Brain Research, 97 (1975) 291-301
29 l
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
RAP1D TRANSPORT OF PROTEINS IN THE SONIC MOTOR SYSTEM OF THE TOADFISH
J. L. BARKER, P. N. H O F F M A N , H. G A I N E R AND R. J. LASEK
Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014, and Department of Anatomy, Case Western Reserve Medical School, Cleveland, Ohio 44106, and Marine Biology Laboratory, Woods Hole, Mass. 02543
(U.S.A.) (Accepted April 27th, 1975)
SUMMARY
A pure cholinergic, motor system of a marine fish has been utilized to study the kinetics and characteristics of proteins rapidly transported from the sonic motor nucleus to the musculature enveloping the swim bladder. Following microinjection of [3H]leucine, [3H]lysine, [35S]methionine, or [3H]fucose into the nucleus a wave of radioactivity was observed moving along the sonic motor nerves with an apparent rate of 96-120 ram/day. Analysis of the rapidly transported methionine-labeled protein using SDS gel electrophoresis revealed at least 9 major peaks of activity. Eight of these proteins were found to incorporate fucose, suggesting that most of the rapidly transported material consists of glycoproteins. These results are consistent with the previously suggested hypothesis relating the function of rapid transport to synaptic vesicles and the maintenance of pre-synaptic terminal membranes.
INTRODUCTION
Transport of material from the nerve cell body by the axon has been well demonstrated in a wide variety of nervous systems (for recent reviews, see Grafstein 10, Lasek 14 and Ochsl7). Analysis of the transported material has for the most part concentrated on characterizing it either in terms of rate of transport, subcellular distribution, and/or solubility in acid. The velocity of the rapidly transported material is directly dependent on temperature u and a proportion of it is considered to be protein since it is insoluble in acid. Further characterization of the amino acid-labeled, rapidly transported protein by electrophoretic mobility or sodium dodecylsulfate-polyacrylamide gels has recently been reported for several sensory and motor systemsl, 7,13,2o. Since the isotopes used differ, it is difficult to compare the patterns obtained in the
292 various systems. The rather complex profile of rapidly transported methionine-labeled protein in the rabbit visual system compared to the simple pattern obtained in other systems when labeled leucine was used suggested that methionine might provide a good precursor for establishing a more complete characterization of rapidly transported protein. We have used a pure motor system unique to certain species of fish to characterize the methionine-labeled, rapidly transported proteins. The cell bodies of the m o t o r system lie in the brain stem and project to, and innervate muscles enveloping the swim bladder. The system consists of pure cholinergic axons s which function to produce sound by causing rapid contractions of the swim bladder musclesS, 19,21. The functional purity of the system, its length and discrete termination would appear to offer an excellent system to investigate the nature and function of rapid axonal transport. The results obtained thusfar indicate (1) that proteins are rapidly transported at 96-120 m m / d a y and (2) that these proteins can be characterized into at least 9 classes, as defined by electrophoretic mobility. Comparison of this data with other electrophoretic profiles of rapidly transported proteins reveal several protein classes common to a variety of systems. METHODS
Preparation The experiments were carried out on 60 toadfish (Opsanus tau) obtained at the Woods Hole Marine Biological Laboratory in Woods Hole, Mass. The general features of the preparation are schematically diagrammed in Fig. 1. The fish were held in tanks continuously perfused with sea-water at 19-20 °C. Anesthesia was induced by
Fig. 1. Sonic motor system of toadfish. Right: picture of toadfish which normally dwells at the bottom of the ocean. Left: schematic diagram of sonic motor nucleus containing nerve cell bodies which project to the swim bladder to innervate muscle enveloping bladder. A glass micropipette used to slowly deliver concentrated isotope under pressure is seen entering nucleus.
293 placing the fish in a bucket containing 0.5 g/1 MS-222 (tricaine methanesulfonate). After several minutes body and gil movements ceased and the fish was placed on a small operating stand kept wet with a sea-water-soaked cloth. The gills were perfused with sea-water containing 0.1 g/1 MS-222. The muscles and bones protecting the dorsal aspect of the midbrain were carefully removed and the dorsal surface of the brain stem visualized with the aid of a dissecting microscope. Preliminary injections using methylene blue showed that the sonic motor nucleus containing the cell bodies of the sonic motor nerves was located approximately 1 mm below the dorsal surface of the brain stem and extended about 3 mm rostro-caudally in the mid-line. Surface landmarks for the nucleus included the mid-line and a shallow, indented area, triangular in extent (the obex), bounded rostrally and caudally by easily visible commissures. Three mid-line injections 1 mm deep were made about 1 mm apart beginning just posterior to the commissure providing the caudal bound to the obex. After the injections the muscles and skin overlying the brain stem were approximated with 3-0 gauge silk in either one or two layers of closure and the fish revived in a holding tank. In 60 ~ of injected fish one of the sonic motor nerves projecting to the swim bladder was exposed through a ventral incision and crushed and ligated at its entrance into the body of swimbladder muscle. The body wall was closed in one layer with 3-0 gauge silk.
Micr oinjection The following isotopic labels were used: (1) L-[4,5-~H]leucine (NEN Chemicals) with a specific activity of 40-60 Ci/mmole, (2) e-[4,5-3H]lysine (NEN), specific activity 31.4 Ci/mmole; (3) L-[35S]methionine (NEN), specific activity 195.5 Ci/mmole, when obtained; (4) L-[3H]fucose (NEN), specific activity 13.4 Ci/mmole. The stock solutions of isotope were dried under nitrogen and the residue redissolved in distilled water to give final concentrations of 10 mCi/ml. In some experiments mixtures of leucine and lysine or methionine and fucose were used. The concentrated isotopic solution was drawn up into a finely tapered glass micropipette (50-1130/~m tip). One ,ul injections of label were made over a 30-rain period at the 3 midline sites using a precision pump coupled to an oil-filled injection system.
Preparation of tissue for radioactivity determination At various times after microinjection into the brain stem the fish were sacrificed by placing them in sea-water containing 0.5 g/1 MS-222 for 15 min. Both sonic motor (SM) nerves were incised as close as possible to their exit from the vertebral column and the SM nerves and swim bladder with enveloping muscle were immediately removed for analysis. For analysis of the transport rate the nerves were cut into 3 mm sections and incubated for 8 h at 60 °C in 0.5 N NaOH. After cooling, Bray's solution was added to the vial and the radioactivity counted either in a Packard or Beckman liquid scintillation counter using the appropriate windows. In the Packard, counting efficiencies of 35S and 3H were 75 ~ and 26 ~ , respectively. An analysis of the distribution of radioactivity between TCA-insoluble and TCA-soluble fractions was not carried out.
294
Polyacrylamide gel electrophoresis For analysis of radioactively labeled proteins the nerves were cut into 5 or 10 mm sections and homogenized by hand using glass-on-glass homogenizers. The material to be homogenized was first ground in 8 M urea and again after adding sodium dodecylsulfate (SDS) to a final concentration of 1 ~ . After the latter treatment the homogenate was brought to 95 °C for 5 min, cooled and then subjected to a final homogenization after making the solution 1 ~ fi-mercaptoethanol. The resuiting homogenate was usually cloudy. An aliquot was taken for radioactivity determination (using Bray's solution as the photofluor). The remainder was analyzed by gel electrophoresis either immediately or after storage at --20 °C. Electrophoresis was carried out using discontinuous, 10 ~ polyacrylamide gels according to the method of Gainer 9. Marker proteins and bromophenol blue were included with each sample. The marker proteins were: myosin (212,(3130 daltons),/3galactosidase (130,(3(30 daltons), bovine serum albumin (68,(3C0 daltons), ovalbumin (43,(3(30 daltons), carbonic anhydrase (29,C(30 daltons), myoglobin (18,C(30 daltons), RNAase (12,13130 daltons) and bacitracin (1,zlC0 daltons). A standard curve was obtained by plotting the logarithm of the molecular weights of the marker proteins as a function of their mobilities relative to myoglobin. After completion of the electrophoresis the gels were stained overnight in 0.25 Coomassie blue, destained in 10 ~ methanol: 7.5 ~ acetic acid solutions and cut into 1.5 mm slices. The slices were digested with H202 and radioactivity was determined using Bray's solution as the photofluor. In double-label experiments the cross-over of aaS counts into the 3H channel (and vice-versa) was minimized by setting the appropriate windows. The cross-over of 35S accounted for 9.6 ~ of the activity present in the aH channel, while the crossover of the 8H into the 35S channel accounted for 0.25 ~ of the activity counted in the latter channel. The 85S contamination of the 3H channel was subtracted from the 3H counts while the negligible 3H contamination of asS was not corrected. RESULTS
Transport rate Radioactive material was present in the sonic motor nerves following injection of isotopic label in the SM nucleus. The time required for the initial appearance of radioactivity was typically 3-4 h after injection. An analysis of radioactivity present in the SM nerves at various intervals during the first 24 h was completed after microinjection of either [aH]leucine, [3H]fucose or [zsS]methionine. Similar results regarding the gross distribution of radioactive material as a function of time were observed with all 3 isotopes. Only those obtained with [aaS]methionine will be presented. Representative examples of the appearance of 35S activity in ligated and unligated SM nerves are illustrated in Fig. 2. An accumulation of radioactivity at the ligature was evident beginning with the 6-h interval. Radioactivity of approximately similar distribution was present in both ligated and unligated nerves of 4-h and 6-h fish. At 9 h the wave front of radioactivity had moved farther down the unligated nerve and
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Fig. 2. The distribution of [zsS]methionine labeled material in the sonic motor nerves is plotted as a function of approximate distance along the nerves at various times after the microinjection of the label into the nuclei of 5 toadfish. The nerves were cut into 3 mm segments and the data expressed as counts/min (cpm)/segment. One sonic motor nerve was crushed and ligated (~') while the other remained untouched. An accumulation of radioactive material at the ligature begins at about the 6th h following intranuclear injection. An apparent wave front of activity is present during the early time periods, disappears with longer intervals post-injection and is replaced by another apparent wave at still longer intervals. showed evidence o f accumulation at the ligature. A suggestion o f a front in the unligated nerve was largely absent from the 14-h fish while a second, later wave of activity had begun to enter the unligated nerve in the 22-h fish. These results indicate that radioactivity is transported along the SM nerves at about 4-5 m m / h (or 96-120 mm/day). Some uncertainty exists regarding the precise rate since the distance from the SM nucleus to the first segment analyzed was estimated at 10 m m (from several complete dissections o f the entire SM nerve). This rate is less than that which would be predicted for 20 °C from data reported for the garfish olfactory nerve 11 (at which temperature rapid transport progresses at about 180 mm/day). The more slowly moving material initially present in the 22-h fish is transported at less than 1 mm/h.
Rapidly transportedproteins The profile o f rapidly transported proteins labeled with [35S]methionine was complex. Similar profiles were obtained from 8 toadfish. At least 9 peaks could be distinguished in the material accumulating at the ligature of both 10-h and 18-h fish (Fig. 3B and C). The peaks had the following approximate molecular weights (esti-
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Fig. 3. Representative electrophoretic profiles of methionine-labeled, rapidly transported proteins. The labeled material was obtained at 10- and 18-h intervals following injections in the nuclei of two toadfish. Ten-ram segmentsjust proximal to the ligature or from the wave front in the unligated nerve (10-h fish) were homogenized in urea, SDS and BME and electrophoresed on 10 ~ polyacrylamide gels, cut into 1.5 mm slices and the radioactivity in each slice plotted above. A set of standard marker proteins was included in the electrophoresis (see text). A number of major peaks can be discerned. mated from comparing their relative mobilities with those of proteins of known molecular weights run on gels during the same electrophoresis run): 190 K, 130 K, 100 K, 68 K, 57 K, 43 K, 34 K, 29 K, 18 K, and several < 1 8 K (where K = 1000). Some variability in the size of the various peaks at ligatures (e.g., the 43 K peak) was observed, but the general pattern was reproducible (see also Fig. 4). A similar, distinct profile of rapidly transported, methionine-labeled proteins was also observed in the wave front of the other (unligated) nerve of the 10-h fish (Fig. 3A). Profiles of rapidly transported proteins labeled with both methionine and fucose were developed for 10-h and 18-h fish. Representative examples are illustrated in Fig. 4. A correspondence of protein labeling with fucose and methionine was found for most of the peaks. However, several fucose-labeled peaks relatively unlabeled with methionine were also found, and vice versa.
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Fig. 4. Representative electrophoretic profiles of doubly-labeled, rapidly transported proteins. The labeled material was obtained 10 and 18 h after injection of a mixture of psS]methionine and [3HIfucose into the sonic motor nuclei of two toadfish. Ten-mm segments immediately proximal to the ligature were utilized to develop the profiles. A number of proteins labeled with both methionine and fucose can be seen (see text for details).
Preliminary experiments using leucine and lysine as radioactive precursors showed a pattern which had many small, labeled peaks. The profile thus appeared to contain fewer distinct, heavily labeled peaks. Further work is required to better compare these proteins with those labeled with methionine or fucose. DISCUSSION
Most of the analysis of material rapidly transported from nerve cell bodies along their axons has provided evidence on its velocity, subcellular distribution, or its solubility in acid 1-7,1°-1s,2°. The velocity of rapid transport is directly dependent on temperature, as has been elegantly demonstrated by Gross 11. Although many laboratories have shown that much of what is rapidly transported is acid-insoluble and hence proteinaceous, only a few have carried the characterization further by
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299 examining the electrophoretic mobility of the transported proteins1,7,13,20. A brief summary of amino acid-labeled, rapidly transported proteins in several sensory systems (Table l) reveals that most of the previous work has been done with leucine as the radioactive precursor. The studies cited have demonstrated a rather simple profile of peaks heavily labeled with leucinel,V,13. In fact, there are only 5 outstanding classes of heavily labeled material in several different systems of two different species (18,C00; 21,1300; 68,000; 75,0130 and 100,CC0 daltons). In contrast, Willard et al. 2° using methionine as the radioactive precursor to label rapidly transported proteins have recently reported a considerable number of distinct labeled proteins. Methionine has labeled 4 of the 5 protein classes labeled by leucine and, in addition, has revealed a number of other well-labeled species. It thus appears that a more complete profile (more heavily labeled peaks relative to background labeling) is obtained when methionine is used. Thus, the use of different amino acid labels has provided quite different expressions of the complexity of rapidly transported proteins. Presumably, the different profiles reflect the differential incorporation of the specific amino acids into those proteins which are rapidly transported. Comparison of the results obtained in the present analysis of methionine-labeled proteins rapidly transported in a pure motor system from the CNS of the fish with the profile developed in the rabbit optic nerve reveal a coincidence of many protein species heavily labeled with methionine (see Table I). Also included in Table I for further comparison are recent unpublished results using methionine in a sensory system in the frog. These latter results help to establish a pattern of rapidly transported proteins common to sensory and motor systems in vertebrates. The methioninelabeled, rapidly transported protein classes common to these systems include those with the following approximate molecular weights: 130,13C0, 1130,1300, 68,13130, 57,13130, 29,0130, and 18,13130.The same classes of proteins are also heavily labeled with leucine in these and other systems, suggesting that these species of proteins may be independent of specific neuronal function. The profile of rapidly transported, methionine- or leucine-labeled proteins is apparently more complex than that of slowly transported, leucine-labeled proteins (in the rat sciatic nerve). The latter consists of 5 heavily labeled peaks: 212 K, 160 K, 68 K, 57 K and 53 K (P. N. Hoffman and R. J. kasek, unpublished observations). The identity of these proteins is yet to be determined, as is the possible similarity between the slowly and rapidly transported 68 K and 57 K classes. It should be apparent that characterization by electrophoretic mobility on an SDS gel only identifies the molecular weight of the protein and does not provide knowledge of charge, amino acid sequence, or configuration. Double-label experiments carried out with methionine and fucose indicate that most of the rapidly transported proteins in the toadfish sonic motor system are fucosylated. The 8 doubly-labeled classes of protein include those with the following approximate molecular weights: 190,13130 daltons, 130,13130 daltons, 1130,13130daltons, 68,0130 daltons, 57,13130 daltons, 34,13C0 daltons, 29,13130 daltons, 18,13130 daltons and peaks < 18,C130daltons. The wide-spread fucosylation of rapidly transported proteins is consistent with the suggestion that most of the rapidly transported proteins are
300 associated with m e m b r a n e s . The presence o f a fairly distinct profile o f fucose-labeled m a t e r i a l contrasts with results o b t a i n e d in the p r i m a r y afferent system o f the frog, where a p o l y d i s p e r s e p a t t e r n was o b s e r v e d 7. The present results c o u p l e d with p r e v i o u s d a t a indicate t h a t there are 6 species o f protein, characterized b y e l e c t r o p h o r e t i c m o b i l i t y , which are r a p i d l y t r a n s p o r t e d in vertebrate sensory a n d m o t o r systems. T h e generizability o f this o b s e r v a t i o n requires further w o r k with other sensory a n d m o t o r systems in vertebrates. The 6 p r o tein classes are also labeled with fucose (in the toadfish sonic m o t o r system), suggesting t h a t they are g l y c o p r o t e i n s p o s s i b l y associated with m e m b r a n e s . The present results are thus consistent with p r e v i o u s studies a n d suggestions o f D r o z 5 a n d others 2-6,12,15,16 who have c o n c l u d e d t h a t m u c h o f the r a p i d l y t r a n s p o r t e d p r o t e i n a n d g l y c o p r o t e i n m a t e r i a l is utilized for the m a i n t e n a n c e o f synaptic vesicles a n d p r e s y n a p t i c p l a s m a m e m b r a n e s . The precise n a t u r e a n d function o f r a p i d l y t r a n s p o r t ed p r o t e i n s in vertebrate n e r v o u s systems requires sub-cellular f r a c t i o n a t i o n a n d electron m i c r o s c o p i c c o r r e l a t i o n to s u p p o r t the general hypothesis coupling r a p i d t r a n s p o r t to c o n t i n u o u s m a i n t e n a n c e o f s y n a p t i c (or a x o n a l terminal) physiology.
REFERENCES 1 ANDERSON,L. E., AND McCLURE, W. O., Differential transport of protein in axons. Comparison between the sciatic nerve and dorsal columns of cats, Proc. nat. Acad. Sci (Wash.), 70 (1973) 15211525. 2 BENNETT,G., DI GIAMBERARDINO,L., KOENIG,H. L., ANDDROZ, B., Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]fucose and [3H]glucosamine, Brain Research, 60 (1973) 129-146. 3 CU~NOD,M., ANDSCHONBACH,J., Synaptic proteins and axonal flow in the pigeon visual pathway, J. Neurochem., 18 (1971) 809-816. 4 DI GIAMBERARDINO,L., BENNETT,G., KOENIG,H. L., ANDDROZ, B., Axonal migration of protein and glycoprotein to nerve endings. III. Cell fraction analysis of chicken ciliary ganglion after intracerebral injection of precursors of proteins and glycoproteins, Brain Research, 69 (1973) 147159. 5 DROZ, B., Renewal of synaptic proteins, Brabt Research, 62 (1973) 383-394. 6 DROZ, B., KOENIG, H. L., AND DI GIAMBERARDINO,L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]lysine, Brain Research, 60 0973) 93-127. 7 EDSTROM,A., AND MATTSSON,H., Electrophoretic characterization of leucine-, glucosamine-, and fucose-labelled proteins rapidly transported in frog sciatic nerve, J. Neurochem., 21 (1973) 14991507. 8 GAINER,H., Multiple innervation of fish skeletal muscle. In J. A. KERKUT(Ed.), Experbnents in Physiology and Biochemistry, Vol. 2, Academic Press, London, 1969, pp. 191-208. 9 GAINER,H., Microdis¢ electrophoresis in sodium dodecyl sulfate: an application to the study of protein synthesis in individual, identified neurons, Anal. Biochem., 44 (1971) 589-605. 10 GRAFSTEIN,B., Axonal transport: communication between soma and synapse. In I. E. COSTAAND P. GREENGARD(Eds.), Advances in Biochemical Psychopharmacology, Vol. 1, Raven Press, New York, 1969, pp. 12-25. 11 GROSS, G. W., The effect of temperature on the rapid axoplasmic transport in C-fibers, Brain Research, 56 0973) 359-363. 12 HENDRICKSON,A. E., Electron microscopic distribution of axoplasmic flow, J. comp. Neurol., 144 (1973) 381-398.
301 13 KARLSSON, J. O., AND SJOSTRAND, J., Electrophoretic characterization of rapidly transported proteins in axons of retinal ganglion cells, FEBS Letters, 16 (1971) 329 332. 14 LASEK,R., Protein transport in neurons. In C. PFEIFFERAND J. R. SMYTHIES(Eds.), International Review of Neurobiology, Iiol. 13, Academic Press, New York, 1970, pp. 289-324. 15 MARKO, P., AND CU~NOD, M., Contribution of the nerve cell body to renewal of axonal and synaptic glycoproteins in the pigeon visual system, Brain Research, 62 (1973) 419-423. 16 MORGAN, I. G., ZANETTA,J.-P., BRECKENRIDGE,W. C., VINCENDON, G., AND GOMBOS, G., The chemical structure of synaptic membranes, Brain Research, 62 (1973) 405 411. 17 OCHS, S., Fast transport of materials in mammalian nerve fibers, Science, 176 (1973) 252-260. 18 SCHONBACH,J., SCHONBACH,CH., AND CU~NOD, M., Rapid phase of axoplasmic flow and synaptic proteins: an electron microscopical and autoradiographic study, J. comp. Neurol., 141 (1971) 485-498. 19 TAVOLGA,W. N., Sonic characteristics and mechanisms in marine fishes. In W. N. TAVOLGA(Ed.), Marine Bioacousties, Syrup., Pergamon, London, 1964, pp. 195-211. 20 WILLARD, M., COWAN, W. M., AND VAGELOS, P. R., The polypeptide composition of intraaxonally transported proteins: evidence for four transport velocities, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 2183-2187. 21 WINN, H. E., The biological significanc~ of fish sounds. In W. N. TAVOLGA(Ed.), Marhze Bioacoustics, Syrup., Pergamon, London, 1964, pp. 213-221.
29 l
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
RAP1D TRANSPORT OF PROTEINS IN THE SONIC MOTOR SYSTEM OF THE TOADFISH
J. L. BARKER, P. N. H O F F M A N , H. G A I N E R AND R. J. LASEK
Behavioral Biology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014, and Department of Anatomy, Case Western Reserve Medical School, Cleveland, Ohio 44106, and Marine Biology Laboratory, Woods Hole, Mass. 02543
(U.S.A.) (Accepted April 27th, 1975)
SUMMARY
A pure cholinergic, motor system of a marine fish has been utilized to study the kinetics and characteristics of proteins rapidly transported from the sonic motor nucleus to the musculature enveloping the swim bladder. Following microinjection of [3H]leucine, [3H]lysine, [35S]methionine, or [3H]fucose into the nucleus a wave of radioactivity was observed moving along the sonic motor nerves with an apparent rate of 96-120 ram/day. Analysis of the rapidly transported methionine-labeled protein using SDS gel electrophoresis revealed at least 9 major peaks of activity. Eight of these proteins were found to incorporate fucose, suggesting that most of the rapidly transported material consists of glycoproteins. These results are consistent with the previously suggested hypothesis relating the function of rapid transport to synaptic vesicles and the maintenance of pre-synaptic terminal membranes.
INTRODUCTION
Transport of material from the nerve cell body by the axon has been well demonstrated in a wide variety of nervous systems (for recent reviews, see Grafstein 10, Lasek 14 and Ochsl7). Analysis of the transported material has for the most part concentrated on characterizing it either in terms of rate of transport, subcellular distribution, and/or solubility in acid. The velocity of the rapidly transported material is directly dependent on temperature u and a proportion of it is considered to be protein since it is insoluble in acid. Further characterization of the amino acid-labeled, rapidly transported protein by electrophoretic mobility or sodium dodecylsulfate-polyacrylamide gels has recently been reported for several sensory and motor systemsl, 7,13,2o. Since the isotopes used differ, it is difficult to compare the patterns obtained in the
292 various systems. The rather complex profile of rapidly transported methionine-labeled protein in the rabbit visual system compared to the simple pattern obtained in other systems when labeled leucine was used suggested that methionine might provide a good precursor for establishing a more complete characterization of rapidly transported protein. We have used a pure motor system unique to certain species of fish to characterize the methionine-labeled, rapidly transported proteins. The cell bodies of the m o t o r system lie in the brain stem and project to, and innervate muscles enveloping the swim bladder. The system consists of pure cholinergic axons s which function to produce sound by causing rapid contractions of the swim bladder musclesS, 19,21. The functional purity of the system, its length and discrete termination would appear to offer an excellent system to investigate the nature and function of rapid axonal transport. The results obtained thusfar indicate (1) that proteins are rapidly transported at 96-120 m m / d a y and (2) that these proteins can be characterized into at least 9 classes, as defined by electrophoretic mobility. Comparison of this data with other electrophoretic profiles of rapidly transported proteins reveal several protein classes common to a variety of systems. METHODS
Preparation The experiments were carried out on 60 toadfish (Opsanus tau) obtained at the Woods Hole Marine Biological Laboratory in Woods Hole, Mass. The general features of the preparation are schematically diagrammed in Fig. 1. The fish were held in tanks continuously perfused with sea-water at 19-20 °C. Anesthesia was induced by
Fig. 1. Sonic motor system of toadfish. Right: picture of toadfish which normally dwells at the bottom of the ocean. Left: schematic diagram of sonic motor nucleus containing nerve cell bodies which project to the swim bladder to innervate muscle enveloping bladder. A glass micropipette used to slowly deliver concentrated isotope under pressure is seen entering nucleus.
293 placing the fish in a bucket containing 0.5 g/1 MS-222 (tricaine methanesulfonate). After several minutes body and gil movements ceased and the fish was placed on a small operating stand kept wet with a sea-water-soaked cloth. The gills were perfused with sea-water containing 0.1 g/1 MS-222. The muscles and bones protecting the dorsal aspect of the midbrain were carefully removed and the dorsal surface of the brain stem visualized with the aid of a dissecting microscope. Preliminary injections using methylene blue showed that the sonic motor nucleus containing the cell bodies of the sonic motor nerves was located approximately 1 mm below the dorsal surface of the brain stem and extended about 3 mm rostro-caudally in the mid-line. Surface landmarks for the nucleus included the mid-line and a shallow, indented area, triangular in extent (the obex), bounded rostrally and caudally by easily visible commissures. Three mid-line injections 1 mm deep were made about 1 mm apart beginning just posterior to the commissure providing the caudal bound to the obex. After the injections the muscles and skin overlying the brain stem were approximated with 3-0 gauge silk in either one or two layers of closure and the fish revived in a holding tank. In 60 ~ of injected fish one of the sonic motor nerves projecting to the swim bladder was exposed through a ventral incision and crushed and ligated at its entrance into the body of swimbladder muscle. The body wall was closed in one layer with 3-0 gauge silk.
Micr oinjection The following isotopic labels were used: (1) L-[4,5-~H]leucine (NEN Chemicals) with a specific activity of 40-60 Ci/mmole, (2) e-[4,5-3H]lysine (NEN), specific activity 31.4 Ci/mmole; (3) L-[35S]methionine (NEN), specific activity 195.5 Ci/mmole, when obtained; (4) L-[3H]fucose (NEN), specific activity 13.4 Ci/mmole. The stock solutions of isotope were dried under nitrogen and the residue redissolved in distilled water to give final concentrations of 10 mCi/ml. In some experiments mixtures of leucine and lysine or methionine and fucose were used. The concentrated isotopic solution was drawn up into a finely tapered glass micropipette (50-1130/~m tip). One ,ul injections of label were made over a 30-rain period at the 3 midline sites using a precision pump coupled to an oil-filled injection system.
Preparation of tissue for radioactivity determination At various times after microinjection into the brain stem the fish were sacrificed by placing them in sea-water containing 0.5 g/1 MS-222 for 15 min. Both sonic motor (SM) nerves were incised as close as possible to their exit from the vertebral column and the SM nerves and swim bladder with enveloping muscle were immediately removed for analysis. For analysis of the transport rate the nerves were cut into 3 mm sections and incubated for 8 h at 60 °C in 0.5 N NaOH. After cooling, Bray's solution was added to the vial and the radioactivity counted either in a Packard or Beckman liquid scintillation counter using the appropriate windows. In the Packard, counting efficiencies of 35S and 3H were 75 ~ and 26 ~ , respectively. An analysis of the distribution of radioactivity between TCA-insoluble and TCA-soluble fractions was not carried out.
294
Polyacrylamide gel electrophoresis For analysis of radioactively labeled proteins the nerves were cut into 5 or 10 mm sections and homogenized by hand using glass-on-glass homogenizers. The material to be homogenized was first ground in 8 M urea and again after adding sodium dodecylsulfate (SDS) to a final concentration of 1 ~ . After the latter treatment the homogenate was brought to 95 °C for 5 min, cooled and then subjected to a final homogenization after making the solution 1 ~ fi-mercaptoethanol. The resuiting homogenate was usually cloudy. An aliquot was taken for radioactivity determination (using Bray's solution as the photofluor). The remainder was analyzed by gel electrophoresis either immediately or after storage at --20 °C. Electrophoresis was carried out using discontinuous, 10 ~ polyacrylamide gels according to the method of Gainer 9. Marker proteins and bromophenol blue were included with each sample. The marker proteins were: myosin (212,(3130 daltons),/3galactosidase (130,(3(30 daltons), bovine serum albumin (68,(3C0 daltons), ovalbumin (43,(3(30 daltons), carbonic anhydrase (29,C(30 daltons), myoglobin (18,C(30 daltons), RNAase (12,13130 daltons) and bacitracin (1,zlC0 daltons). A standard curve was obtained by plotting the logarithm of the molecular weights of the marker proteins as a function of their mobilities relative to myoglobin. After completion of the electrophoresis the gels were stained overnight in 0.25 Coomassie blue, destained in 10 ~ methanol: 7.5 ~ acetic acid solutions and cut into 1.5 mm slices. The slices were digested with H202 and radioactivity was determined using Bray's solution as the photofluor. In double-label experiments the cross-over of aaS counts into the 3H channel (and vice-versa) was minimized by setting the appropriate windows. The cross-over of 35S accounted for 9.6 ~ of the activity present in the aH channel, while the crossover of the 8H into the 35S channel accounted for 0.25 ~ of the activity counted in the latter channel. The 85S contamination of the 3H channel was subtracted from the 3H counts while the negligible 3H contamination of asS was not corrected. RESULTS
Transport rate Radioactive material was present in the sonic motor nerves following injection of isotopic label in the SM nucleus. The time required for the initial appearance of radioactivity was typically 3-4 h after injection. An analysis of radioactivity present in the SM nerves at various intervals during the first 24 h was completed after microinjection of either [aH]leucine, [3H]fucose or [zsS]methionine. Similar results regarding the gross distribution of radioactive material as a function of time were observed with all 3 isotopes. Only those obtained with [aaS]methionine will be presented. Representative examples of the appearance of 35S activity in ligated and unligated SM nerves are illustrated in Fig. 2. An accumulation of radioactivity at the ligature was evident beginning with the 6-h interval. Radioactivity of approximately similar distribution was present in both ligated and unligated nerves of 4-h and 6-h fish. At 9 h the wave front of radioactivity had moved farther down the unligated nerve and
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Fig. 2. The distribution of [zsS]methionine labeled material in the sonic motor nerves is plotted as a function of approximate distance along the nerves at various times after the microinjection of the label into the nuclei of 5 toadfish. The nerves were cut into 3 mm segments and the data expressed as counts/min (cpm)/segment. One sonic motor nerve was crushed and ligated (~') while the other remained untouched. An accumulation of radioactive material at the ligature begins at about the 6th h following intranuclear injection. An apparent wave front of activity is present during the early time periods, disappears with longer intervals post-injection and is replaced by another apparent wave at still longer intervals. showed evidence o f accumulation at the ligature. A suggestion o f a front in the unligated nerve was largely absent from the 14-h fish while a second, later wave of activity had begun to enter the unligated nerve in the 22-h fish. These results indicate that radioactivity is transported along the SM nerves at about 4-5 m m / h (or 96-120 mm/day). Some uncertainty exists regarding the precise rate since the distance from the SM nucleus to the first segment analyzed was estimated at 10 m m (from several complete dissections o f the entire SM nerve). This rate is less than that which would be predicted for 20 °C from data reported for the garfish olfactory nerve 11 (at which temperature rapid transport progresses at about 180 mm/day). The more slowly moving material initially present in the 22-h fish is transported at less than 1 mm/h.
Rapidly transportedproteins The profile o f rapidly transported proteins labeled with [35S]methionine was complex. Similar profiles were obtained from 8 toadfish. At least 9 peaks could be distinguished in the material accumulating at the ligature of both 10-h and 18-h fish (Fig. 3B and C). The peaks had the following approximate molecular weights (esti-
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Fig. 3. Representative electrophoretic profiles of methionine-labeled, rapidly transported proteins. The labeled material was obtained at 10- and 18-h intervals following injections in the nuclei of two toadfish. Ten-ram segmentsjust proximal to the ligature or from the wave front in the unligated nerve (10-h fish) were homogenized in urea, SDS and BME and electrophoresed on 10 ~ polyacrylamide gels, cut into 1.5 mm slices and the radioactivity in each slice plotted above. A set of standard marker proteins was included in the electrophoresis (see text). A number of major peaks can be discerned. mated from comparing their relative mobilities with those of proteins of known molecular weights run on gels during the same electrophoresis run): 190 K, 130 K, 100 K, 68 K, 57 K, 43 K, 34 K, 29 K, 18 K, and several < 1 8 K (where K = 1000). Some variability in the size of the various peaks at ligatures (e.g., the 43 K peak) was observed, but the general pattern was reproducible (see also Fig. 4). A similar, distinct profile of rapidly transported, methionine-labeled proteins was also observed in the wave front of the other (unligated) nerve of the 10-h fish (Fig. 3A). Profiles of rapidly transported proteins labeled with both methionine and fucose were developed for 10-h and 18-h fish. Representative examples are illustrated in Fig. 4. A correspondence of protein labeling with fucose and methionine was found for most of the peaks. However, several fucose-labeled peaks relatively unlabeled with methionine were also found, and vice versa.
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Fig. 4. Representative electrophoretic profiles of doubly-labeled, rapidly transported proteins. The labeled material was obtained 10 and 18 h after injection of a mixture of psS]methionine and [3HIfucose into the sonic motor nuclei of two toadfish. Ten-mm segments immediately proximal to the ligature were utilized to develop the profiles. A number of proteins labeled with both methionine and fucose can be seen (see text for details).
Preliminary experiments using leucine and lysine as radioactive precursors showed a pattern which had many small, labeled peaks. The profile thus appeared to contain fewer distinct, heavily labeled peaks. Further work is required to better compare these proteins with those labeled with methionine or fucose. DISCUSSION
Most of the analysis of material rapidly transported from nerve cell bodies along their axons has provided evidence on its velocity, subcellular distribution, or its solubility in acid 1-7,1°-1s,2°. The velocity of rapid transport is directly dependent on temperature, as has been elegantly demonstrated by Gross 11. Although many laboratories have shown that much of what is rapidly transported is acid-insoluble and hence proteinaceous, only a few have carried the characterization further by
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299 examining the electrophoretic mobility of the transported proteins1,7,13,20. A brief summary of amino acid-labeled, rapidly transported proteins in several sensory systems (Table l) reveals that most of the previous work has been done with leucine as the radioactive precursor. The studies cited have demonstrated a rather simple profile of peaks heavily labeled with leucinel,V,13. In fact, there are only 5 outstanding classes of heavily labeled material in several different systems of two different species (18,C00; 21,1300; 68,000; 75,0130 and 100,CC0 daltons). In contrast, Willard et al. 2° using methionine as the radioactive precursor to label rapidly transported proteins have recently reported a considerable number of distinct labeled proteins. Methionine has labeled 4 of the 5 protein classes labeled by leucine and, in addition, has revealed a number of other well-labeled species. It thus appears that a more complete profile (more heavily labeled peaks relative to background labeling) is obtained when methionine is used. Thus, the use of different amino acid labels has provided quite different expressions of the complexity of rapidly transported proteins. Presumably, the different profiles reflect the differential incorporation of the specific amino acids into those proteins which are rapidly transported. Comparison of the results obtained in the present analysis of methionine-labeled proteins rapidly transported in a pure motor system from the CNS of the fish with the profile developed in the rabbit optic nerve reveal a coincidence of many protein species heavily labeled with methionine (see Table I). Also included in Table I for further comparison are recent unpublished results using methionine in a sensory system in the frog. These latter results help to establish a pattern of rapidly transported proteins common to sensory and motor systems in vertebrates. The methioninelabeled, rapidly transported protein classes common to these systems include those with the following approximate molecular weights: 130,13C0, 1130,1300, 68,13130, 57,13130, 29,0130, and 18,13130.The same classes of proteins are also heavily labeled with leucine in these and other systems, suggesting that these species of proteins may be independent of specific neuronal function. The profile of rapidly transported, methionine- or leucine-labeled proteins is apparently more complex than that of slowly transported, leucine-labeled proteins (in the rat sciatic nerve). The latter consists of 5 heavily labeled peaks: 212 K, 160 K, 68 K, 57 K and 53 K (P. N. Hoffman and R. J. kasek, unpublished observations). The identity of these proteins is yet to be determined, as is the possible similarity between the slowly and rapidly transported 68 K and 57 K classes. It should be apparent that characterization by electrophoretic mobility on an SDS gel only identifies the molecular weight of the protein and does not provide knowledge of charge, amino acid sequence, or configuration. Double-label experiments carried out with methionine and fucose indicate that most of the rapidly transported proteins in the toadfish sonic motor system are fucosylated. The 8 doubly-labeled classes of protein include those with the following approximate molecular weights: 190,13130 daltons, 130,13130 daltons, 1130,13130daltons, 68,0130 daltons, 57,13130 daltons, 34,13C0 daltons, 29,13130 daltons, 18,13130 daltons and peaks < 18,C130daltons. The wide-spread fucosylation of rapidly transported proteins is consistent with the suggestion that most of the rapidly transported proteins are
300 associated with m e m b r a n e s . The presence o f a fairly distinct profile o f fucose-labeled m a t e r i a l contrasts with results o b t a i n e d in the p r i m a r y afferent system o f the frog, where a p o l y d i s p e r s e p a t t e r n was o b s e r v e d 7. The present results c o u p l e d with p r e v i o u s d a t a indicate t h a t there are 6 species o f protein, characterized b y e l e c t r o p h o r e t i c m o b i l i t y , which are r a p i d l y t r a n s p o r t e d in vertebrate sensory a n d m o t o r systems. T h e generizability o f this o b s e r v a t i o n requires further w o r k with other sensory a n d m o t o r systems in vertebrates. The 6 p r o tein classes are also labeled with fucose (in the toadfish sonic m o t o r system), suggesting t h a t they are g l y c o p r o t e i n s p o s s i b l y associated with m e m b r a n e s . The present results are thus consistent with p r e v i o u s studies a n d suggestions o f D r o z 5 a n d others 2-6,12,15,16 who have c o n c l u d e d t h a t m u c h o f the r a p i d l y t r a n s p o r t e d p r o t e i n a n d g l y c o p r o t e i n m a t e r i a l is utilized for the m a i n t e n a n c e o f synaptic vesicles a n d p r e s y n a p t i c p l a s m a m e m b r a n e s . The precise n a t u r e a n d function o f r a p i d l y t r a n s p o r t ed p r o t e i n s in vertebrate n e r v o u s systems requires sub-cellular f r a c t i o n a t i o n a n d electron m i c r o s c o p i c c o r r e l a t i o n to s u p p o r t the general hypothesis coupling r a p i d t r a n s p o r t to c o n t i n u o u s m a i n t e n a n c e o f s y n a p t i c (or a x o n a l terminal) physiology.
REFERENCES 1 ANDERSON,L. E., AND McCLURE, W. O., Differential transport of protein in axons. Comparison between the sciatic nerve and dorsal columns of cats, Proc. nat. Acad. Sci (Wash.), 70 (1973) 15211525. 2 BENNETT,G., DI GIAMBERARDINO,L., KOENIG,H. L., ANDDROZ, B., Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]fucose and [3H]glucosamine, Brain Research, 60 (1973) 129-146. 3 CU~NOD,M., ANDSCHONBACH,J., Synaptic proteins and axonal flow in the pigeon visual pathway, J. Neurochem., 18 (1971) 809-816. 4 DI GIAMBERARDINO,L., BENNETT,G., KOENIG,H. L., ANDDROZ, B., Axonal migration of protein and glycoprotein to nerve endings. III. Cell fraction analysis of chicken ciliary ganglion after intracerebral injection of precursors of proteins and glycoproteins, Brain Research, 69 (1973) 147159. 5 DROZ, B., Renewal of synaptic proteins, Brabt Research, 62 (1973) 383-394. 6 DROZ, B., KOENIG, H. L., AND DI GIAMBERARDINO,L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]lysine, Brain Research, 60 0973) 93-127. 7 EDSTROM,A., AND MATTSSON,H., Electrophoretic characterization of leucine-, glucosamine-, and fucose-labelled proteins rapidly transported in frog sciatic nerve, J. Neurochem., 21 (1973) 14991507. 8 GAINER,H., Multiple innervation of fish skeletal muscle. In J. A. KERKUT(Ed.), Experbnents in Physiology and Biochemistry, Vol. 2, Academic Press, London, 1969, pp. 191-208. 9 GAINER,H., Microdis¢ electrophoresis in sodium dodecyl sulfate: an application to the study of protein synthesis in individual, identified neurons, Anal. Biochem., 44 (1971) 589-605. 10 GRAFSTEIN,B., Axonal transport: communication between soma and synapse. In I. E. COSTAAND P. GREENGARD(Eds.), Advances in Biochemical Psychopharmacology, Vol. 1, Raven Press, New York, 1969, pp. 12-25. 11 GROSS, G. W., The effect of temperature on the rapid axoplasmic transport in C-fibers, Brain Research, 56 0973) 359-363. 12 HENDRICKSON,A. E., Electron microscopic distribution of axoplasmic flow, J. comp. Neurol., 144 (1973) 381-398.
301 13 KARLSSON, J. O., AND SJOSTRAND, J., Electrophoretic characterization of rapidly transported proteins in axons of retinal ganglion cells, FEBS Letters, 16 (1971) 329 332. 14 LASEK,R., Protein transport in neurons. In C. PFEIFFERAND J. R. SMYTHIES(Eds.), International Review of Neurobiology, Iiol. 13, Academic Press, New York, 1970, pp. 289-324. 15 MARKO, P., AND CU~NOD, M., Contribution of the nerve cell body to renewal of axonal and synaptic glycoproteins in the pigeon visual system, Brain Research, 62 (1973) 419-423. 16 MORGAN, I. G., ZANETTA,J.-P., BRECKENRIDGE,W. C., VINCENDON, G., AND GOMBOS, G., The chemical structure of synaptic membranes, Brain Research, 62 (1973) 405 411. 17 OCHS, S., Fast transport of materials in mammalian nerve fibers, Science, 176 (1973) 252-260. 18 SCHONBACH,J., SCHONBACH,CH., AND CU~NOD, M., Rapid phase of axoplasmic flow and synaptic proteins: an electron microscopical and autoradiographic study, J. comp. Neurol., 141 (1971) 485-498. 19 TAVOLGA,W. N., Sonic characteristics and mechanisms in marine fishes. In W. N. TAVOLGA(Ed.), Marine Bioacousties, Syrup., Pergamon, London, 1964, pp. 195-211. 20 WILLARD, M., COWAN, W. M., AND VAGELOS, P. R., The polypeptide composition of intraaxonally transported proteins: evidence for four transport velocities, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 2183-2187. 21 WINN, H. E., The biological significanc~ of fish sounds. In W. N. TAVOLGA(Ed.), Marhze Bioacoustics, Syrup., Pergamon, London, 1964, pp. 213-221.
