J o u r w l o/ h'nwrii hu,srrr>. VoI 31. pp. 217-224. Pcrpmon Press Ltd. 1978 Printed in Grext Britilin. @lnirm.mon.ri Society for Neurnchemlstry Lid.
0022-)042 78'0701-0217 SJ2.W 0
TWO FORMS OF NEURONAL ACTIN Q. L. CHOO'and D. BRAY MRC Cell Biophysics Unit, King's College, 26-29 Drury Lane, London W.C.Z., U.K. ( R e w i r e d 8 September 1977. Reaised 23 Nooember 1977. Accepted 8 December 1977)
Abstract-Cultures of neurons essentially free of non-neuronal cells were prepared from chick sympathetic neurons and from sensory neurons that had been enriched on a simple density gradient. The proteins of these cultures were examined by two-dimensional gel electrophoresis and two species found in each type of nerve cell that ran close to, but not precisely with, muscle actin. They comigrated with the fi and 7 actins previously seen in developing myoblasts (WHALENet al., 1976). Peptide patterns obtained from the two neuronal proteins by limited papain digestion, as well as from three analogous proteins of cultured fibroblasts and purified chicken muscle actin, were extremely similar. The same two species, in similar amounts, were found in soluble and residual fractions of cultured neurons produced by brief detergent treatment; in fractions enriched for neuronal processes or cell soma from cultured sensory ganglia; and in purified actin recovered from material released upon gentle homogenisation of embryonic chick brains.
Sweden) in L 15. The cells from 12 to 24 ganglia were then layered on to distinct both chemically and functionally from the protein of myofibrils. Small differences in the amino a linear 5-20% gradient of Ficoll in L15 contained in a acid sequence of platelet actin and muscle actin have 3in x 4in polycarbonate centrifuge tube (Beckmann Instruments Co.). The gradient was conveniently prepared from a been found (ELZINGA et al., 1976), while some prestepwise gradient of 1.51111 lots of 20%, IS%, 10% and 5% parations of platelet actin have unusual polymerisation Ficoll that was covered and left to stand overnight at room properties (ABRAMOWITZ et a/., 1975). Similarly, the temperature. The tubes were inserted into llOx 70mm actin from whole brain has a different mobility to conical based culture tubes (Nunc, Denmark) and centrimuscle actin in gelscontaining urea (PUSZKIN& BERL, fuged at l500rev/min (330g) for 10 min in a swing-bucket 1972; STORTI& RICH, 1976), and much of it is readily desk top centrifuge. Two or three bands could usually be seen after centrireleased in a monomeric form upon gentle homofugation, the lower of which was composed of neurons. genisation (BRAY& THOMAS,1976). Recently, the technique of two-dimensional gel The tubes were capped with a sterile rubber stopper carrying a tube and adjustable clamp, and pierced at the base with electrophoresis described by OFARRELL (1975), was a sterile 23G syringe needle. Drops were collected and the used to show microheterogeneity in actin. Both deneuronal fraction located by light microscopy, this was then veloping muscle and cultured fibroblasts show three seeded into the culture medium. closely migrating species called a, /Iand j, actin Cultures. Sensory and sympathetic neurons were cultured (WHALEN f t Uf., 1976; RUBENSTEIN& SPUDICH, 1977). in supplemented L15 medium in a normal atmosphere W e have now applied this technique t o the actin of (SHAW& BRAY,1977). For radioactive labelling, the basal cultured neurons and purified brain actin t o see what medium was changed to Minimum Essential Medium species they contain and what relation these have t o the (GIBCo Bio-Cult, Glasgow, Scotland) and incubation was in state of polymerisation of the actin within the cell. 5% COz, 957; air. Secondary cultures of chick skin fibro1975. While this work was in progress analyses of the actin blasts were prepared as described by BRAY& THOMAS, Preparationsenriched in neuronal cell bodies, or i n neuronal of three nerve cell tumour lines (CARRELS& GIBSON, processes were obtained from long term cultures of sensory 1976), and of brain synaptosomes (KELLY& COTMAN. ganglia (ESTRIDGE, 1977). These were grown on collagen1977) were briefly described. covered dishes and fed daily with the above medium supplemented on every second day with 10- M-fluorodeoxyuridine. M ETH 0DS Cultures were harvested after 24-36 h incubation at 37 C. E w ; d i m w f o/',w.risr~ryttetfrorws. Dorsal root ganglia from They were washed with buffered saline and either lifted by the thornco-lumbar region of 11 - I3 day chick embryos were gentle pipetting or scraped from the dish. Cultures to be trypsinised in 0.25;,, trypsin in calcium/magnesium-free detergent-treated were incubated at 37-C in a 0.5"" solution balanced salt solution for 25 min at 37 C. They were of Nonidet P-40 (BDH Chemicals Ltd.) in balanced salt washed once in saline, once in a solution of O.1'Y0 trypsin solution for 5 min, either in a dish (for fibroblasts). or inhibitor (Sigma Chemical Co.) i n balanced salt solution, following transfer to a centrifuge tube (neurons). They were and once in Leibovitz medium L15 (GIBCo Bio-Cult. pooled and centrifuged for 30 min at 100,ooOg at 4 C. Glasgow. Scotland). They were suspended in 1 mI L15 Pellets were dissolved in 0.1""SDS in 0.01 M-Tris-HCI pH medium and dissociated by pipetting with a pasteur pipette 7.4 and supernatants dialysed against 0.01 M-Tris-HCI and mixed with 0.5", Ficoll 400 (Pharmacia. Uppsaba. pH 7.4. and their volume reduced by ultrafiltration. The protein concentration ofall samples was adjusted to 1 mg,'ml before analysis on two-dimensional gels. I To whom all correspondence should be'addressed. L I17
THEACTIN of non-muscle cells has been shown t o be
Q. L. CHOOand D. BRAY
218
B i d t t w t i c t r l [oiolysic-. Samples were analysed by twodimensional gel electrophoresis exactly as described by OFARRELL 11975) except that a linear gradient of 4- 17",, acrylamide was used for the second dimension. The pH gradient was established by 2"" (w/v)Ampholines of which 80",wsre pH 5-7 and 20", were pH 3.5-10. About 10 pg of protein. and 10- 100nCi of protein labelled with [>'S] methionine were usually applied. Autoradiographs were prepared by exposing the dry gel to Kodirex X-ray film. usually for 4-8 days; densitometry of these was carried out o n a Joyce-Loebl Microdensitometer. Proteins were analysed by limited cleavage with papain as described by CLEVELAND et al. (1977). Samples labelled with ["S]methionine were pooled from 3-6 twodimensional gels before analysis; each specimen contained about 0.6pg protein and was mixed with I O N papain (SigmaChemical Co.)at 4 ngiml. The peptides were analysed on a linear gradient of 5-23?,; acrylamide.
RESULTS Sympathetic ganglia in early embryos have relatively few satellite cells and if they are grown in culture at high density the neuronal network may be harvested selectively, however, this procedure is not in general applicable t o other tissues. For chick dorsal root ganglia dissociated with trypsin the simple gradient separation described in the Methods section was developed. The large sensory neurons, diameter about 2 0 p , sediment much more quickly than the satellite cells, whose diameter after trypsin treatment is under 10 pn. The distribution of cell types following a short centrifugation a t low speed is shown in Fig. I. Neurons made up !90:, of the cells in the leading peak and must comprise an even greater fraction of the cellular protein because of their size. Cultures prepared from the neuronal peak had a much reduced satellite cell population as judged by light microscopy (Fig. 1). Furthermore, three distinctive protein species seen in cultures of fibroblasts and sensory ganglion satellite cells by two-dimensional gel electrophoresis (Fig. 2 ) were not detectable in the cultured sympathetic or sensory neurons. The proteins of both sympathetic and sensory neurons, whether detected by autoradiography or by staining with Coomassie Brilliant Blue, showed two components running close to muscle actin (Fig. 2 and 3). Comparison with a standard of chicken muscle actin prepared in a conventional manner showed that this had the same mobility on SDS acrylamide, but ran to the acidic side of the two neuronal spots (Fig. 3). Cultured skin fibroblasts from embryos of the same age show three spots in this region, the most acidic of which (a)comigrated with muscle actin and the remaining two (fi and 7) which migrated with the neuronal doublet. Cultures of satellite cells from the dorsal root ganglia showed a similar pattern. Although theidentityoftheseproteinspotsa s actin is implied by their close relation to muscle actin o n the gels, independent confirmation of this was sought. CLEVELAND~C al. (1977) have described a fingerprinting technique suitable for single bands o n one dimensional
Fraction
No
FIG. la. Separation of embryonic chick sensory ganglionic
neurones from satellite cells. Dorsal root ganglia from II day chick embryos were dissociated with trypsin and applied to a small Ficoll gradient as described in Methods. Aftercentrifugationfor6minat 350gthe tubewas punctured and fractions of4 drops collected.These were examined in ii hemocytometer and the cells counted. Neurons were identified as large round cells (diameter about 20pm) with a prominent nucleus and. usually, a residual axonal process: satellite cells had a diameter of less than 10 pm and were phase-dense with an irregular contour. ---D---; neurons. - C;satellite cells. SDSgels, and we haveapplied this to the proteins of the two-dimensional gel electropherograms. The procedure gave a simple and distinctive pattern of peptides from chicken muscle actin with 4 major species with molecular weights very roughly 35,000; 33,000; 23,000 and 21,000 (Fig. 4). Autoradiographs of samples from two-dimensional gels of cultures showed the same 4 major species, and in the same relative proportion, together with a larger number of minor species (Fig. 4). The patterns from the two kinds of actin species (only sympathetic neurons were examined in this way), and the three fibroblast species were indistinguishable and we conclude from this that they are all forms of actin. The possibility that the various actins had a different distribution within the cell was examined. Cellular 'ghosts'wereprepared bya brieftreatment with thenonionic detergent Nonidet P-40 and both the solubilized and residual components analysed. The two fractions of the neuronal cultures did not differ in composition and densitometry of the autoradiographs of the gels indicated a ratio of approx 1.2:l for B : y in both the soluble and residual fractions (Table 1). The distribution of fibroblast actins showed more variation, but this was not thought to be significant. These ratios were unaffected by changes in the amount of protein loaded on to the gels, even within a ten-fold range (Table 1). A second approach was to examine the unpolymerised actin that is readily released from chick brain. This was purified by a previously described procedure in which embryonic brain is homogenised and applied to a Sephadex GI50 column, and the fractionated monomeric actin caused to aggregate by
219
FIG.lb. Cultures prepared from a Ficoll gradient. Those on the left were from the neuronal peak; those on the right from the major peak of satellite cells ( x 300).
FIG. 2a. An autoradiograph of a two-dimensional gel of the pellet fraction of cultured sjmpdthctic neurons labelled with ["S]mcthionine. The autoradiograph was exposed for 4 days. The first dimension. with isoelectric focusing wiik performed from right (cathode) to left (anode). approximate pH value\ are indicated: the second dimcn5iun w i t h S I X polydcrylamide gradient electrophoresis was from 4"" acrjlamide (top) to 17"". Molccular weights are i n kilodaltons IK).The position ofmusclc actin (2-actin) is indicated by an arrow. FIG.2b. Gel ofthe pellet fraction from cultured skin fibroblasts. The three protein spccics on the left (curly arrow J arc prescnt in fibroblasts and satellite cells but absent in neurons.
221
FIG. 3. Comparison of neuronal and muscle actins. The central areas of three gels stained with Coomassie Vrilliant Blue and one autoradiograph are shown. (a), a samples of muscle actin prepared by conventional procedures from an acetone powder of chicken breast muscle. (b), a sample of the pellet fraction of sympathetic neurons prepared as described in the text. (c), a mixture of equal amounts of (a) and (b); the position of the neuronal proteins was confirmed by autoradiography (d). The arrow indicates the position of a actin.
FIG.4. Papain fingerprints of actin species. Proteins from the two-dimensional gels were excised and digested with er al. (1977). (a), muscle actin: stained gel. (b) neuronal actin: autoradiograph. papain by the method of CLEVELAND (c) fibrolast actin: autoradiograph. a, b, and c, are from the same slab gel. The position of undigested muscle actin is indicated by an arrow.
Two forms of neuronal actin TAHLE 1. DENSITOMETRY OF ACTIN
SPOTS
~
Source of actin
Fraction of total actin ?
B
Y
n.d. n.d. n.d.
0.53 0.51 0.57 0.57
0.47 0.49 0.43 0.43
Brain actin, column prep.
n.d.
0.59
0.41
Fibroblast S Fibroblast P Fibroblast sonicated S Fibroblast sonicated P
0.28 0.39 0.20 0.21
0.44 0.31 0.50 0.52
0.28 0.30 0.30 0.27
Fibroblast P ( x 9/10) Fibroblast P ( x 1/2) Fibroblast P ( x 1/4) Fibroblast P ( x 1/10)
0.34 0.36 0.32 0.31
0.33
0.33 0.30 0.32 0.28
Sympathetic neuron S Sympathetic neron P Sensory neuron S Sensory neuron P
n.d.
0.34
0.36 0.41
Autoradiographs of radioactive gels or, in the case of the brain actin, a positive print on Ilford SP352 film of stained gels were scanned. The areas of the peaks recorded for the different actins were measured and expressed above as fractions of the total. S and P were supernatant and pellet fractions obtained following Nonidet P-40 treatment as described in the text; sonication of fibroblasts was performed as described by BRAY& THOMAS (1976).The last four entries in the table refer to a control experiment in which a fibroblast sample was loaded onto gels in various amounts. n.d.-not detectable. ultrafiltration(BRAu &THOMAS, 1976).This showed the same two species that comigrated with the neuronal actins, and in similar proportions (Table 1). It is worth noting that the species corresponding to (I actin was undetectable in these preparations, and also from a whole homogenate of 12 day embryonic brain. Finally, long term culturesofsensory ganglia were prepared and dissected, as described by ESTRIDGE(l977), into fractions enriched in cell bodies and processes. Once again, both fractions showed a similar pattern of actin sub-species. DISCUSSION
Two kinds of cultured neuron and whole chick embryonic brain, each contain two actin species corresponding to the 8 and y actins of developing et NI.,1976).It is possible that this myoblasts (WHALEN is true of all neurons, especially since the actin of three nervecell tumour lines (GARRELS & GJBSON, 1976) & COTMAN.1977) and of brain synaptosomes (KELLY show the same two species. In the present experiments the separation ofneurons and satellite cells was accomplished either by gently lifting the network of neuronal fibres from the culture. or-in the case of dorsal root ganglia-by separating most of the non-neuronal cells by centrifugation prior to culturing. This was found to be a fast and gentle procedure that presents an alternative to methods that use mitotic poisons (WOOD,1976).selective attachment on to glass (MCCARTHY & PARTLOW,1975). or prolonged centrifugation in gradients of albumin
223
(DIZEREGA et a/., 1970).Thepresent method wasgentle, could be performed in less than 15 min, and gave cultures that were, by light microscopic observation, predominantly neuronal. This was corroborated by the two-dimensional gel analyses which showed neither 3 actin nor three distinctive protein species (Fig. 2), all of which were prominent features of the patterns from satellite cells. The pattern shown by the neuronal actin was distinct from cultured skin fibroblasts and cultured satellite cells, which show three species, and from chicken muscle actin which shows a single species in the a position. The identity of a actin with the protein from striated muscle has been shown previously, while 7 has been suggested to be typical of smooth muscle on the basis ofits preponderance in actin from chicken gizzard et al., 1976; RUBENSTEIN & SPUDICH,1977; (WHALEN IZANT & LAZARIDES, 1977).In work as yet unpublished we have found that human erythrocyte ghosts contain only fr actin. The identity of these spots as actin has been implied in the past by their close migration to muscle actin, and, for chick embryo fibroblasts and a muscle line by a close similarity in tryptic peptides (RUBENSTEIN & SPUDICH,~ ~ ~ ~ ; G A R RGIESON, E L s &1976).We used the method of CLEVELAND et al. (1977), of fingerprinting proteins from gels and found a set of identically migrating peptides from muscle actin and from each of the radioactive neuronal and fibroblastic specis. This is unambiguous evidence that they are all forms of actin. Since only the molecular weights of the largest proteolytic fragments are examined in this method, the absence ofdifferences in the patterns of Fig. 4 should not be taken to indicate identity of sequence. The reason for the differences in migration is unknown, but we believe that they are unlikely to be artefactual Muscle, nerve and fibroblasts in our work, and other tissues in other laboratories, consistently give distinct patterns of actin species: muscle actin and radioactive neuronal actin migrate to their characteristic positions even if mixed beforehand and fractionated on the same gel. The production of multiple closely migrating spots is restricted t o actin and a few other proteins on the gels and is not typical of most of the proteins on the two-dimensional gel. The relative amounts of the different actins d o not change from preparation to preparation and are not affected by the size of the sample within a ten-fold range (Table 1). From the established differences in amino acid er ul.. sequence of platelet and muscle actins (ELZ~NGA 1976)andfrom the previous evidence that the difference between muscle and brain actin is expressed in a RNA fraction from the two tissues (STORTI & RICH. 1976). it seems most probable that they are the products of different genes. This is supported by differences in twodimensional peptide maps between z and /( actins (RUBENSTEIN & SPUDICH. 1977; GARRFU & GIRSON, 1976). The need for the three species of actin is not clear. There was no indication in our work that they are re-
224
Q. L. C ~ o and o D. BRAY
BRAYD. & THOMAS C. (1976) J. molec. Biol. 105,527-544. CLEVELAND D. W., FISCHER S . G., KIRSCHNER M. W. & LAEMMLI U. K . (1977) J . biol. Chem. 252, 1102- 1106. DIZEREGA G., JOHNSONL., MORROWJ. & KASTENF. H. (1970) E x p l . Cell Res. 63, 189- 192. ELZINGAM., MARONB. J. & ADELSTEIN R. S. (1976) Science. N.Y. 191,94495. ESTRIDGE M. (1977) Nuture, Lond. 268, 60-63. GARRELS J. 1. & GILLSON W. (1977) Cell 9, 793-805. IZANT J. G. & LAZARIDES E. (1977) Proc. nain. Acud. Sci.. U.S.A. 74. 1450-1454. KELLYP. 1.& COTMAN C. W. (1977) 6rh Int. Meer. Int. Sac. Neurochem. Abstr. 79a. MCCARTHY K. D. & PARTLOW L. M. (1975) Trans. Am. Soc. Neurochem. 6,96. OFARRELL P. H. (1975) J. biol. Chem. 250,4007-4021. PUSZKINS . & BERLS. (1972) Biochim. biophys. Acta 256, Acknowledgements-We thank DAVID GILBERT, CLIVE 695-709. THOMAS and ZOLLY GABOR for their advice and help. RUBENSTEINP. A. & SPUDICHJ. A. (1977) Proc. natn. Acad. Sci., U.S.A. 74, 120-123. SHAWG. & BRAYD. (1977) E x p l . Cell Res. 104, 55-62. STORTIR. V. & RICH A. (1976) Proc. nutn. Arud. Sci., REFERENCES U.S.A. 13.2346-2350. G. S. & GROSF. (1976) ABRAMOWITZ J. W., STRACHERA. & DETWILLER T. C. WHALENR. G., BUTLER-BROWNE (1975)Archs Biochem. Bwphys. 167,230-237. Proc. natn. Acad. Sci.. U.S.A. 73, 2018-2022. BRAYD. & THOMASC. (1975) Biochem. J . 147,221-228. WOOD P. M. (1976) Brain Res. 115, 361-375.
lated to the state of aggregations of actin in the cell, and a similar observation was made by RUBENSTEIN& SPUDICH(1977). If we assume that the /J and 7 actins from different cell types are the same proteins, then their varying amounts suggest that they are not subunits of adefined polymer-as with the two classesof tubulinbut rather independent proteins. The fact that the readily released form of brain actin previously studied in this laboratory (BRAY& THOMAS,1976), contains j3 and y actin but not a is consistent with the different properties this has to muscle actin. This suggests, furthermore, that B and y actins may exist within the cell in a monomeric or labile form.
0022-)042 78'0701-0217 SJ2.W 0
TWO FORMS OF NEURONAL ACTIN Q. L. CHOO'and D. BRAY MRC Cell Biophysics Unit, King's College, 26-29 Drury Lane, London W.C.Z., U.K. ( R e w i r e d 8 September 1977. Reaised 23 Nooember 1977. Accepted 8 December 1977)
Abstract-Cultures of neurons essentially free of non-neuronal cells were prepared from chick sympathetic neurons and from sensory neurons that had been enriched on a simple density gradient. The proteins of these cultures were examined by two-dimensional gel electrophoresis and two species found in each type of nerve cell that ran close to, but not precisely with, muscle actin. They comigrated with the fi and 7 actins previously seen in developing myoblasts (WHALENet al., 1976). Peptide patterns obtained from the two neuronal proteins by limited papain digestion, as well as from three analogous proteins of cultured fibroblasts and purified chicken muscle actin, were extremely similar. The same two species, in similar amounts, were found in soluble and residual fractions of cultured neurons produced by brief detergent treatment; in fractions enriched for neuronal processes or cell soma from cultured sensory ganglia; and in purified actin recovered from material released upon gentle homogenisation of embryonic chick brains.
Sweden) in L 15. The cells from 12 to 24 ganglia were then layered on to distinct both chemically and functionally from the protein of myofibrils. Small differences in the amino a linear 5-20% gradient of Ficoll in L15 contained in a acid sequence of platelet actin and muscle actin have 3in x 4in polycarbonate centrifuge tube (Beckmann Instruments Co.). The gradient was conveniently prepared from a been found (ELZINGA et al., 1976), while some prestepwise gradient of 1.51111 lots of 20%, IS%, 10% and 5% parations of platelet actin have unusual polymerisation Ficoll that was covered and left to stand overnight at room properties (ABRAMOWITZ et a/., 1975). Similarly, the temperature. The tubes were inserted into llOx 70mm actin from whole brain has a different mobility to conical based culture tubes (Nunc, Denmark) and centrimuscle actin in gelscontaining urea (PUSZKIN& BERL, fuged at l500rev/min (330g) for 10 min in a swing-bucket 1972; STORTI& RICH, 1976), and much of it is readily desk top centrifuge. Two or three bands could usually be seen after centrireleased in a monomeric form upon gentle homofugation, the lower of which was composed of neurons. genisation (BRAY& THOMAS,1976). Recently, the technique of two-dimensional gel The tubes were capped with a sterile rubber stopper carrying a tube and adjustable clamp, and pierced at the base with electrophoresis described by OFARRELL (1975), was a sterile 23G syringe needle. Drops were collected and the used to show microheterogeneity in actin. Both deneuronal fraction located by light microscopy, this was then veloping muscle and cultured fibroblasts show three seeded into the culture medium. closely migrating species called a, /Iand j, actin Cultures. Sensory and sympathetic neurons were cultured (WHALEN f t Uf., 1976; RUBENSTEIN& SPUDICH, 1977). in supplemented L15 medium in a normal atmosphere W e have now applied this technique t o the actin of (SHAW& BRAY,1977). For radioactive labelling, the basal cultured neurons and purified brain actin t o see what medium was changed to Minimum Essential Medium species they contain and what relation these have t o the (GIBCo Bio-Cult, Glasgow, Scotland) and incubation was in state of polymerisation of the actin within the cell. 5% COz, 957; air. Secondary cultures of chick skin fibro1975. While this work was in progress analyses of the actin blasts were prepared as described by BRAY& THOMAS, Preparationsenriched in neuronal cell bodies, or i n neuronal of three nerve cell tumour lines (CARRELS& GIBSON, processes were obtained from long term cultures of sensory 1976), and of brain synaptosomes (KELLY& COTMAN. ganglia (ESTRIDGE, 1977). These were grown on collagen1977) were briefly described. covered dishes and fed daily with the above medium supplemented on every second day with 10- M-fluorodeoxyuridine. M ETH 0DS Cultures were harvested after 24-36 h incubation at 37 C. E w ; d i m w f o/',w.risr~ryttetfrorws. Dorsal root ganglia from They were washed with buffered saline and either lifted by the thornco-lumbar region of 11 - I3 day chick embryos were gentle pipetting or scraped from the dish. Cultures to be trypsinised in 0.25;,, trypsin in calcium/magnesium-free detergent-treated were incubated at 37-C in a 0.5"" solution balanced salt solution for 25 min at 37 C. They were of Nonidet P-40 (BDH Chemicals Ltd.) in balanced salt washed once in saline, once in a solution of O.1'Y0 trypsin solution for 5 min, either in a dish (for fibroblasts). or inhibitor (Sigma Chemical Co.) i n balanced salt solution, following transfer to a centrifuge tube (neurons). They were and once in Leibovitz medium L15 (GIBCo Bio-Cult. pooled and centrifuged for 30 min at 100,ooOg at 4 C. Glasgow. Scotland). They were suspended in 1 mI L15 Pellets were dissolved in 0.1""SDS in 0.01 M-Tris-HCI pH medium and dissociated by pipetting with a pasteur pipette 7.4 and supernatants dialysed against 0.01 M-Tris-HCI and mixed with 0.5", Ficoll 400 (Pharmacia. Uppsaba. pH 7.4. and their volume reduced by ultrafiltration. The protein concentration ofall samples was adjusted to 1 mg,'ml before analysis on two-dimensional gels. I To whom all correspondence should be'addressed. L I17
THEACTIN of non-muscle cells has been shown t o be
Q. L. CHOOand D. BRAY
218
B i d t t w t i c t r l [oiolysic-. Samples were analysed by twodimensional gel electrophoresis exactly as described by OFARRELL 11975) except that a linear gradient of 4- 17",, acrylamide was used for the second dimension. The pH gradient was established by 2"" (w/v)Ampholines of which 80",wsre pH 5-7 and 20", were pH 3.5-10. About 10 pg of protein. and 10- 100nCi of protein labelled with [>'S] methionine were usually applied. Autoradiographs were prepared by exposing the dry gel to Kodirex X-ray film. usually for 4-8 days; densitometry of these was carried out o n a Joyce-Loebl Microdensitometer. Proteins were analysed by limited cleavage with papain as described by CLEVELAND et al. (1977). Samples labelled with ["S]methionine were pooled from 3-6 twodimensional gels before analysis; each specimen contained about 0.6pg protein and was mixed with I O N papain (SigmaChemical Co.)at 4 ngiml. The peptides were analysed on a linear gradient of 5-23?,; acrylamide.
RESULTS Sympathetic ganglia in early embryos have relatively few satellite cells and if they are grown in culture at high density the neuronal network may be harvested selectively, however, this procedure is not in general applicable t o other tissues. For chick dorsal root ganglia dissociated with trypsin the simple gradient separation described in the Methods section was developed. The large sensory neurons, diameter about 2 0 p , sediment much more quickly than the satellite cells, whose diameter after trypsin treatment is under 10 pn. The distribution of cell types following a short centrifugation a t low speed is shown in Fig. I. Neurons made up !90:, of the cells in the leading peak and must comprise an even greater fraction of the cellular protein because of their size. Cultures prepared from the neuronal peak had a much reduced satellite cell population as judged by light microscopy (Fig. 1). Furthermore, three distinctive protein species seen in cultures of fibroblasts and sensory ganglion satellite cells by two-dimensional gel electrophoresis (Fig. 2 ) were not detectable in the cultured sympathetic or sensory neurons. The proteins of both sympathetic and sensory neurons, whether detected by autoradiography or by staining with Coomassie Brilliant Blue, showed two components running close to muscle actin (Fig. 2 and 3). Comparison with a standard of chicken muscle actin prepared in a conventional manner showed that this had the same mobility on SDS acrylamide, but ran to the acidic side of the two neuronal spots (Fig. 3). Cultured skin fibroblasts from embryos of the same age show three spots in this region, the most acidic of which (a)comigrated with muscle actin and the remaining two (fi and 7) which migrated with the neuronal doublet. Cultures of satellite cells from the dorsal root ganglia showed a similar pattern. Although theidentityoftheseproteinspotsa s actin is implied by their close relation to muscle actin o n the gels, independent confirmation of this was sought. CLEVELAND~C al. (1977) have described a fingerprinting technique suitable for single bands o n one dimensional
Fraction
No
FIG. la. Separation of embryonic chick sensory ganglionic
neurones from satellite cells. Dorsal root ganglia from II day chick embryos were dissociated with trypsin and applied to a small Ficoll gradient as described in Methods. Aftercentrifugationfor6minat 350gthe tubewas punctured and fractions of4 drops collected.These were examined in ii hemocytometer and the cells counted. Neurons were identified as large round cells (diameter about 20pm) with a prominent nucleus and. usually, a residual axonal process: satellite cells had a diameter of less than 10 pm and were phase-dense with an irregular contour. ---D---; neurons. - C;satellite cells. SDSgels, and we haveapplied this to the proteins of the two-dimensional gel electropherograms. The procedure gave a simple and distinctive pattern of peptides from chicken muscle actin with 4 major species with molecular weights very roughly 35,000; 33,000; 23,000 and 21,000 (Fig. 4). Autoradiographs of samples from two-dimensional gels of cultures showed the same 4 major species, and in the same relative proportion, together with a larger number of minor species (Fig. 4). The patterns from the two kinds of actin species (only sympathetic neurons were examined in this way), and the three fibroblast species were indistinguishable and we conclude from this that they are all forms of actin. The possibility that the various actins had a different distribution within the cell was examined. Cellular 'ghosts'wereprepared bya brieftreatment with thenonionic detergent Nonidet P-40 and both the solubilized and residual components analysed. The two fractions of the neuronal cultures did not differ in composition and densitometry of the autoradiographs of the gels indicated a ratio of approx 1.2:l for B : y in both the soluble and residual fractions (Table 1). The distribution of fibroblast actins showed more variation, but this was not thought to be significant. These ratios were unaffected by changes in the amount of protein loaded on to the gels, even within a ten-fold range (Table 1). A second approach was to examine the unpolymerised actin that is readily released from chick brain. This was purified by a previously described procedure in which embryonic brain is homogenised and applied to a Sephadex GI50 column, and the fractionated monomeric actin caused to aggregate by
219
FIG.lb. Cultures prepared from a Ficoll gradient. Those on the left were from the neuronal peak; those on the right from the major peak of satellite cells ( x 300).
FIG. 2a. An autoradiograph of a two-dimensional gel of the pellet fraction of cultured sjmpdthctic neurons labelled with ["S]mcthionine. The autoradiograph was exposed for 4 days. The first dimension. with isoelectric focusing wiik performed from right (cathode) to left (anode). approximate pH value\ are indicated: the second dimcn5iun w i t h S I X polydcrylamide gradient electrophoresis was from 4"" acrjlamide (top) to 17"". Molccular weights are i n kilodaltons IK).The position ofmusclc actin (2-actin) is indicated by an arrow. FIG.2b. Gel ofthe pellet fraction from cultured skin fibroblasts. The three protein spccics on the left (curly arrow J arc prescnt in fibroblasts and satellite cells but absent in neurons.
221
FIG. 3. Comparison of neuronal and muscle actins. The central areas of three gels stained with Coomassie Vrilliant Blue and one autoradiograph are shown. (a), a samples of muscle actin prepared by conventional procedures from an acetone powder of chicken breast muscle. (b), a sample of the pellet fraction of sympathetic neurons prepared as described in the text. (c), a mixture of equal amounts of (a) and (b); the position of the neuronal proteins was confirmed by autoradiography (d). The arrow indicates the position of a actin.
FIG.4. Papain fingerprints of actin species. Proteins from the two-dimensional gels were excised and digested with er al. (1977). (a), muscle actin: stained gel. (b) neuronal actin: autoradiograph. papain by the method of CLEVELAND (c) fibrolast actin: autoradiograph. a, b, and c, are from the same slab gel. The position of undigested muscle actin is indicated by an arrow.
Two forms of neuronal actin TAHLE 1. DENSITOMETRY OF ACTIN
SPOTS
~
Source of actin
Fraction of total actin ?
B
Y
n.d. n.d. n.d.
0.53 0.51 0.57 0.57
0.47 0.49 0.43 0.43
Brain actin, column prep.
n.d.
0.59
0.41
Fibroblast S Fibroblast P Fibroblast sonicated S Fibroblast sonicated P
0.28 0.39 0.20 0.21
0.44 0.31 0.50 0.52
0.28 0.30 0.30 0.27
Fibroblast P ( x 9/10) Fibroblast P ( x 1/2) Fibroblast P ( x 1/4) Fibroblast P ( x 1/10)
0.34 0.36 0.32 0.31
0.33
0.33 0.30 0.32 0.28
Sympathetic neuron S Sympathetic neron P Sensory neuron S Sensory neuron P
n.d.
0.34
0.36 0.41
Autoradiographs of radioactive gels or, in the case of the brain actin, a positive print on Ilford SP352 film of stained gels were scanned. The areas of the peaks recorded for the different actins were measured and expressed above as fractions of the total. S and P were supernatant and pellet fractions obtained following Nonidet P-40 treatment as described in the text; sonication of fibroblasts was performed as described by BRAY& THOMAS (1976).The last four entries in the table refer to a control experiment in which a fibroblast sample was loaded onto gels in various amounts. n.d.-not detectable. ultrafiltration(BRAu &THOMAS, 1976).This showed the same two species that comigrated with the neuronal actins, and in similar proportions (Table 1). It is worth noting that the species corresponding to (I actin was undetectable in these preparations, and also from a whole homogenate of 12 day embryonic brain. Finally, long term culturesofsensory ganglia were prepared and dissected, as described by ESTRIDGE(l977), into fractions enriched in cell bodies and processes. Once again, both fractions showed a similar pattern of actin sub-species. DISCUSSION
Two kinds of cultured neuron and whole chick embryonic brain, each contain two actin species corresponding to the 8 and y actins of developing et NI.,1976).It is possible that this myoblasts (WHALEN is true of all neurons, especially since the actin of three nervecell tumour lines (GARRELS & GJBSON, 1976) & COTMAN.1977) and of brain synaptosomes (KELLY show the same two species. In the present experiments the separation ofneurons and satellite cells was accomplished either by gently lifting the network of neuronal fibres from the culture. or-in the case of dorsal root ganglia-by separating most of the non-neuronal cells by centrifugation prior to culturing. This was found to be a fast and gentle procedure that presents an alternative to methods that use mitotic poisons (WOOD,1976).selective attachment on to glass (MCCARTHY & PARTLOW,1975). or prolonged centrifugation in gradients of albumin
223
(DIZEREGA et a/., 1970).Thepresent method wasgentle, could be performed in less than 15 min, and gave cultures that were, by light microscopic observation, predominantly neuronal. This was corroborated by the two-dimensional gel analyses which showed neither 3 actin nor three distinctive protein species (Fig. 2), all of which were prominent features of the patterns from satellite cells. The pattern shown by the neuronal actin was distinct from cultured skin fibroblasts and cultured satellite cells, which show three species, and from chicken muscle actin which shows a single species in the a position. The identity of a actin with the protein from striated muscle has been shown previously, while 7 has been suggested to be typical of smooth muscle on the basis ofits preponderance in actin from chicken gizzard et al., 1976; RUBENSTEIN & SPUDICH,1977; (WHALEN IZANT & LAZARIDES, 1977).In work as yet unpublished we have found that human erythrocyte ghosts contain only fr actin. The identity of these spots as actin has been implied in the past by their close migration to muscle actin, and, for chick embryo fibroblasts and a muscle line by a close similarity in tryptic peptides (RUBENSTEIN & SPUDICH,~ ~ ~ ~ ; G A R RGIESON, E L s &1976).We used the method of CLEVELAND et al. (1977), of fingerprinting proteins from gels and found a set of identically migrating peptides from muscle actin and from each of the radioactive neuronal and fibroblastic specis. This is unambiguous evidence that they are all forms of actin. Since only the molecular weights of the largest proteolytic fragments are examined in this method, the absence ofdifferences in the patterns of Fig. 4 should not be taken to indicate identity of sequence. The reason for the differences in migration is unknown, but we believe that they are unlikely to be artefactual Muscle, nerve and fibroblasts in our work, and other tissues in other laboratories, consistently give distinct patterns of actin species: muscle actin and radioactive neuronal actin migrate to their characteristic positions even if mixed beforehand and fractionated on the same gel. The production of multiple closely migrating spots is restricted t o actin and a few other proteins on the gels and is not typical of most of the proteins on the two-dimensional gel. The relative amounts of the different actins d o not change from preparation to preparation and are not affected by the size of the sample within a ten-fold range (Table 1). From the established differences in amino acid er ul.. sequence of platelet and muscle actins (ELZ~NGA 1976)andfrom the previous evidence that the difference between muscle and brain actin is expressed in a RNA fraction from the two tissues (STORTI & RICH. 1976). it seems most probable that they are the products of different genes. This is supported by differences in twodimensional peptide maps between z and /( actins (RUBENSTEIN & SPUDICH. 1977; GARRFU & GIRSON, 1976). The need for the three species of actin is not clear. There was no indication in our work that they are re-
224
Q. L. C ~ o and o D. BRAY
BRAYD. & THOMAS C. (1976) J. molec. Biol. 105,527-544. CLEVELAND D. W., FISCHER S . G., KIRSCHNER M. W. & LAEMMLI U. K . (1977) J . biol. Chem. 252, 1102- 1106. DIZEREGA G., JOHNSONL., MORROWJ. & KASTENF. H. (1970) E x p l . Cell Res. 63, 189- 192. ELZINGAM., MARONB. J. & ADELSTEIN R. S. (1976) Science. N.Y. 191,94495. ESTRIDGE M. (1977) Nuture, Lond. 268, 60-63. GARRELS J. 1. & GILLSON W. (1977) Cell 9, 793-805. IZANT J. G. & LAZARIDES E. (1977) Proc. nain. Acud. Sci.. U.S.A. 74. 1450-1454. KELLYP. 1.& COTMAN C. W. (1977) 6rh Int. Meer. Int. Sac. Neurochem. Abstr. 79a. MCCARTHY K. D. & PARTLOW L. M. (1975) Trans. Am. Soc. Neurochem. 6,96. OFARRELL P. H. (1975) J. biol. Chem. 250,4007-4021. PUSZKINS . & BERLS. (1972) Biochim. biophys. Acta 256, Acknowledgements-We thank DAVID GILBERT, CLIVE 695-709. THOMAS and ZOLLY GABOR for their advice and help. RUBENSTEINP. A. & SPUDICHJ. A. (1977) Proc. natn. Acad. Sci., U.S.A. 74, 120-123. SHAWG. & BRAYD. (1977) E x p l . Cell Res. 104, 55-62. STORTIR. V. & RICH A. (1976) Proc. nutn. Arud. Sci., REFERENCES U.S.A. 13.2346-2350. G. S. & GROSF. (1976) ABRAMOWITZ J. W., STRACHERA. & DETWILLER T. C. WHALENR. G., BUTLER-BROWNE (1975)Archs Biochem. Bwphys. 167,230-237. Proc. natn. Acad. Sci.. U.S.A. 73, 2018-2022. BRAYD. & THOMASC. (1975) Biochem. J . 147,221-228. WOOD P. M. (1976) Brain Res. 115, 361-375.
lated to the state of aggregations of actin in the cell, and a similar observation was made by RUBENSTEIN& SPUDICH(1977). If we assume that the /J and 7 actins from different cell types are the same proteins, then their varying amounts suggest that they are not subunits of adefined polymer-as with the two classesof tubulinbut rather independent proteins. The fact that the readily released form of brain actin previously studied in this laboratory (BRAY& THOMAS,1976), contains j3 and y actin but not a is consistent with the different properties this has to muscle actin. This suggests, furthermore, that B and y actins may exist within the cell in a monomeric or labile form.
