the particulate fraction of embryonic cell culture and e f a/., 1971 ; NORTON, 1976: BLOOM,1972). We have recently shown that dissociated cells from discusses its possible relevance to normal brain develthe fetal rat forebrain express in culture several dis- opment. tinct features of the developing nervous tissue such as a n abundant neuritic aborization. establishment of MATERIALS AND METHODS synaptic contacts and appearance of myelin (YAWN Cell culture. Cerebral hemispheres of 16-day-old rat & YAVIN, 1977). While considerable information embryos were dissociated and cultured on plastic Petri exists on the protein pattern during brain ontogenesis dishes precoated with poly L-lysine under the same growth (GROSSFELD & SHOOTER,197 1 ; WAEHNELDT & NEU- conditions as previously described (YAVIN& YAWN, 1974). HOFF, 1974; SCHMITT rt a / . , 1977) no systematic studies Prrpuratiori OJ the particulate protein fraction. Prior to are available on the protein composition in relation- harvesting, cells were washed three times with 2 ml each ship t o brain cell maturation in cultwe. To our of 0.01 M-Tris -CI buffer, p H 7.5, scraped off and centrifuged knowledge, no attempts havc been made to correlate for 1 min in an Eppendorf 3200 microcentrifuge. The cell the ample ultrastructural evidence for synapses in cul- pellct was stored at -20°C and re-homogenized in the tured cells and the presence of membrane specific pro- above buffer for subsequent biochemical analysis.
’
Present address: Department of Neuroscience, The Children’s Hospital Medical Center. Boston, MA, U.S.A. * T o whom all correspondence should be addressed. Abhretiiatioris used: SDS. sodium dodecyl sulphate; CNP, 2‘3’-cyclic nucleotide phosphohydrolase; K, Kilodalton. N.C 32.1
I
133
Rut bruin Jructioriarion. Rat forebrains were taken out after decapitation, homogenized in 0.01 M-Tris-C1 buffer, p H 7.5 (10% homogenate w/v) and centrifuged as above. The resulting pellet was re-homogenized and centrifuged under the same conditions, yielding the particulate fraction of the total homogenate. Synaptosomes and synaptic membranes were prepared from adult rat brain according to MORGANet al. (1971). Rat brain myelin was prepared as
134
C. RICHTER-LANDSBERG and E. YAVIN
described by WAEHNELDT & MANDEL (1972) with somc by seeding cells at a relatively high density on polymodifications. described elsewhere (WAEHNELDT, 1975). All lysine-coated surfaces (YAVIN & YAWN, 1977). As a protein fractions were stored at -20°C until used for result of establishment of early contacts between further biochemical studies. homogeneously distributed cells at close proximities, SDS-polyacrylarnide gel electrophorrsis. Protein samplcs the appearance of typical fibroblastic elements was ( I mg proteinjml) were mixed with concentrated sample buffer (2:1, v/v) to yield a final concentration of 10% gly- greatly reduced for extended periods of growth thus cerol, 5% 2-mercaptoethanol, 3% SDS, 0.001 bromophenol enabling cells of neural origin to express some of their blue, 60 mM-Tris-CI (pH 6.8) and then hcated for 5 min characteristic properties in oitro. Figure 1 depicts the changes in the D N A content at 100°C. Aliquots of about 13pg protein in 20pI volume were routinely subjected to electrophoresis. Electrophoretic and the rate of C3H]thymidine incorporation under separation was carried out in 1G20% gradient slab gels these experimental conditions. It is evident that the unless otherwise stated as described by LAEMMLI ( I 970) daily D N A increment reaches a value of 8% by the with 0.025 ~-Tris-Cilycine,pH 8.3, containing 0.1% SDS as second day, and is subsequently reduced to a value electrophoresis buffer, at constant voltage of 120 V at 18°C of 2.5% per day by the end of the second week in for 5 h. Gels were stained with Coomassie brilliant blue culture. The daily increment, cxpressed as percent of (0.2% in methanolLacetic acid-water, 5 :1 :4, by vol) and the amount of DNA/plate on day 1, probably sugdestained with 10% acetic acid containing 20% methanol. Scanning of the photographic transparencies was carried gests that a stable population of relatively long-lived cells is obtained. This is further corroborated by the out in a Gilford 24003 spectrophotometer at 560 nm: the densitometric tracings were analysed with a Keuffler and finding that L3H]thyrnidine incorporation into cellular DNA is markcdly reduced after 1 week in culture, Esser planimeter. Metabolic studies. About 20 pCi ~-[~~S]methionine.indicating that there is little or no cell turnover, which (400 mCi/mmol, New England Nuclear) in 2 ml meth- would be measured by synthesis of new DNA. Thereionine-free medium was added per dish and cells incubated fore, changes in the protein profiles to be subsefor 5 h at 37'C. For glycoprotein labeling either quently described are presumably related to a popula25 pCi r-[6-'H]fucose (5.3Ci/mrnol New England Nuclear) tion of cells that is preponderantly non-dividing. or 4 pci AcetyI-o-[l-'4C]mannosamine (54.5 mCi/mmol, New England Nuclear) was added per plate. Cells were Alterations in protein prqfiles with culture rnaturatioii harvested after 6 days incubation. DNA labeling was The electrophoretic pattern of the particulate fracaccomplished by pulsing cells with 3 pCiC3H]thymidinc tion of the fetal rat brain is shown in Fig. 2a. Apart (6.58 Ci/mmol, Kamag, Dimona) for 1 h. Procedural details for DNA quantitation and radioactivity determination are from a fast migrating group of polypeptides (I 1-15 K) described elsewhere (YAVIN,1977). which presumably represent the cellular histones, a Derermiizarion of toral proteirt-bound radioactivity. Ali- 56 K and a 42 K band, coinciding with tubulin and quots of the cell homogenate was precipitated at 4°C o n actin markers, respectively, were the predominant Whatman 3 M M paper discs with 7.5% trichloracetic acid components of the complex protein pattern. During containing 2 mg/ml of Casamino acids (Difco). The filters subsequent culture, characteristic changes in the prowere heated at 90°C in 7.5% TCA for 20 min, washed three times in cold TCA, rinsed with ethanol and diethyl-ether tein composition of the fetal brain cells were seen. and dried. Radioactivity was measured in a Packard Tri- In contrast to the fairly steady level of the 42 K component and other minor proteins, a slight decrease carb liquid scintillator spectrometer. Autoradiography For [35S]methionine autoradiography in the concentration of the 56K component and a the stained gels were dried and exposed to an X-ray film marked decrease of the nuclear histones is evident. (Kodak XRP 54) for 48 h. In order to enhance the effi- With progressive maturation in culture, a significant ciency of the autoradiography of [3H]fucose or [14C]N- increasc in a polypeptide (band I), migrating slightly acetyl-mannosamine-labeled proteins, the gels were im- slower than the 56 K component, was apparent. The pregnated with a scintillation cocktail (BONNEK & LASKEY, protein components (bands 3 and 4) migrating with 1974) and aRer drying exposed to the film for 5-6 weeks a relative mobility between the 56 K and 42 K bands at - 70°C. Developed films were scanned and evaluated were also more pronounced at 15 days than at 2 days as described above. Miscellaneous analytical methods. Protein was deter- in culture. These findings are consistent in different mined according to LOWRYet al. (1951) using bovine gel systems as clearly demonstrated by the use of 7.5% serum albumin as standard. 2',3'-cycIic nucleotide 3'-phos- homogeneous (Fig. 2b) and 5-150/, polyacrylamide phohydrolase (CNP, EC 3.1.4.16) activity was measured by gradient gels (Fig. 2c). et al. (1973). the method of PROHASKA The relative pcrcentage of the various proteins was assessed by densitometric measurements of the Coomassie blue stained gels. As shown in Fig. 3, a deRESULTS crease of the histonal-like components from 35% at The phenomenon of fibroblast proliferation is one day one t o 20% after three weeks in culturc was of the major problems encountered in adapting pri- apparent. A decline of the 5 6 K band from 15% to mary cultures originating from highly specialized tis- a constant level of approximately 10% is seen after sues to grow and express their specific properties in the first week in oitro. As already noticed, the relative oitro. In our attempts to grow dissociated fetal rat amount of the 42 K component was practically uncerebral cells, this problem has been largely curtailed changed whereas the proteins migrating between the
Protein profiles in cultured brain cells
135 10
0 A TI L
a
a
s +
5
=
E
2
c a
z n
0
6
12
18
24
Days in culture
FIG.I . Incorporation of C3H]thymidine and changes in the DNA content during cell.growth in culturc. Experimental details were described under Materials and Methods. [3H]Thymidine incorporation (-@) is expressed as c.p.m./pg DNA. DNA daily increment (o----
