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Rapid important paper
Neurochem. Int. Vol. 8, No. 3, pp. 435-442, 1986 Printed in Great Britain
0197-0186/86 $3.00 + 0.00 Pergamon Journals Ltd
RAPID IMPORTANT PAPER MESSENGER RNA IN SQUID AXOPLASM Antonio Giuditta, Tim Hunt I and Luigia Santella 2 Department of General a n d Environmental Physiology, University of Naples, Via Mezzocannone 8, 80134 Naples, Italy. 1Department of Biochemistry, University of Cambridge, Cambridge, U.K. ; 2Zoological Station, Naples, Italy. (Received 5 January 1986; accepted 24 February 1986) ABSTRACT Using a translation assay we have shown that the axoplasm of the squid giant axon contains significant amounts of mRNA coding for a heterogeneous group of proteins. The axoplasmic translation products overlap with but differ from the sets of proteins specified by glial and neuronal perikaryai mRNA. Messenger RNA is associated with the "microsomal" fraction of the axoplasm. The possible involvement of axoplasmic mRNA in protein synthesis remains to be ascertained. It is known that axoplasmic proteins are synthesized by the isolated giant axon, presumably by the surrounding glia cells.
The axonal territory and its related synaptic periphery may attain volumes considerably larger than the perinuclear cytoplasm. Yet, the occurrence of protein synthesis in axons and synaptic terminals has not been conclusively demonstrated (Barondes, 1974). A notable exception in this regard is the squid giant axon. Work on this system has convincingly demonstrated that the isolated axon can synthesize its own proteins (Giuditta et al., 1968; Lasek et al., 1974). However, a sizable body of evidence suggests that this synthesis originates in the periaxonal glia cells, and that proteins are transferred to axoplasm by a process which is still to be defined (Lasek et al., 1977). It is known, on the other hand, that squid axoplasm contains all soluble factors necessary for protein synthesis (Giuditta et al., 1977), minor amounts of rRNA (Giuditta et al., 1980) and tRNA (Lasek et al., 1973; Ingoglia et al., 1983). In this paper we report that squid axoplasm also contains an hitherto unexpected family of mRNAs (Giuditta et al., 1983). EXPERIMENTAL P R O C E D U R E S Squid (Loligo pealii) were obtained at the Marine Biological Laboratory, Woods Hole, Mass., and kept in running sea water. The most medial stellate nerves were dissected from cleaned mantles, tied at both ends and kept in ice-cold sea water. The proximal ends of the giant fibres were carefully cleaned of the surroundings small axons for a length of about 5 mm, and the axoplasm was then extruded onto parafilm sheet over ice with a tiny roller. Samples of axoplasm were quick-frozen at -80* and stored at this temperature. In one experiment, the giant axons were completely cleaned of their surroundings thin fibres before extrusion. Giant fibre lobes were dissected from the stellate ganglia and immediately frozen at -80*. Subcellular fractions from the axoplasm were obtained by homogenizing 100 mg axoplasm (about 15 axons) in 1 ml of 1 M sucrose and centrifuging at 13,000 g for 20 min to yield a mitochondrial pellet. The 435
436
'~N'[()NIO ( j [ [ I ) I l I A ~'1 ~1].
s u p e r n a t a n t was c e n t r i f u g e d at 170,000 g for 3 hr, g i v i n g a pellet o5 "microsomes". S u b c e l l u l a r f r a c t i o n s from the squid optic lobe were prep a r e d a c c o r d i n g to P o l l a r d and Pappas (1979). R N A was e x t r a c t e d from tissue samples by d i l u t i n g w i t h 3 volumes of a b u f f e r c o n t a i n i n g 200 mM NaCl, 20 mM Tris-Cl, 2 m M EDTA, 1% w / v SDS, pH 7.5, and a d d i n g an equal volume of w a t e r - s a t u r a t e d phenol c o n t a i n i n g 0.1% w / v 8 - h y d r o x y q u i n o l i n e . After v i g o r o u s m i x i n g , t h e tubes w e r e incubated at 60 ° for 30 sec. The aqueous phase i n c l u d i n g the i n t e r p h a s e was then e x t r a c t e d w i t h chloroform, and the r e s u l t i n g aqueous phase extracted a second time w i t h phenol. The RNA was p r e c i p i t a t e d by a d d i t i o n of 2.5 v o l u m e s of ethanol, and h a r v e s t e d by c e n t r i f u g a t i o n after s t a n d i n g for several hours at -20 ° It was d i s s o l v e d in 20 ~i of water at conc e n t r a t i o n s b e t w e e n 1 and i0 mg/ml, as m e a s u r e d by a b s o r b a n c e at 260 rim.
The a c t i v i t y of m R N A was a s s a y e d using the m e t h o d of P e l h a m and J a c k s o n (1976), w i t h [ 3 5 S ] m e t h i o n i n e (Amersham International; 1400 Ci/mM) at a final c o n c e n t r a t i o n of 500 ~Ci/ml. After 1 hr at 30 ° the samples w e r e d i l u t e d w i t h an equal volume of a s o l u t i o n c o n t a i n i n g i00 ~g/ml RMase A, i0 m M E D T A pH 7.5, and left at 20 ° for I0 m i n (Jackson and Hunt, 1983). T C A - i n s o l u b l e r a d i o a c t i v i t y was m e a s u r e d in samples of 2 ~i d i l u t e d w i t h 1 ml of water and b l e a c h e d by a d d i t i o n of o.5 ml of 1 N NaOH, 0.5 M H202, 1 m g / m l methionine. To p r e p a r e samples for analysis on a c r y l a m i d e gels, 6 v o l u m e s of SDS gel sample buffer were added to the i n c u b a t i o n samples, h e a t e d at 90 ° for 1 min before loading the e q u i v a l e n t of 2 ~i of the o r i g i n a l t r a n s l a t i o n m i x t u r e in a 3.5 x 0.8 mm gel slot. The gels w e r e about 80 mm long, and were run at 150 v for about 1.5 hr. T h e y c o n t a i n e d 15% a c r y l a m i d e (Anderson et al., 1973) and w e r e fixed, s t a i n e d and f l u o r o g r a p h e d by the p r o c e d u r e of L a s k e y and Mills (1975). The gel in Fig. 2 was f l u o r o g r a p h e d u s i n g A m p l i f y from Amersham International. Controls o m i t t i n g m R N A and w i t h a s a t u r a t i n g dose (i00 ~g/ml) of t o b a c c o m o s a i c virus RNA were run in p a r a l l e l in e v e r y experiment. B a c k g r o u n d s w e r e b e t w e e n 500 and i000 cpm/ul, and the s t i m u l a t i o n by added T M V b e t w e e n i00 and 200-fold. L a b e l i n g of a x o p l a s m i c p r o t e i n s was o b t a i n e d by i n c u b a t i n g up to 5 giant axons (stripped of their s u r r o u n d i n g small axons) or up to 2 intact (i.e. not stripped) s t e l l a t e nerves (which c o n t a i n 1 giant axon) in 1 ml of M i l l i p o r e - f i l t e r e d sea w a t e r c o n t a i n i n g i00 ~Ci/ml [35S]met h i o n i n e at 18 ° for 4 hr. Giant fibre lobes were i n c u b a t e d under similar conditions. At the end of the i n c u b a t i o n the tissues were e x t e n s i v e l y w a s h e d in cold sea water, and the a x o p l a s m was p r e p a r e d as described above; it and other labeled f r a c t i o n s w e r e stored at -80 ° until r e a d y for a n a l y s i s by a c r y l a m i d e gel, at w h i c h time they were m i x e d w i t h a s u i t a b l e volume of SDS gel sample b u f f e r and h e a t e d b r i e f l y to help e x t r a c t the proteins. Several d i l u t i o n s of the v a r i o u s samples w e r e run in order to get the c o r r e c t loadings.
RESULTS A x o p l a s m i c mRNA. A x o p l a s m i c RNA gave a v e r y s i g n i f i c a n t s t i m u l a t i o n of i n c o r p o r a t i o n of [ 35S]methionine in the t r a n s l a t i o n assays, a l t h o u g h the d e g r e e of s t i m u l a t i o n d i f f e r e d in d i f f e r e n t e x p e r i m e n t s (Table i). A c o m p a r a b l e v a r i a b i l i t y was seen in the a c t i v i t y of the RNA o b t a i n e d from the other tissue samples. Since m o s t of the R N A in a x o p l a s m is 4S, w h e r e a s the R N A from the other tissues is p r e d o m i n a n t l y r i b o s o m a l (La-
mRNA in squid axoplasm
TABLE
437
1
Translation assay of axoplasmic and related RNAs Protein Radioactivity
RNA source
(cpm/mg wet weight x 10 -3 ) (i) Axoplasm (100 mg) Axonal Sheath a (4 mg) Stellate Nerve b (250 mg) Giant Fibre Lobe (50 mg)
Experiment (2) (3)
15.6
4.6
25.1 386.8
5.1 27.2
5.0
(4) 3.2 25.0
In parenthesis, approximate wet weight of samples used for RNA extraction, a, remaining after extrusion of axoplasm from isolated giant axons; b, remaining after extrusion of the giant axon axoplasm from stellate nerves. sek et al., 1973; Giuditta et al., 1980), we normalized the data on the basis of wet weight of starting material, assuming a reasonably similar efficiency of RNA extraction. On this basis, mRNA is considerably less abundant in axoplasm than in the giant fibre lobe (cell bodies of the giant axon), but the residual nerve (largely glia cells) contains a comparable amount. The higher mRNA content of the giant fibre lobe is to be expected in view of its high total RNA content (Giuditta et al.,
1980). The translation patterns resolved by ID gel electrophoresis were recognizably distinct for the three RNA preparations (Fig 1, A). Both lobe and axoplasm mRNA gave patterns dominated by 3 bands of 42, 56 and 60 kD, presumably representing actin and the a and ~ subunits of tubulin. Two proteins in the axoplasmic set appear to be lacking in the pattern of the giant fibre lobe and of the nerve (at about 117 and 18 kD). Conversely, 3 proteins in the lobe pattern are absent in the axoplasm and nerve patterns. Two of these proteins are of low MW (in the 17-20 kD range); the third protein migrates just ahead of the tubulin subunit. By contrast, the nerve RNA gave a strikingly simple pattern dominated by a set of high MW polypeptides, by a 65 kD protein and by actin. These high MW polypeptides presumably represent neurofilament proteins, which is intriguing in view of their absence from the set of proteins directed by mRNA from the nerve cell bodies. The very different translation patterns obtained with axoplasmic and nerve RNAs argue strongly against the possibility of contamination of the axoplasmic sample by nerve RNA. The possibility of contamination was further explored by carefully stripping all the surrounding thin axons from the giant axon before extrusion, and then preparing RNA from both axoplasm and the residual sheath. The sheath RNA was not very active, although on a wet weight basis it was considerably more active than the axoplasmic mRNA (experiment 4 of Table 1). Comparison of the translation patterns was made difficult by the low activity of the sheath sample; it required a very long exposure to detect only the
438
A n f o l i o (_}ItiDITTA e[ al.
presumed actin band (Fig.l, B), whereas the axoplasmic mRNA gave a pattern similar to that shown in Fig.l, A. This does not lend support to the idea that the mRNA activity found in axoplasm arises from contamination from adjacent nucleated cells. Rather it would seem that axoplasm contains a distinct subset of mRNAs.
A b
116
-
68-
n
a
B
I
b
n
s
a
116
68
56
-
56
42
-
42 40
35
-
35
14
-
14
Figure i. A. Electrophoretic patterns of the translation products specified by axoplasmic and related RNAs. b, blank without added RNA; n, stellate nerve remaining after extrusion of axoplasm; a, axoplasm; i, giant fibre lobe. The samples were appropriately diluted with unlabeled lysate mixture to give patterns of approximately comparable intensity. The MW of the marker proteins are indicated in kD. Exposure time, 68 hr at -80 ° on Fuji RX X-ray film. B. Translation patterns obtained with axoplasm extruded from isolated giant axons (a) and from the corresponding axonal sheath (s); b, blank without added RNA. Exposure time, 6 wk on Fuji RX.
mRNA in squid axoplasm
439
Subcellular studies. The subcellular distribution of axoplasmic RNA was examined by preparing RNA from axoplasmic mitochondrial and microsoma1 fractions, starting from approximately 100 mg tissue. No mRNA activity was detectable in either the mitochondrial fraction or the high speed sunp~srnatant. All the activity sedimented with the "microsomes" (4.2 x 10 cpm). Similarly, the synaptosomal mitochondria prepan
n'
s
I'
I
o'
O
116
68
56
42 40 35 31
21
14
Figure 2. Comparison of the translation patterns obtained with axoplasmic and related RNAs and the patterns of proteins synthesized by the corresponding regions of the stellate ganglion and nerve. Translation pattern of the remaining stellate nerve (n'), of the giant fibre lobe (1') and of the axoplasm (a') are shown side by side with the patterns of proteins synthesized by the remaining stellate nerve (n) and by the giant fibre lobe (i) and with the patterns of axoplasmic proteins (a) and of sheath proteins (s) synthesized by the giant axon. Exposure time, 48 hr on Fuji RX.
440
ANTONIO G l [ DITrA el ~1l.
red from 4 g of optic lobes (courtesy of Dr. R. Cohen) were completely inactive as a source of mRNA. These mitochondria are available in larger yield than axoplasmic mitochondria. The RNA of the other subcellular fractions of the optic lobe were quite active (up to 127 x l0 s total cpm in the microsomal fraction) and gave similar translation patterns (data not shown). Comparison with newly-synthesized protein s . We made some preliminary studies to see how the patterns of proteins specified by mRNA corresponded with the patterns of protein synthesis in the various tissues associated with the giant axon. Figure 2 shows a side-by-side comparison of the labeling pattern obtained with the extruded stellate nerve (n), giant axon sheath (s), axoplasm (a) or giant fibre lobe (i) with the corresponding translation patterns obtained in the reticulocyte lysate (n', i' and a'). It is striking that while the giant fibre lobe tracks 1 and i' show a high degree of correspondence, the a and a' lanes show poor agreement, as would be expected if most of the axoplasmic proteins were synthesized by surrounding glia cells. Indeed, the pattern of newly-synthesized axoplasmic proteins most closely resembles that shown by whole nerve and the isolated sheath, although they are not identical. DISCUSSION The possibility that the mRNA activity of the axoplasm arose by contamination from extraneous tissue during the extrusion step may be excluded. The stellate nerves were carefully dissected, and the proximal ends of the giant axons were cleaned of surrounding small axons before gently squeezing the axoplasm. More compellingly, the translation patterns of axoplasm and of the surrounding axonal sheath or the remaining stellate nerve are quite different. In addition, preliminary in situ hybridization analyses using [3H]poly(U) have demonstrated the presence of significant amounts of poly(A)RNA in the axoplasm (Grassi Zucconi G. et al., unpublished data). The presence of poly(A)RNA in the extruded axoplasm has been confirmed by a hybridization method using [3H]poly(U) (Perrone Capano et al., unpublished results). The mRNA in axoplasm is not derived from mitochondria, but is associated with the microsomal fraction. This is not surprising in view of the inability of the cytoplasmic protein synthesis machinery to translate mitochondrial RNA (Barrel et al., 1979). We were unable to detect any mRNA activity in the mitochondrial fraction of the axoplasm, or in the analogous mitochondria derived from optic lobe synaptosomes. There is considerably less mRNA in axoplasm, judged on a wet weight basis, than in the corresponding cell bodies of the giant fibre lobe. One should bear in mind the large size of the axonal territory in making comparisons, however. Taken as a whole, the axoplasmic mRNA becomes a significant fraction of the total mRNA present in these unusual cells. Similar considerations would apply to other neurons, if mRNA were shown to occur in axons and nerve endings of other invertebrate and vertebrate nerve cells, as future analyses should ascertain. The apparent segregation of neuronal mRNA into two sets, one to be translated in the cytoplasm surrounding the nucleus, the other to be found in cytoplasm that is almost completely or possibly entirely lacking ribosomes, raises several intriguing questions. First, what is the origin
mRNA in squid axoplasm
441
of the axoplasmic mRNA? It seems likely that it mostly originates in the nerve cell body, and the greater similarity between the translation patterns of giant fibre lobe and axoplasmic mRNA tends to support this idea. We cannot however exclude the possibility that some may come from the surrounding glia cells. Indeed, the synthesis of axoplasmic RNA has been detected in isolated giant axons (Cutillo et al., 1983). Wherever it comes from, there must be some kind of specificity in either its delivery to the axon, or in the stability of particular messages as they pass down the axon. Probably the most intriguing question concerns the role of axoplasmic mRNA. We cannot tell whether it is actively translated in axoplasm, and hence associated with polyribosomes, as it has been proposed (Giuditta, 1980), or whether it is stored in the axon in an untranslated form, perhaps en route to remote sites of protein synthesis either at nerve terminals or even beyond. The limited degree of similarity between the in vitro translation pattern of axoplasmic mRNA and the pattern of axoplasmic proteins synthesized by the giant axon suggests that little if any translation of the mRNA goes within the axon, in accord with the glia transfer hypothesis of Lasek et al. (1977). However, the differences might be attributed to the inability of the reticulocyte cell-free lysate to effect post-translational modifications of the translated proteins. Finally, it is of interest that the complexity of axoplasmic mRNA is much lower than that of the giant fibre lobe mRNA (Perrone Capano et al., unpublished results). It has been known for some time that maumnalian brain RNA contains a much wider set of sequences than other tissues of the body (Chaudary and Hahn, 1983; Kaplan, 1983). Similar results have been obtained recently in the squid (Perrone Capano et al, in press). The fact that a single nerve cell type like the squid giant , axon has a relatively simple set of sequences suggests that the reason for the high complexity found in the brain is because it contains so many different types of cells. ACKOWLEDGEMENTS We thank the permanent staff of the Marine Biological Laboratory, Woods Hole, Mass., for providing facilities and particularly supplies of animals. We also thank J. Rosenbaum and J. Ruderman for their generous hospitality during these studies. A.G. ackowledges finantial support from NATO grant 18781, from the Ministero della Pubblica Istruzione and the Progetto Finalizzato di Medicina Preventiva e Riabilitativa del CNR. T.H. was supported in part by NIH training grant GM-31136-04 and 05.
Anderson virus Barondes lism. Barrell de in Chaudari brain.
REFERENCES C.W., Baum P.R. and Gesteland R.F. (1973) Processing of adeno2-induced proteins. J.Virol. 12, 241-252. S. (1974) Synaptic macromolecules: identification and metaboAnn.Rev.Biochem. 43, 147-168. B.G., Bankier A.T. and Drouin J. (1979) A different genetic cohuman mitochondria. Nature 282, 189-194. N. and Hahn W.E. (1983) Genetic expression in the developing Science 220, 924-928.
442
ANTONIO G~t.urrT'a et a/.
Cutillo V., Montagnese P., Gremo F., Casola L. and Giuditta A. (1983> Origin of axoplasmic RNA in the squid giant fibre. Neurochem. Res. 8, 1621-1634. Giuditta A., Dettbarn W.-D. and Brzin M. (1968) Protein synthesis in the isolated giant axon of the squid. Proc.Natl.Acad. Sci. USA 59, 1284-1287. Giuditta A., Metafora S., Felsani A. and Del Rio A. (1977) Factors for protein synthesis in the axoplasm of squid giant axons. J.Neurochem. 28, 1393-1395. Giuditta A. (1980) Origin of axoplasmic proteins in the squid giant axon. Riv. Biol. 7_33, 35-49. Giuditta A., Cupello A. and Lazzarini G. (1980) Ribosomal RNA in the axoplasm of the squid giant axon. J.Neurochem. 3_~4, 1757-1760. Giuditta A., Hunt T. and Santella L. (1983) Messenger RNA in squid axoplasm. Biol.Bull. 165, 526. Jackson R.J. and Hun£ T. (1983) Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukariotic messenger RNA. Meth.Enzymol. 9_66, 50-74. Kaplan B.B. (1983) RNA-DNA hybridization: analysis of gene expression. In Handbook of Neurochemistry (Lajtha A. ed.) 2nd edition, Vol. 6, pp. 1-26, Plenum Press, New York. Ingoglia N., Giuditta A., Zanakis M.S., Babigian A., Tasaki I., Chakraborty G. and Sturman J. (1983) Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA mediated, post-translational protein modification in squid giant axon. J.Neurosc. 3, 2463-2473. Lasek R.J., Dabrowski J.C. and Nordlander R. (1973) Analysis of axoplasmic RNA from invertebrate giant axons. Nature 244, 162-165. Lasek R.J., Gainer H. and Przybylski (1974) Transfer of newly-synthesized proteins from Schwann cells to the squid giant axon. Proc.Natl. Acad. Sci. USA 7_~I, 1188-1192. Lasek R.J., Gainer H. and Barker J.L. (1977) Cell to cell transfer of glial proteins to the squid giant axon. The glia-neuron protein transfer hypothesis. J.Cell Biol. 74, 501-523. Laskey R.A. and Mills A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Europ. J.Biochem. 56, 335-341. Pelham H.R.B. and Jackson R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Europ. J.Biochem. 6_/7, 247256. Perrone Capano C., Giuditta A., Gioio A. and Kaplan B.B. (1986) Complexity of nuclear and polysomal RNA from squid optic lobe and gill. J.Neurochem. "In press". Pollard H.B. and Pappas G.B. (1979) Veratridine-activated release of adenosine-5'-tmiphosphate from synaptosomes: evidence for calcium dependence and blockade by tetrodotoxin. Biochem.Biophys.Res. Comm. 88, 1315-1321.
0197-0186/86 $3.00 + 0.00 Pergamon Journals Ltd
RAPID IMPORTANT PAPER MESSENGER RNA IN SQUID AXOPLASM Antonio Giuditta, Tim Hunt I and Luigia Santella 2 Department of General a n d Environmental Physiology, University of Naples, Via Mezzocannone 8, 80134 Naples, Italy. 1Department of Biochemistry, University of Cambridge, Cambridge, U.K. ; 2Zoological Station, Naples, Italy. (Received 5 January 1986; accepted 24 February 1986) ABSTRACT Using a translation assay we have shown that the axoplasm of the squid giant axon contains significant amounts of mRNA coding for a heterogeneous group of proteins. The axoplasmic translation products overlap with but differ from the sets of proteins specified by glial and neuronal perikaryai mRNA. Messenger RNA is associated with the "microsomal" fraction of the axoplasm. The possible involvement of axoplasmic mRNA in protein synthesis remains to be ascertained. It is known that axoplasmic proteins are synthesized by the isolated giant axon, presumably by the surrounding glia cells.
The axonal territory and its related synaptic periphery may attain volumes considerably larger than the perinuclear cytoplasm. Yet, the occurrence of protein synthesis in axons and synaptic terminals has not been conclusively demonstrated (Barondes, 1974). A notable exception in this regard is the squid giant axon. Work on this system has convincingly demonstrated that the isolated axon can synthesize its own proteins (Giuditta et al., 1968; Lasek et al., 1974). However, a sizable body of evidence suggests that this synthesis originates in the periaxonal glia cells, and that proteins are transferred to axoplasm by a process which is still to be defined (Lasek et al., 1977). It is known, on the other hand, that squid axoplasm contains all soluble factors necessary for protein synthesis (Giuditta et al., 1977), minor amounts of rRNA (Giuditta et al., 1980) and tRNA (Lasek et al., 1973; Ingoglia et al., 1983). In this paper we report that squid axoplasm also contains an hitherto unexpected family of mRNAs (Giuditta et al., 1983). EXPERIMENTAL P R O C E D U R E S Squid (Loligo pealii) were obtained at the Marine Biological Laboratory, Woods Hole, Mass., and kept in running sea water. The most medial stellate nerves were dissected from cleaned mantles, tied at both ends and kept in ice-cold sea water. The proximal ends of the giant fibres were carefully cleaned of the surroundings small axons for a length of about 5 mm, and the axoplasm was then extruded onto parafilm sheet over ice with a tiny roller. Samples of axoplasm were quick-frozen at -80* and stored at this temperature. In one experiment, the giant axons were completely cleaned of their surroundings thin fibres before extrusion. Giant fibre lobes were dissected from the stellate ganglia and immediately frozen at -80*. Subcellular fractions from the axoplasm were obtained by homogenizing 100 mg axoplasm (about 15 axons) in 1 ml of 1 M sucrose and centrifuging at 13,000 g for 20 min to yield a mitochondrial pellet. The 435
436
'~N'[()NIO ( j [ [ I ) I l I A ~'1 ~1].
s u p e r n a t a n t was c e n t r i f u g e d at 170,000 g for 3 hr, g i v i n g a pellet o5 "microsomes". S u b c e l l u l a r f r a c t i o n s from the squid optic lobe were prep a r e d a c c o r d i n g to P o l l a r d and Pappas (1979). R N A was e x t r a c t e d from tissue samples by d i l u t i n g w i t h 3 volumes of a b u f f e r c o n t a i n i n g 200 mM NaCl, 20 mM Tris-Cl, 2 m M EDTA, 1% w / v SDS, pH 7.5, and a d d i n g an equal volume of w a t e r - s a t u r a t e d phenol c o n t a i n i n g 0.1% w / v 8 - h y d r o x y q u i n o l i n e . After v i g o r o u s m i x i n g , t h e tubes w e r e incubated at 60 ° for 30 sec. The aqueous phase i n c l u d i n g the i n t e r p h a s e was then e x t r a c t e d w i t h chloroform, and the r e s u l t i n g aqueous phase extracted a second time w i t h phenol. The RNA was p r e c i p i t a t e d by a d d i t i o n of 2.5 v o l u m e s of ethanol, and h a r v e s t e d by c e n t r i f u g a t i o n after s t a n d i n g for several hours at -20 ° It was d i s s o l v e d in 20 ~i of water at conc e n t r a t i o n s b e t w e e n 1 and i0 mg/ml, as m e a s u r e d by a b s o r b a n c e at 260 rim.
The a c t i v i t y of m R N A was a s s a y e d using the m e t h o d of P e l h a m and J a c k s o n (1976), w i t h [ 3 5 S ] m e t h i o n i n e (Amersham International; 1400 Ci/mM) at a final c o n c e n t r a t i o n of 500 ~Ci/ml. After 1 hr at 30 ° the samples w e r e d i l u t e d w i t h an equal volume of a s o l u t i o n c o n t a i n i n g i00 ~g/ml RMase A, i0 m M E D T A pH 7.5, and left at 20 ° for I0 m i n (Jackson and Hunt, 1983). T C A - i n s o l u b l e r a d i o a c t i v i t y was m e a s u r e d in samples of 2 ~i d i l u t e d w i t h 1 ml of water and b l e a c h e d by a d d i t i o n of o.5 ml of 1 N NaOH, 0.5 M H202, 1 m g / m l methionine. To p r e p a r e samples for analysis on a c r y l a m i d e gels, 6 v o l u m e s of SDS gel sample buffer were added to the i n c u b a t i o n samples, h e a t e d at 90 ° for 1 min before loading the e q u i v a l e n t of 2 ~i of the o r i g i n a l t r a n s l a t i o n m i x t u r e in a 3.5 x 0.8 mm gel slot. The gels w e r e about 80 mm long, and were run at 150 v for about 1.5 hr. T h e y c o n t a i n e d 15% a c r y l a m i d e (Anderson et al., 1973) and w e r e fixed, s t a i n e d and f l u o r o g r a p h e d by the p r o c e d u r e of L a s k e y and Mills (1975). The gel in Fig. 2 was f l u o r o g r a p h e d u s i n g A m p l i f y from Amersham International. Controls o m i t t i n g m R N A and w i t h a s a t u r a t i n g dose (i00 ~g/ml) of t o b a c c o m o s a i c virus RNA were run in p a r a l l e l in e v e r y experiment. B a c k g r o u n d s w e r e b e t w e e n 500 and i000 cpm/ul, and the s t i m u l a t i o n by added T M V b e t w e e n i00 and 200-fold. L a b e l i n g of a x o p l a s m i c p r o t e i n s was o b t a i n e d by i n c u b a t i n g up to 5 giant axons (stripped of their s u r r o u n d i n g small axons) or up to 2 intact (i.e. not stripped) s t e l l a t e nerves (which c o n t a i n 1 giant axon) in 1 ml of M i l l i p o r e - f i l t e r e d sea w a t e r c o n t a i n i n g i00 ~Ci/ml [35S]met h i o n i n e at 18 ° for 4 hr. Giant fibre lobes were i n c u b a t e d under similar conditions. At the end of the i n c u b a t i o n the tissues were e x t e n s i v e l y w a s h e d in cold sea water, and the a x o p l a s m was p r e p a r e d as described above; it and other labeled f r a c t i o n s w e r e stored at -80 ° until r e a d y for a n a l y s i s by a c r y l a m i d e gel, at w h i c h time they were m i x e d w i t h a s u i t a b l e volume of SDS gel sample b u f f e r and h e a t e d b r i e f l y to help e x t r a c t the proteins. Several d i l u t i o n s of the v a r i o u s samples w e r e run in order to get the c o r r e c t loadings.
RESULTS A x o p l a s m i c mRNA. A x o p l a s m i c RNA gave a v e r y s i g n i f i c a n t s t i m u l a t i o n of i n c o r p o r a t i o n of [ 35S]methionine in the t r a n s l a t i o n assays, a l t h o u g h the d e g r e e of s t i m u l a t i o n d i f f e r e d in d i f f e r e n t e x p e r i m e n t s (Table i). A c o m p a r a b l e v a r i a b i l i t y was seen in the a c t i v i t y of the RNA o b t a i n e d from the other tissue samples. Since m o s t of the R N A in a x o p l a s m is 4S, w h e r e a s the R N A from the other tissues is p r e d o m i n a n t l y r i b o s o m a l (La-
mRNA in squid axoplasm
TABLE
437
1
Translation assay of axoplasmic and related RNAs Protein Radioactivity
RNA source
(cpm/mg wet weight x 10 -3 ) (i) Axoplasm (100 mg) Axonal Sheath a (4 mg) Stellate Nerve b (250 mg) Giant Fibre Lobe (50 mg)
Experiment (2) (3)
15.6
4.6
25.1 386.8
5.1 27.2
5.0
(4) 3.2 25.0
In parenthesis, approximate wet weight of samples used for RNA extraction, a, remaining after extrusion of axoplasm from isolated giant axons; b, remaining after extrusion of the giant axon axoplasm from stellate nerves. sek et al., 1973; Giuditta et al., 1980), we normalized the data on the basis of wet weight of starting material, assuming a reasonably similar efficiency of RNA extraction. On this basis, mRNA is considerably less abundant in axoplasm than in the giant fibre lobe (cell bodies of the giant axon), but the residual nerve (largely glia cells) contains a comparable amount. The higher mRNA content of the giant fibre lobe is to be expected in view of its high total RNA content (Giuditta et al.,
1980). The translation patterns resolved by ID gel electrophoresis were recognizably distinct for the three RNA preparations (Fig 1, A). Both lobe and axoplasm mRNA gave patterns dominated by 3 bands of 42, 56 and 60 kD, presumably representing actin and the a and ~ subunits of tubulin. Two proteins in the axoplasmic set appear to be lacking in the pattern of the giant fibre lobe and of the nerve (at about 117 and 18 kD). Conversely, 3 proteins in the lobe pattern are absent in the axoplasm and nerve patterns. Two of these proteins are of low MW (in the 17-20 kD range); the third protein migrates just ahead of the tubulin subunit. By contrast, the nerve RNA gave a strikingly simple pattern dominated by a set of high MW polypeptides, by a 65 kD protein and by actin. These high MW polypeptides presumably represent neurofilament proteins, which is intriguing in view of their absence from the set of proteins directed by mRNA from the nerve cell bodies. The very different translation patterns obtained with axoplasmic and nerve RNAs argue strongly against the possibility of contamination of the axoplasmic sample by nerve RNA. The possibility of contamination was further explored by carefully stripping all the surrounding thin axons from the giant axon before extrusion, and then preparing RNA from both axoplasm and the residual sheath. The sheath RNA was not very active, although on a wet weight basis it was considerably more active than the axoplasmic mRNA (experiment 4 of Table 1). Comparison of the translation patterns was made difficult by the low activity of the sheath sample; it required a very long exposure to detect only the
438
A n f o l i o (_}ItiDITTA e[ al.
presumed actin band (Fig.l, B), whereas the axoplasmic mRNA gave a pattern similar to that shown in Fig.l, A. This does not lend support to the idea that the mRNA activity found in axoplasm arises from contamination from adjacent nucleated cells. Rather it would seem that axoplasm contains a distinct subset of mRNAs.
A b
116
-
68-
n
a
B
I
b
n
s
a
116
68
56
-
56
42
-
42 40
35
-
35
14
-
14
Figure i. A. Electrophoretic patterns of the translation products specified by axoplasmic and related RNAs. b, blank without added RNA; n, stellate nerve remaining after extrusion of axoplasm; a, axoplasm; i, giant fibre lobe. The samples were appropriately diluted with unlabeled lysate mixture to give patterns of approximately comparable intensity. The MW of the marker proteins are indicated in kD. Exposure time, 68 hr at -80 ° on Fuji RX X-ray film. B. Translation patterns obtained with axoplasm extruded from isolated giant axons (a) and from the corresponding axonal sheath (s); b, blank without added RNA. Exposure time, 6 wk on Fuji RX.
mRNA in squid axoplasm
439
Subcellular studies. The subcellular distribution of axoplasmic RNA was examined by preparing RNA from axoplasmic mitochondrial and microsoma1 fractions, starting from approximately 100 mg tissue. No mRNA activity was detectable in either the mitochondrial fraction or the high speed sunp~srnatant. All the activity sedimented with the "microsomes" (4.2 x 10 cpm). Similarly, the synaptosomal mitochondria prepan
n'
s
I'
I
o'
O
116
68
56
42 40 35 31
21
14
Figure 2. Comparison of the translation patterns obtained with axoplasmic and related RNAs and the patterns of proteins synthesized by the corresponding regions of the stellate ganglion and nerve. Translation pattern of the remaining stellate nerve (n'), of the giant fibre lobe (1') and of the axoplasm (a') are shown side by side with the patterns of proteins synthesized by the remaining stellate nerve (n) and by the giant fibre lobe (i) and with the patterns of axoplasmic proteins (a) and of sheath proteins (s) synthesized by the giant axon. Exposure time, 48 hr on Fuji RX.
440
ANTONIO G l [ DITrA el ~1l.
red from 4 g of optic lobes (courtesy of Dr. R. Cohen) were completely inactive as a source of mRNA. These mitochondria are available in larger yield than axoplasmic mitochondria. The RNA of the other subcellular fractions of the optic lobe were quite active (up to 127 x l0 s total cpm in the microsomal fraction) and gave similar translation patterns (data not shown). Comparison with newly-synthesized protein s . We made some preliminary studies to see how the patterns of proteins specified by mRNA corresponded with the patterns of protein synthesis in the various tissues associated with the giant axon. Figure 2 shows a side-by-side comparison of the labeling pattern obtained with the extruded stellate nerve (n), giant axon sheath (s), axoplasm (a) or giant fibre lobe (i) with the corresponding translation patterns obtained in the reticulocyte lysate (n', i' and a'). It is striking that while the giant fibre lobe tracks 1 and i' show a high degree of correspondence, the a and a' lanes show poor agreement, as would be expected if most of the axoplasmic proteins were synthesized by surrounding glia cells. Indeed, the pattern of newly-synthesized axoplasmic proteins most closely resembles that shown by whole nerve and the isolated sheath, although they are not identical. DISCUSSION The possibility that the mRNA activity of the axoplasm arose by contamination from extraneous tissue during the extrusion step may be excluded. The stellate nerves were carefully dissected, and the proximal ends of the giant axons were cleaned of surrounding small axons before gently squeezing the axoplasm. More compellingly, the translation patterns of axoplasm and of the surrounding axonal sheath or the remaining stellate nerve are quite different. In addition, preliminary in situ hybridization analyses using [3H]poly(U) have demonstrated the presence of significant amounts of poly(A)RNA in the axoplasm (Grassi Zucconi G. et al., unpublished data). The presence of poly(A)RNA in the extruded axoplasm has been confirmed by a hybridization method using [3H]poly(U) (Perrone Capano et al., unpublished results). The mRNA in axoplasm is not derived from mitochondria, but is associated with the microsomal fraction. This is not surprising in view of the inability of the cytoplasmic protein synthesis machinery to translate mitochondrial RNA (Barrel et al., 1979). We were unable to detect any mRNA activity in the mitochondrial fraction of the axoplasm, or in the analogous mitochondria derived from optic lobe synaptosomes. There is considerably less mRNA in axoplasm, judged on a wet weight basis, than in the corresponding cell bodies of the giant fibre lobe. One should bear in mind the large size of the axonal territory in making comparisons, however. Taken as a whole, the axoplasmic mRNA becomes a significant fraction of the total mRNA present in these unusual cells. Similar considerations would apply to other neurons, if mRNA were shown to occur in axons and nerve endings of other invertebrate and vertebrate nerve cells, as future analyses should ascertain. The apparent segregation of neuronal mRNA into two sets, one to be translated in the cytoplasm surrounding the nucleus, the other to be found in cytoplasm that is almost completely or possibly entirely lacking ribosomes, raises several intriguing questions. First, what is the origin
mRNA in squid axoplasm
441
of the axoplasmic mRNA? It seems likely that it mostly originates in the nerve cell body, and the greater similarity between the translation patterns of giant fibre lobe and axoplasmic mRNA tends to support this idea. We cannot however exclude the possibility that some may come from the surrounding glia cells. Indeed, the synthesis of axoplasmic RNA has been detected in isolated giant axons (Cutillo et al., 1983). Wherever it comes from, there must be some kind of specificity in either its delivery to the axon, or in the stability of particular messages as they pass down the axon. Probably the most intriguing question concerns the role of axoplasmic mRNA. We cannot tell whether it is actively translated in axoplasm, and hence associated with polyribosomes, as it has been proposed (Giuditta, 1980), or whether it is stored in the axon in an untranslated form, perhaps en route to remote sites of protein synthesis either at nerve terminals or even beyond. The limited degree of similarity between the in vitro translation pattern of axoplasmic mRNA and the pattern of axoplasmic proteins synthesized by the giant axon suggests that little if any translation of the mRNA goes within the axon, in accord with the glia transfer hypothesis of Lasek et al. (1977). However, the differences might be attributed to the inability of the reticulocyte cell-free lysate to effect post-translational modifications of the translated proteins. Finally, it is of interest that the complexity of axoplasmic mRNA is much lower than that of the giant fibre lobe mRNA (Perrone Capano et al., unpublished results). It has been known for some time that maumnalian brain RNA contains a much wider set of sequences than other tissues of the body (Chaudary and Hahn, 1983; Kaplan, 1983). Similar results have been obtained recently in the squid (Perrone Capano et al, in press). The fact that a single nerve cell type like the squid giant , axon has a relatively simple set of sequences suggests that the reason for the high complexity found in the brain is because it contains so many different types of cells. ACKOWLEDGEMENTS We thank the permanent staff of the Marine Biological Laboratory, Woods Hole, Mass., for providing facilities and particularly supplies of animals. We also thank J. Rosenbaum and J. Ruderman for their generous hospitality during these studies. A.G. ackowledges finantial support from NATO grant 18781, from the Ministero della Pubblica Istruzione and the Progetto Finalizzato di Medicina Preventiva e Riabilitativa del CNR. T.H. was supported in part by NIH training grant GM-31136-04 and 05.
Anderson virus Barondes lism. Barrell de in Chaudari brain.
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442
ANTONIO G~t.urrT'a et a/.
Cutillo V., Montagnese P., Gremo F., Casola L. and Giuditta A. (1983> Origin of axoplasmic RNA in the squid giant fibre. Neurochem. Res. 8, 1621-1634. Giuditta A., Dettbarn W.-D. and Brzin M. (1968) Protein synthesis in the isolated giant axon of the squid. Proc.Natl.Acad. Sci. USA 59, 1284-1287. Giuditta A., Metafora S., Felsani A. and Del Rio A. (1977) Factors for protein synthesis in the axoplasm of squid giant axons. J.Neurochem. 28, 1393-1395. Giuditta A. (1980) Origin of axoplasmic proteins in the squid giant axon. Riv. Biol. 7_33, 35-49. Giuditta A., Cupello A. and Lazzarini G. (1980) Ribosomal RNA in the axoplasm of the squid giant axon. J.Neurochem. 3_~4, 1757-1760. Giuditta A., Hunt T. and Santella L. (1983) Messenger RNA in squid axoplasm. Biol.Bull. 165, 526. Jackson R.J. and Hun£ T. (1983) Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukariotic messenger RNA. Meth.Enzymol. 9_66, 50-74. Kaplan B.B. (1983) RNA-DNA hybridization: analysis of gene expression. In Handbook of Neurochemistry (Lajtha A. ed.) 2nd edition, Vol. 6, pp. 1-26, Plenum Press, New York. Ingoglia N., Giuditta A., Zanakis M.S., Babigian A., Tasaki I., Chakraborty G. and Sturman J. (1983) Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA mediated, post-translational protein modification in squid giant axon. J.Neurosc. 3, 2463-2473. Lasek R.J., Dabrowski J.C. and Nordlander R. (1973) Analysis of axoplasmic RNA from invertebrate giant axons. Nature 244, 162-165. Lasek R.J., Gainer H. and Przybylski (1974) Transfer of newly-synthesized proteins from Schwann cells to the squid giant axon. Proc.Natl. Acad. Sci. USA 7_~I, 1188-1192. Lasek R.J., Gainer H. and Barker J.L. (1977) Cell to cell transfer of glial proteins to the squid giant axon. The glia-neuron protein transfer hypothesis. J.Cell Biol. 74, 501-523. Laskey R.A. and Mills A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Europ. J.Biochem. 56, 335-341. Pelham H.R.B. and Jackson R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Europ. J.Biochem. 6_/7, 247256. Perrone Capano C., Giuditta A., Gioio A. and Kaplan B.B. (1986) Complexity of nuclear and polysomal RNA from squid optic lobe and gill. J.Neurochem. "In press". Pollard H.B. and Pappas G.B. (1979) Veratridine-activated release of adenosine-5'-tmiphosphate from synaptosomes: evidence for calcium dependence and blockade by tetrodotoxin. Biochem.Biophys.Res. Comm. 88, 1315-1321.