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Axonal transport of ribonucleoprotein particles (Vaults)
Pergamon PII:
Neuroscience Vol. 91, No. 3, pp. 1055–1065, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00622-8
AXONAL TRANSPORT OF RIBONUCLEOPROTEIN PARTICLES (VAULTS) J.-Y. LI,*† W. VOLKNANDT,‡ A. DAHLSTROM,* C. HERRMANN,‡ J. BLASI,§ B. DASk and H. ZIMMERMANN‡ *Department of Anatomy and Cell Biology, Goteborg University, Box 420, SE-405 30 Goteborg, Sweden ‡Biozentrum der J. W. Goethe-Universitat, AK Neurochemie, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany §Department de Biologia y Anatomia Patologia, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain k State University of New York, Health Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203-2098, U.S.A.
Abstract—RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the bsubunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure—in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: axonal transport, ribonucleoprotein particle, RNA, Torpedo, vault, SV2.
Translocation of RNA to specific destinations in the cytoplasm has recently received increasing attention. This concerns in particular specific species of mRNA that may be translocated to their sites of subsequent translation. There is evidence that mRNA is transported as part of a larger structure. 10,18,53 Such a possibility is of particular interest for the highly polarized nerve cell. In dendritic domains the presence of mRNA encoding specific †To whom correspondence should be addressed. Abbreviations: CLSM, confocal laser scanning microscope; FITC, fluorescein isothiocyanate; LRP, lung resistancerelated protein; MVP100, major vault protein 100; SDS, sodium dodecyl sulfate; SV2, synaptic vesicle protein 2.
proteins such as the microtubule-associated protein, 1,3,8,33 the a subunit of Ca 2⫹/calmodulindependent protein kinase type II, 4 type I inositol trisphosphate receptor, 19 protein L7, 5 the activityregulated cytoskeleton-associated protein (Arc), 42 or the a-subunit of the glycin receptor 49 has been identified. The presence of the translational machinery close to the site of function such as the synapse would provide the possibility for a local regulation of synthesis and thus synaptic function. There is evidence that a number of mRNAs can be found in axons of invertebrates. 56 The axonal compartment of mammalian axons has long been thought to be devoid of mRNA. But at least in the neurosecretory axons of the hypothalamo–neurohypophyseal
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system, mRNAs encoding specific protein species have been identified. These include mRNAs for vasopressin and oxytocin, prodynorphin and, interestingly, also for the neurofilament protein. 27,36,43,44,46 In addition, non-translated RNA can be transported. BCl RNA, a small polymerase III transcript that is expressed almost exclusively in the nervous system is transported in hypothalamo– neurohypophyseal axons. 54 BCl RNA which is a component of a ribonucleoprotein particle has also been located in the dendrites and somata of subsets of neurons in the central and peripheral nervous system. 55 We used the cholinergic electromotor system of the electric ray Torpedo 59 to study the axonal transport of a ribonucleoprotein particle referred to as vault. Vaults represent barrel-shaped ubiquitous cytoplasmic ribonucleoprotein particles originally identified in preparations of coated vesicles from rat liver. 30 They are widely distributed throughout eukaryotes and have been purified from mammals, amphibians, avians and the slime mold Dictyostelium discoideum. 29 Vaults are also contained in the cholinergic nerve terminals of electric ray electric organ. 22 The dimension of the isolated particles is about 2/3 of the size of synaptic vesicles from the same source. 21 Vaults are multimeric protein complexes with the major vault protein of about 100,000 mol. wt as the predominant member. 32 Major vault proteins are highly conserved during evolution and the major vault protein of the electric ray (MVP100) reveals 69% amino acid identity with those of human and rat origin and 52% with the two isoforms identified in the slime mold. 22 An interesting feature of vault particles is their content in untranslated small RNA (vault RNA) which, like BCl, represents a RNA polymerase III transcript. 31 It constitutes about 5% of the vault particle mass in the rat. The length of vault RNAs is in the order of 100 bases. Their levels vary between tissues. In the electric ray Torpedo MVP100 is highly expressed in neural tissue, in particular in the electric lobe that contains the neurons innervating the electric organs. 23 Western blots detect the protein not only in the electric lobe but also in the electric nerve and the electric organ. 22 This suggests that vaults are transported from their site of synthesis in the electric lobe via the axons to the nerve terminals in the electric organ. In the present investigation we crushed the electromotor nerves and studied the timedependent accumulation of MVP100 by immunofluorescence. The accumulation of MVP100 was compared to that of SV2, a synaptic vesicle proteoglycan and putative transporter protein 17 and to mitochondria. EXPERIMENTAL PROCEDURES
Preparation of nerves Electric rays Torpedo marmorata were caught at the
Mediterranean coast and maintained in fresh sea water. Fishes were anesthetized in seawater containing 0.05% ethyl maminobenzoate ethanesulfonate until respiratory movements of spiracles ceased. They were then transferred into a shallow dissection tank and the gills were superfused with 0.01% of the above anesthetic as previously described. 62 Electric nerves were exposed between the skull and gill arches 15 and the first and fourth nerve were firmly double-crushed for 5 s with fine watchmaker’s forceps. The distance between the two crushes was 1– 2 mm. After closing the incision, the fish was revived by exchange with fresh seawater and returned to the tank at room temperature (21⬚C). Zero, 1, 3, 6, 9, 12, 24, 48 and 72 h after operation fishes were anesthetized as above and killed by removing the brain. At least two animals were investigated for each time-point. Electric nerves were dissected and fixed by immersion in sodium cacodylatebuffered (0.4 M, pH 7.4) 4% paraformaldehyde for 7–10 h at 4⬚C. After washing in the above cacodylate buffer they were rinsed in cacodylate buffer containing 20% sucrose and 0.1% sodium azide. Subsequently, nerves were frozen with compressed C02, cryostat sectioned longitudinally at 10 mm and placed on gelatinized glass slides. All efforts were made to minimize animal suffering, and to reduce the number of animals used. Antibodies The following primary antibodies were used: (1) AntiMVP100 protein, rabbit polyclonal antibody raised against the amino acid sequence KDPVLDRNARQT (amino acids 429–440) coupled to keyhole limpet hemocyanin via an additional cysteine (Eurogentec, Seraing, Belgium). The amino acid sequence was determined by direct microsequencing of MVP100-peptide fragments derived from Torpedo marmorata electric organ. 21 (2) Polyclonal antibody raised against a 21-mer peptide (amino acids Y311 to A331) in the conserved ATP-binding region of the bsubunit of human mitochondrial H-ATPase. 14 (3) Mouse monoclonal anti-SV2 (clone CKKlOH). 9 Dilutions of antibodies in indirect immunofluorescence were: anti-MVP100, 1:20; anti-b-subunit, 1:20; anti-SV2, 1:400. Indirect immunofluorescence Single and double immunofluorescence of tissue sections was performed as previously described. 39 Briefly, sections were double-labeled using two different fluorophores. After preincubation with a mixture of normal horse and goat sera for 1 h, a mixture of monoclonal (SV2) and polyclonal antibody (anti-MVP100, or anti-13-subunit) was added for incubation overnight. After rinsing, a mixture of biotinylated goat anti-rabbit IgG (dilution 1:200) and Texas Redlabeled horse anti-mouse IgG (dilution 1:50) (Vector Lab, Burlingame, CA, U.S.A.), was added followed by incubation with streptavidin–fluorescein isothiocyanate (FITC, Amersham, Buckinghamshire, U.K.). All incubations were carried out in a moist chamber at room temperature and all solutions contained 1% bovine serum albumin, 0.1% sodium azide and 0.2% Triton X-100. In control experiments, the primary antibody was substituted by normal sera from rabbit, horse or goat. Controls were always negative for immunofluorescence. The sections were first viewed in a Nikon epifluorescence microscope, registered with cytofluorimetric scanning techniques, and then studied in a confocal laser scanning microscope (CLSM; BioRad, MRC 600, Richmond, VA, U.S.A.), using single or dual channel scanning with exciting wavelengths for FITC (488 nm) and for Texas Red (568 nm). For cytofluorimetric scanning, sections are passed at a constant speed with a motor-driven cross-table, under the objective. The fluorescence intensity along the nerve is registered as a curve, and the areas under the peaks, registered as number of pixels, are relative to the
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amount of immunofluorescence in the section. 11,35 Photographs were taken using Kodak Plus 125 film. Electrophoretical techniques and western blotting Small pieces of electric nerve were sonicated for 10 s in a Branson Sonifier 250 in 1% sodium dodecyl sulfate (SDS) (10 ml/mg wet weight) prior to addition of sample buffer. Polyacrylamide gel electrophoresis in the presence of SDS was carried out on minigels (10% acrylamide). 57 Immunoblotting was performed using the Amersham enhanced chemiluminescence system according to the manufacturers instructions. RESULTS
The axons belonging to the electromotor neurons situated within the electric lobe are large and myelinated. The diameter of the axon cylinder is about 7 mm. 61 The axons form voluminous ramifications within the electric organ requiring an intense exchange of material between the soma and the axon. The accumulation of immunoreactive material at the site of ligation was investigated between 0 min and 72 h after ligation. Immunofluorescence in frozen sections was analysed qualitatively and quantitatively using a confocal laser microscope and dual channel scanning. Crushing the nerve alone did not cause an accumulation of immunoreactive axonal material (MVP100, Fig. 1A; SV2, Fig. 1A 0 ). No significant accumulation of MVP100 immunoreactivity was detected up to 6 h following the crush operation (1h, Fig. 1B; 3 h, Fig. 5A), but weakly fluorescent traces of accumulated MVP100 were detected in the confocal microscope at 3 h. From 12 h onwards vault immunoreactivity was accumulated significantly on either side of the crush (Fig. 1C) and the accumulation increased markedly with time (48 h, Fig. 1D). In fact, the cytofluorimetric scanning curve indicated an acceleration in accumulating amounts with time after 24 h. In contrast, an accumulation of the synaptic vesicle protein SV2 could first be detected 1 h after the crush operation both, proximal and distal to the crush (Fig. 1B 0 ). The accumulation was increased during the following hours (12 h, Fig. 1C 0 ) and days (48 h, Fig. 1D 0 ), but appeared to decelerate during the time after the initial 24 h. For both, SV2 and MVP100 the accumulation at the proximal side of the crush was always larger than at the distal side (Fig. 1). After 48 h about twice as much material had accumulated at the proximal side for either protein. Figure 2 reveals the distribution of the two proteins at higher magnification. Generally, all MVP100-containing axons also revealed SV2, except that the immunostaining for SV2 was more intense throughout the axon. This is also the case for axon profiles proximal and distal to the crush at time-point zero (Fig. 2A). The fast transport of SV2 is reflected by a significant increase in immunoreactivity of axon profiles close to the crush already after 1 h (Fig. 2B). After 12 h axonal profiles
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proximal and distal to the crush are clearly double labeled for MVP100 and SV2 even though the immunofluorescence was more intense for SV2 (Fig. 2C). The two proteins were essentially colocalized. After more than 24 h the immunofluorescence for MVP100 had also become very intense and axonal profiles were equally labeled (Fig. 2D, 48 h). The antibody against MVP100 stained also some non-axonal structures that presumably represent connective tissue surrounding axon bundles (Fig. 2D). This labeling was also observed on some occasions in uncrushed nerves. In contrast to SV2, expression of MVP100 is not neuron-specific. An analysis of the immunoreactive structures at high magnification revealed an association of MVP100 with granular organellar structures, indicating that MVP100 is associated with particulate structures. The subcellular distribution was identical proximal (Fig. 3A) and distal (Fig. 3B) to the crush. In contrast, immunoreactivity of SV2 at the sites of the crush was rather evenly distributed revealing a very fine punctuate pattern. The immunolabeling of SV2 was different at sites distal to the crush or in non-crushed axons. There, distinct organellar structures of round or elongated shape were labeled (Fig. 3C, D). MPV100, however, was not detected in the normal, non-accumulated part of the axons. Cytofluorimetric scanning studies showed that the increase in SV2 gradually decelerated between 24 and 48 h, and further between 48 and 72 h (Fig. 4), in agreement with early observations in longterm crush-operated rat sciatic nerves. 6,7 In contrast, the accumulations of MVP100-immunoreactive material accelerated (Fig. 4). Thus, while during the early phase after crushing the SV2-carrying vesicles accumulated at a rapid rate and the vault protein appeared to increase very slowly, the reverse was apparent during the interval between 48 and 72 h. We also analysed the crush-induced accumulation of mitochondria as an additional organelle, using an antibody directed against the b-subunit of the mitochondrial ATPase. An accumulation of immunoreactivity could be observed as early as 3 h after application of the crush (Fig. 5B). The immunoreactivity for mitochondria was thereafter increased on either side of the crush. The accumulation of immunoreactive material increased further with time at an apparently steady rate, and was always more pronounced than that of MVP100. The specificity of the antibodies applied for immunocytochemistry was investigated using total tissue homogenates of electric nerves (Fig. 6). All antibodies proved to be monospecific. The antibody against MVP100 recognized a single band at 100,000 mol. wt that against the proteoglycan SV2 revealed the known broad band between 90 and 110,000 mol. wt, and the antibody against the b-subunit bound to the expected band at 52,000 mol. wt.
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Fig. 1. Nerve sections and their cytofluorimetric scanning curves after varying times following the crush operation. Longitudinal sections were double labeled for MVP100 (FITC; A, B, C, D) and SV2 (Texas Red; A 0 , B 0 , C 0 , D 0 ). Nerves were analysed after 0 h (A, A 0 ), 1 h (B, B 0 ), 12 h (C, C 0 ) and 48 h (D, D 0 ) following the crush. Arrowheads indicate sites of the crush. The proximal is to the left. The peaks of the upper scanning curves can be identified in the microscope photographs. The pixel numbers under the curves, representing the amounts of accumulation, are shown in the left boxes, below the photographs; the upper right boxes show the percentage between the distal and proximal accumulation.
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Fig. 2. CLSM photographs of bundles of electromotor axons double labeled for MVP100 and SV2 0 h (A), 1 h (B), 12 h (C) and 48 h (D) after the crush operation. M, labeling for MVP (FITC); S, labeling for SV2 (Texas Red). The site of the crush is indicated by large arrows with the proximal side above and the distal side below. The vertical line in the middle indicates the border between the two channels. Proximal accumulation of SV2 becomes apparent after 1 h and is then further increased. Accumulation of MVP100 is distinct at 12 h and 48 h. At these time-points the two proteins are co-localized within the axons proximal and distal to the crush. Small arrows indicate immunostaining for MVP100 of connective tissue. Scale bars 100 mm.
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Fig. 3. CLSM photographs of axons 48 h after the crush operation at high resolution. Photographs were taken closely proximal (A) or distal (B) to the crush. M, labeling for MVP (FITC); S, labeling for SV2 (Texas Red). The distribution of SV2 immunoreactivity is shown in addition at large distance either proximal (C) or distal (D) to the crush. The immunoreactivity for SV2 in close proximity to the crush reveals largely a very fine punctuate pattern, however, at larger distance away from the crush it is associated with large organelles of varying shape. Scale bars 5 mm. DISCUSSION
Vaults represent granular structures High resolution immunofluorescence analysis revealed that MVP100 is associated with distinct granular structures in the axon. In principle, a granular structure is in agreement with previous biochemical findings. MVP100 is associated with a granular structure on sucrose density gradient or glycerol velocity gradient centrifugation. 22 It cosediments with synaptic vesicles on sucrose gradients but can be separated from vesicles by gel filtration where it elutes with a smaller particle size. Negative staining of the MVP100 containing subcellular fractions derived from Torpedo electric organ revealed the presence of barrel-shaped particles with 45 nm width and 65 nm length. For comparison, synaptic vesicles in the Torpedo electric organ have a diameter of about 90 nm. 21 As revealed by immunoelectron microscopy, vaults are highly enriched within the nerve terminal, and appear to be closely associated with the SV2containing synaptic vesicles. 22 Although it is difficult to derive the size of a particle from immunofluorescence data the size of many of the MVP100-immunopositive structures is
presumably larger than that expected for the size of vaults. This suggests that vaults are clustered together in the axon or are associated with larger organelles driven by axonal transport. Little is known concerning the subcellular distribution of vaults in other tissues. A punctuate distribution of the major vault protein has been observed in fibroblasts where the protein can colocalize also with the tips of actin filaments at the edge of the cultured cells. 28 Synaptic vesicle protein 2 can be associated with a variety of axonal organelles In previous immunoelectron microscopic studies of electromotor axons the synaptic vesicle protein SV2 was found in association with a variety of organelles including multivesicular bodies, vesiculotubular structures, lamellar bodies, electron-dense granules and also electron-lucent vesicles of the size of synaptic vesicles. Some of these structures may represent organelles moving in anterograde direction, others organelles directed to the cell body. 26 The association of SV2 with distinct organellar structures of round or elongated shape as revealed by immunofluorescence corresponds to
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Fig. 4. Cytofluorimetric scanning curves of crush-operated Torpedo nerves, showing the accumulation of immunoreactive MVP100 and SV2 up to 72 h after the operation. Note that with time after the initial 24 h the accumulations of MVP100 increases at an accelerated speed, while the accumulation of SV2 appears to decelerate. The intensity of immunoreactive material cannot directly be compared between the two antigens, since the settings for the confocal scanning equipment was different for SV2 and MVP100. Values represent means ^ S.E.M. from two independent experiments. Acc, accumulation.
the electron microscopic observations. Close to the crush the subcellular distribution of SV2 was different from that of MVP100 and revealed a much more intense and fine punctuate immunofluorescence. This could represent the crush-induced formation of very small vesicular organelles. Alternatively, it may be due to the pile-up of large numbers of small organelles in the short segment proximal and distal to the crush. This could preclude the identification of individual organelles. In unligated electromotor axons, SV2-containing organelles are accumulated proximally and distally of each individual node of Ranvier suggesting that this structure represents a natural site of organelle accumulation. 26,60,61 The accumulation of immunofluorescent material in individual axons near the crush observed in the present study is discontinuous. It presumably reflects the intense accumulation of organelles at individual nodes. The crush-induced accumulation of SV2 in the electromotor nerves corresponds to that previously observed with the cytofluorimetric scanning method in mammalian peripheral axons. Similar results were obtained also for the synaptic vesicle membrane proteins synaptophysin and synaptobrevin that reveal significant anterograde and retrograde
transport. 12,38 In contrast, proteins such as the synapsins, rabphilin 3A or the small GTP-binding protein rab3A that are loosely associated with the synaptic vesicle membrane reveal a fast anterograde but only a marginal retrograde component. 12,37,40 Dynamics of axonal transport of vaults may differ from those of vesicle proteins and mitochondria Vault particles can be transported in either direction but about half of the material transported to the synapse appears to be degraded there. Significant accumulation of vaults can be observed first 6–9 h after application of the crush, but traces were observed in the confocal microscope already 3–6 h after crush. Transport appeared initially considerably slower than that of the organelles carrying the synaptic vesicle protein SV2 (significant after 1 h) and also slower than that of mitochondria (significant after 3 h). However, the accumulation was faster than would be expected for material susceptible to slow axonal transport. 13 It was interesting to observe that the increase in accumulation proximal to a crush of vault protein appeared to accelerate with time after crush, while the increase in SV2 protein decelerated (Fig. 4). This behaviour of
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Fig. 6. Analysis of the antibodies by immunoblotting using homogenates of electric nerve. Equal amounts of protein (40 mg) were loaded per lane. 1, MVP100 antibody; 2, SV2 antibody; 3, antibody against F1-subunit of Fl-ATPase. KD, relative molecular masses of standard proteins.
Fig. 5. Comparison of the localization of MVP100 (A) and the F1-subunit of mitochondrial Fl-ATPase (B) 3 h after the crush operation. The site of the crush is indicated by arrows with the proximal side above and the distal side below. Labelling for MVP100 (M) and FlATPase (Fl) (both FITC) was performed on different sections derived from the same nerve. Whereas MVP100 reveals very little accumulation Fl-ATPase is significantly accumulated on either side of the crush. Scale bars 100 mm.
synaptic vesicle protein-immunoreactive material has been observed earlier in rat sciatic nerve; Bo¨o¨j and colleagues 6,7 observed that the accumulation of RASVA immunoreactivity (rabbit antisynaptic vesicle antibody, 6 the first antibody that recognized SV2 in cholinergic vesicles 51) and of 10H-immunoreactive material, 7 decelerated during periods later than 12 h after the crush. Both these antibody preparations recognize the proteoglycan in synaptic vesicle that is labeled by the SV2 antibody. This indicates that the soma production of synaptic vesicle proteins, as a reaction to the axonal trauma, reduce the synthesis of material involved in transmission. In contrast, an axotomized neuron increases the production of reparative material. Thus, the differences in initial accumulation rate in the Torpedo nerves demonstrated here during the first 24 h after crush need not necessarily indicate dramatic differences in normal rate of anterograde axonal transport, but may be due to differences in the normal levels of vault protein on the one hand, and SV2-containing organelles on the other. At later intervals, beyond the 48 h period, the situation is altered, in that the amount of vault protein in transport is increased while the SV2 production and
amount in transport is down-regulated. For this reason it is difficult to estimate the rate of anterograde transport of the vault protein. One may speculate, based on the observations here, that it is slower than that of the SV2-carrying organelles, but, at least during the late phase (48–72 h) as fast as, or possibly faster, than that of the large mitochondria. It is interesting to consider the mechanisms of fast axonal transport of different components in the axon. An in vitro association of sea urchin vaults with microtubules has recently been observed. 20 If vaults were linked to molecular motors for transport these may be different from motors associated with synaptic vesicle precursors or mitochondria. In extracts of axonal organelles from adult rat cauda equina KIFlA, a monomeric motor for anterograde axonal transport, is associated with organelles containing the synaptic vesicle proteins synaptotagmin, synaptophysin and rab3A, but not SV2. This suggests that synaptic vesicle proteins may be transported into the axon terminal by differing organellar carriers. 16,48 Mitochondria are transported considerably more slowly than vesicular organelles (up to 70 mm/ d). 16,58 Purified KIFlB can transport mitochondria along microtubules in vitro. 47 Cross-bridges have been observed between mitochondria and microtubules. 24,50 But filamentous actin has also been implicated in axonal transport of mitochondria in vivo. 45 Little is known concerning the mechanisms of RNA-particle transport. Messenger RNA anchoring to actin filaments (fibroblasts) 2,52 and also to microtubules (neurons) 3 has been demonstrated. Using the membrane-permeable nucleic acid stain SYTO 14 the translocation of endogenous RNA could be visualized in neurites of cultured nerve cells. RNA
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was found to be associated with granules. Anti-kinesin antisense-oligonucleotides inhibit the transport of myelin basic protein mRNA in processes of cultured oligodendrocytes. 10 In cultured neurons RNA granules were found to be transported in anterograde and also in a retrograde direction. The motility had characteristics of an actively transported organelle that might be transported along cytoskeletal tracks. 34 Our observations demonstrate the anterograde and retrograde axonal transport of ribonucleoprotein particles in situ. At present the functional role of vaults is unknown. The only functional implication supported by experimental evidence so far is a potential involvement of vaults in multi drug-resistance. It is well documented that the lung resistance-related protein (LRP), the human homologue of MVP100, correlates with a poor response to chemotherapy. Multi-drug-resistant cancer cells frequently overexpress the LRP protein and LRP over-expression has been found to predict a multi-drug-resistance phenotype in acute leukemia and ovarian carcinoma. 25,41,52 Furthermore, vault particles interact with estrogen receptor in MCF-7 breast cancer cells. The functional role of the association with nuclear receptors is not known but it could relate to the intracellular traffic. 1 Whatever its function, major vault protein may be modulated by protein
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kinases. MVP100 is phosphorylated in vivo by protein tyrosine kinase and it can be phosphorylated in vitro by protein kinase C and casein kinase II. 21,22 CONCLUSIONS
Our studies demonstrate that vault particles are transported in the cholinergic electromotor axons of the electric ray. Transport occurs both in an anterograde and retrograde direction and transport velocity appears slower than that of the synaptic vesicle membrane protein SV2. An up-regulation of the vault protein appears to take place late after crushing and a simultaneous down-regulation of the SV2 protein. High resolution immunofluorescence reveals a granular distribution of the protein distinct from that of SV2. At present the functional role of vaults is unknown. Studies of the transport of this organelle could reveal insights into the transport mechanisms of ribonucleoprotein particles in general whether in neurons or in other cells.
Acknowledgements—This work was supported by an EUproject No. BMH4-CT 96-1586, by the Swedish MRC (2207) (A. D.), by the Swedish Medical Society (J.Y.L) and by a grant from the Deutsche Forschungsgemeinschaft to W.V. We are grateful to Andrea Winter for expert technical support.
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(1997) Characterization of the sea urchin major vault protein: a possible role for vault ribonucleoprotein particles in nucleocytoplasmic transport. Devl Biol. 190, 117–128. 21. Herrmann C., Kellner R. and Volknandt W. (1998) Major vault protein of electric ray is a phosphoprotein. Neurochem. Res. 23, 39–46. 22. Herrmann C., Volknandt W., Wittich B., Kellner R. and Zimmermann H. (1996) The major vault protein (MVP100) is contained in cholinergic nerve terminals of electric ray electric organ. J. biol. Chem. 271, 13,908–13,915. 23. Herrmann C., Zimmermann H. and Volknandt W. (1997) Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata. Gene 188, 85–90. 24. Hirokawa N. (1982) Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J. Cell Biol. 94, 129–142. 25. Izquierdo M. A., van der Zee A. G., Vermoorken J. B., van der Valk P., Belien J. A., Giaconne G., Scheffer G. L., Flens M. J., Pinedo H. M., Kenemans P., Meijer C. J. L. M., De Vries E. G. E. and Scheper R. J. (1995) Drug resistance-associated marker LRP for predication of response to chemotherapy and prognoses in advanced ovarian carcinoma. J. natn. Cancer Inst. 87, 1230–1237. 26. Janetzko A., Zimmermann H. and Volknandt W. (1989) Intraneuronal distribution of a synaptic vesicle membrane protein: antibody binding sites at axonal membrane compartments and trans-Golgi network and accumulation at nodes of Ranvier. Neuroscience 32, 65–77. 27. Jirikowsky G. F., Sanna P. P. and Bloom F. E. (1990) mRNA coding for oxytocin is present in axons of the hypothalamohypophyseal tract. Proc. natn. Acad. Sci. U.S.A. 87, 7400–7404. 28. Kedersha N. and Rome L. H. (1990) Vaults: large cytoplasmic RNPs that associate with cytoskeletal elements. Molec. biol. Reprod. 14, 121–122. 29. Kedersha N. L., Miquel M.-C., Bittner D. and Rome L. H. (1990) Vaults. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes. J. Cell Biol. 110, 895–901. 30. Kedersha N. L. and Rome L. H. (1986) Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA. J. Cell Biol. 103, 699–709. 31. Kickhoefer V. A., Searles R. P., Kedersha N. L., Garber M. E., Johnson D. L. and Rome L. H. (1993) Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III. J. biol. Chem. 268, 7868–7873. 32. Kickhoefer V. A., Vasu S. K. and Rome L. H. (1996) Vaults are the answer, what is the question? Trends Cell Biol. 6, 174–178. 33. Kleiman R., Banker G. A. and Steward O. (1990) Differential subcellular localization of particular mRNAs in hippocampal neurons in culture. Neuron 5, 812–830. 34. Knowles R. B., Sabry J. H., Martone M. E., Deerinck T. J., Ellisman M. H., Bassell G. J. and Kosik K. S. (1996) Translocation of RNA granules in living neurons. J. Neurosci. 16, 7812–7820. 35. Larsson P.-A., Bo¨o¨j S., Lundmark K., Goldstein M. and Dahlstrom A. (1987) Cytofluorimetric scanning studies on axonal transport in reserpinized adrenergic nerves. Expl Brain Res. 16, 282–287. 36. Lehmann E., Hanze J., Pauschinger M., Ganten D. and Lang R. E. (1990) Vasopressin mRNA in the neurolobe of the rat pituitary. Neurosci. Lett. 111, 170–175. 37. Li J.-Y. (1996) Rabphilin-3A is transported with fast anterograde axonal transport and associated with synaptic vesicles. Synapse 23, 79–88. 38. Li J.-Y., Edelmann L., Jahn R. and Dahlstrom A. (1996) Axonal transport and distribution of synaptobrevin I and II in the rat peripheral nervous system. J. Neurosci. 16, 137–147. 39. Li J.-Y., Jahn R. and Dahlstrom A. (1994) Synaptotagmin I is present mainly in autonomic and sensory neurons of the rat peripheral nervous system. Neuroscience 63, 837–850. 40. Li J.-Y., Jahn R. and Dahlstrom A. (1995) Rab3a, a small GTP-binding protein, undergoes fast anterograde transport but not retrograde transport in neurons. Eur. J. Cell Biol. 67, 297–307. 41. List A. F., Spier C. S., Grogan T. M., Johnson C., Roe D. J., Greer J. P., Wolff S. N., Broxterman H. J., Scheffer G. L., Scheper R. J. and Dalton W. S. (1996) Over expression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia. Blood 87, 2464–2469. 42. Lyford G. L., Yamagata K., Kaufmann W. E., Barnes C. A., Sanders L. K., Copeland N. G., Gilbert D. J., Jenkins N. A., Lanahan A. A. and Worley P. F. (1995) Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeletonassociated protein that is enriched in neuronal dendrites. Neuron 14, 433–445. 43. McCabe J. T., Lehman E., Chastrette N., Ha¨nze J., Lang R. E., Ganten D. and Pfaff D. W. (1990) Detection of vasopressin mRNA in the neurointermediate lobe of the rat pituitary. Molec. Brain Res. 8, 325–329. 44. Mohr E. and Richter D. (1992) Diversity of mRNAs in the axonal compartment of peptidergic neurons in the rat. Eur. J. Neurosci. 4, 870–876. 45. Morris R. L. and Hollenbeck P. J. (1995) Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. J. Cell Biol. 131, 1315–1326. 46. Murphy D., Levy A., Lightman S. and Carter D. (1989) Vasopressin RNA in the neural lobe of the pituitary: dramatic accumulation in response to salt loading. Proc. natn. Acad. Sci. U.S.A. 86, 9002–9005. 47. Nangaku M., Satoyoshitake R., Okada Y., Noda Y., Takemura R., Yamazaki H. and Hirokawa N. (1994) KIFlB, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209–1220. 48. Okada Y., Yamazaki H., Sekineaizawa Y. and Hirokawa N. (1995) The neuron-specific kinesin superfamily protein KIFla is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors. Cell 81, 769–780.
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49. Racca C., Gardiol A. and Triller A. (1997) Dendritic and postsynaptic localizations of glycine receptor a subunit mRNAs. J. Neurosci. 17, 1691–1700. 50. Raine C. S., Roytta M. and Dolich M. (1987) Microtubule-mitochondria associations in regenerating axons after taxol intoxications. J. Neurocytol. 16, 461–468. 51. Sanes J. B., Carlson S. S., von Weden R. J. and Kelly R. B. (1979) Antiserum specific for motor nerve terminals in skeletal muscle. Nature 280, 403–404. 52. Scheffer G. L., Wijngaard P. L. J., Flens M. J., Izquierdo M. A., Slovak M. L., Pinedo H. M., Meijer C. J. L. M., Clevers H. C. and Scheper R. J. (1995) The drug resistance-related protein LRP is the human major vault protein. Nature Med. 1, 578–582. 53. Taneja K. L., Lifshitz L. M., Fay F. S. and Singer R. H. (1992) Poly(A ⫹ ) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J. Cell Biol. 119, 1245–1260. 54. Tiedge H., Chen W. and Brosius J. (1993) Primary structure, neural-specific expression, and denritic location of human BC200 RNA. J. Neurosci. 13, 2382–2390. 55. Tiedge H., Fremeau R. T., Weinstock P. H., Arancio O. and Brosius J. (1991) Dendritic localization of neural BCl RNA. Proc. natn. Acad. Sci. U.S.A. 88, 2093–2097. 56. Van Minnen J., Bergman J. J., Van Kesteren E. R., Smit A. B., Geraerts W. P. M., Lukowiak K., Hasan U. and Syed N. I. (1997) De novo protein synthesis in isolated axons of identified neurons. Neuroscience 80, 1–7. 57. Volknandt W., Henkel A. and Zimmermann H. (1988) Heterogenous distribution of synaptophysin and protein 65 in synaptic vesicles isolated from rat cerebral cortex. Neurochem. Int. 12, 337–345. 58. Weiss D. G. (1982) General properties of axoplasmic transport. In Axoplasmic Transport in Physiology and Pathology (eds. Weiss D. G. and Gorio A.), pp. 1–14. Springer, Berlin, Heidelberg. 59. Whittaker V. P. (1992) The Cholinergic Neuron and its Target: The Electromotor Innervation of the Electric Ray Torpedo as a Model, Birkhauser, Boston, Basel, Berlin. 60. Zimmermann H. (1995) Accumulation of synaptic vesicle proteins and cytoskeletal specializations at the peripheral node of Ranvier. Microsc. Res. Techn. 34, 462–473. 61. Zimmermann H. and Vogt M. (1989) Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat. Cell Tiss. Res. 258, 617–629. 62. Zimmermann H. and Whittaker V. P. (1974) The effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo: a combined biochemical, electrophysiological and morphological study. J. Neurochem. 22, 435–450. (Accepted 8 October 1998)
Neuroscience Vol. 91, No. 3, pp. 1055–1065, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00622-8
AXONAL TRANSPORT OF RIBONUCLEOPROTEIN PARTICLES (VAULTS) J.-Y. LI,*† W. VOLKNANDT,‡ A. DAHLSTROM,* C. HERRMANN,‡ J. BLASI,§ B. DASk and H. ZIMMERMANN‡ *Department of Anatomy and Cell Biology, Goteborg University, Box 420, SE-405 30 Goteborg, Sweden ‡Biozentrum der J. W. Goethe-Universitat, AK Neurochemie, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany §Department de Biologia y Anatomia Patologia, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain k State University of New York, Health Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203-2098, U.S.A.
Abstract—RNA was previously shown to be transported into both dendritic and axonal compartments of nerve cells, presumably involving a ribonucleoprotein particle. In order to reveal potential mechanisms of transport we investigated the axonal transport of the major vault protein of the electric ray Torpedo marmorata. This protein is the major protein component of a ribonucleoprotein particle (vault) carrying a non-translatable RNA and has a wide distribution in the animal kingdom. It is highly enriched in the cholinergic electromotor neurons and similar in size to synaptic vesicles. The axonal transport of vaults was investigated by immunofluorescence, using the anti-vault protein antibody as marker, and cytofluorimetric scanning, and was compared to that of the synaptic vesicle membrane protein SV2 and of the bsubunit of the F1-ATPase as a marker for mitochondria. Following a crush significant axonal accumulation of SV2 proximal to the crush could first be observed after 1 h, that of mitochondria after 3 h and that of vaults after 6 h, although weekly fluorescent traces of accumulations of vault protein were observed in the confocal microscope as early as 3 h. Within the time-period investigated (up to 72 h) the accumulation of all markers increased continuously. Retrograde accumulations also occurred, and the immunofluorescence for the retrograde component, indicating recycling, was weaker than that for the anterograde component, suggesting that more than half of the vaults are degraded within the nerve terminal. High resolution immunofluorescence revealed a granular structure—in accordance with the biochemical characteristics of vaults. Of interest was the observation that the increase of vault immunoreactivity proximal to the crush accelerated with time after crushing, while that of SV2-containing particles appeared to decelerate, indicating that the crush procedure with time may have induced perikaryal alterations in the production and subsequent export to the axon of synaptic vesicles and vault protein. Our data show that ribonucleoprotein-immunoreactive particles can be actively transported within axons in situ from the soma to the nerve terminal and back. The results suggest that the transport of vaults is driven by fast axonal transport motors like the SV2-containing vesicles and mitochondria. Vaults exhibit an anterograde and a retrograde transport component, similar to that observed for the vesicular organelles carrying SV2 and for mitochondria. Although the function of vaults is still unknown studies of the axonal transport of this organelle may reveal insights into the mechanisms of cellular transport of ribonucleoprotein particles in general. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: axonal transport, ribonucleoprotein particle, RNA, Torpedo, vault, SV2.
Translocation of RNA to specific destinations in the cytoplasm has recently received increasing attention. This concerns in particular specific species of mRNA that may be translocated to their sites of subsequent translation. There is evidence that mRNA is transported as part of a larger structure. 10,18,53 Such a possibility is of particular interest for the highly polarized nerve cell. In dendritic domains the presence of mRNA encoding specific †To whom correspondence should be addressed. Abbreviations: CLSM, confocal laser scanning microscope; FITC, fluorescein isothiocyanate; LRP, lung resistancerelated protein; MVP100, major vault protein 100; SDS, sodium dodecyl sulfate; SV2, synaptic vesicle protein 2.
proteins such as the microtubule-associated protein, 1,3,8,33 the a subunit of Ca 2⫹/calmodulindependent protein kinase type II, 4 type I inositol trisphosphate receptor, 19 protein L7, 5 the activityregulated cytoskeleton-associated protein (Arc), 42 or the a-subunit of the glycin receptor 49 has been identified. The presence of the translational machinery close to the site of function such as the synapse would provide the possibility for a local regulation of synthesis and thus synaptic function. There is evidence that a number of mRNAs can be found in axons of invertebrates. 56 The axonal compartment of mammalian axons has long been thought to be devoid of mRNA. But at least in the neurosecretory axons of the hypothalamo–neurohypophyseal
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system, mRNAs encoding specific protein species have been identified. These include mRNAs for vasopressin and oxytocin, prodynorphin and, interestingly, also for the neurofilament protein. 27,36,43,44,46 In addition, non-translated RNA can be transported. BCl RNA, a small polymerase III transcript that is expressed almost exclusively in the nervous system is transported in hypothalamo– neurohypophyseal axons. 54 BCl RNA which is a component of a ribonucleoprotein particle has also been located in the dendrites and somata of subsets of neurons in the central and peripheral nervous system. 55 We used the cholinergic electromotor system of the electric ray Torpedo 59 to study the axonal transport of a ribonucleoprotein particle referred to as vault. Vaults represent barrel-shaped ubiquitous cytoplasmic ribonucleoprotein particles originally identified in preparations of coated vesicles from rat liver. 30 They are widely distributed throughout eukaryotes and have been purified from mammals, amphibians, avians and the slime mold Dictyostelium discoideum. 29 Vaults are also contained in the cholinergic nerve terminals of electric ray electric organ. 22 The dimension of the isolated particles is about 2/3 of the size of synaptic vesicles from the same source. 21 Vaults are multimeric protein complexes with the major vault protein of about 100,000 mol. wt as the predominant member. 32 Major vault proteins are highly conserved during evolution and the major vault protein of the electric ray (MVP100) reveals 69% amino acid identity with those of human and rat origin and 52% with the two isoforms identified in the slime mold. 22 An interesting feature of vault particles is their content in untranslated small RNA (vault RNA) which, like BCl, represents a RNA polymerase III transcript. 31 It constitutes about 5% of the vault particle mass in the rat. The length of vault RNAs is in the order of 100 bases. Their levels vary between tissues. In the electric ray Torpedo MVP100 is highly expressed in neural tissue, in particular in the electric lobe that contains the neurons innervating the electric organs. 23 Western blots detect the protein not only in the electric lobe but also in the electric nerve and the electric organ. 22 This suggests that vaults are transported from their site of synthesis in the electric lobe via the axons to the nerve terminals in the electric organ. In the present investigation we crushed the electromotor nerves and studied the timedependent accumulation of MVP100 by immunofluorescence. The accumulation of MVP100 was compared to that of SV2, a synaptic vesicle proteoglycan and putative transporter protein 17 and to mitochondria. EXPERIMENTAL PROCEDURES
Preparation of nerves Electric rays Torpedo marmorata were caught at the
Mediterranean coast and maintained in fresh sea water. Fishes were anesthetized in seawater containing 0.05% ethyl maminobenzoate ethanesulfonate until respiratory movements of spiracles ceased. They were then transferred into a shallow dissection tank and the gills were superfused with 0.01% of the above anesthetic as previously described. 62 Electric nerves were exposed between the skull and gill arches 15 and the first and fourth nerve were firmly double-crushed for 5 s with fine watchmaker’s forceps. The distance between the two crushes was 1– 2 mm. After closing the incision, the fish was revived by exchange with fresh seawater and returned to the tank at room temperature (21⬚C). Zero, 1, 3, 6, 9, 12, 24, 48 and 72 h after operation fishes were anesthetized as above and killed by removing the brain. At least two animals were investigated for each time-point. Electric nerves were dissected and fixed by immersion in sodium cacodylatebuffered (0.4 M, pH 7.4) 4% paraformaldehyde for 7–10 h at 4⬚C. After washing in the above cacodylate buffer they were rinsed in cacodylate buffer containing 20% sucrose and 0.1% sodium azide. Subsequently, nerves were frozen with compressed C02, cryostat sectioned longitudinally at 10 mm and placed on gelatinized glass slides. All efforts were made to minimize animal suffering, and to reduce the number of animals used. Antibodies The following primary antibodies were used: (1) AntiMVP100 protein, rabbit polyclonal antibody raised against the amino acid sequence KDPVLDRNARQT (amino acids 429–440) coupled to keyhole limpet hemocyanin via an additional cysteine (Eurogentec, Seraing, Belgium). The amino acid sequence was determined by direct microsequencing of MVP100-peptide fragments derived from Torpedo marmorata electric organ. 21 (2) Polyclonal antibody raised against a 21-mer peptide (amino acids Y311 to A331) in the conserved ATP-binding region of the bsubunit of human mitochondrial H-ATPase. 14 (3) Mouse monoclonal anti-SV2 (clone CKKlOH). 9 Dilutions of antibodies in indirect immunofluorescence were: anti-MVP100, 1:20; anti-b-subunit, 1:20; anti-SV2, 1:400. Indirect immunofluorescence Single and double immunofluorescence of tissue sections was performed as previously described. 39 Briefly, sections were double-labeled using two different fluorophores. After preincubation with a mixture of normal horse and goat sera for 1 h, a mixture of monoclonal (SV2) and polyclonal antibody (anti-MVP100, or anti-13-subunit) was added for incubation overnight. After rinsing, a mixture of biotinylated goat anti-rabbit IgG (dilution 1:200) and Texas Redlabeled horse anti-mouse IgG (dilution 1:50) (Vector Lab, Burlingame, CA, U.S.A.), was added followed by incubation with streptavidin–fluorescein isothiocyanate (FITC, Amersham, Buckinghamshire, U.K.). All incubations were carried out in a moist chamber at room temperature and all solutions contained 1% bovine serum albumin, 0.1% sodium azide and 0.2% Triton X-100. In control experiments, the primary antibody was substituted by normal sera from rabbit, horse or goat. Controls were always negative for immunofluorescence. The sections were first viewed in a Nikon epifluorescence microscope, registered with cytofluorimetric scanning techniques, and then studied in a confocal laser scanning microscope (CLSM; BioRad, MRC 600, Richmond, VA, U.S.A.), using single or dual channel scanning with exciting wavelengths for FITC (488 nm) and for Texas Red (568 nm). For cytofluorimetric scanning, sections are passed at a constant speed with a motor-driven cross-table, under the objective. The fluorescence intensity along the nerve is registered as a curve, and the areas under the peaks, registered as number of pixels, are relative to the
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amount of immunofluorescence in the section. 11,35 Photographs were taken using Kodak Plus 125 film. Electrophoretical techniques and western blotting Small pieces of electric nerve were sonicated for 10 s in a Branson Sonifier 250 in 1% sodium dodecyl sulfate (SDS) (10 ml/mg wet weight) prior to addition of sample buffer. Polyacrylamide gel electrophoresis in the presence of SDS was carried out on minigels (10% acrylamide). 57 Immunoblotting was performed using the Amersham enhanced chemiluminescence system according to the manufacturers instructions. RESULTS
The axons belonging to the electromotor neurons situated within the electric lobe are large and myelinated. The diameter of the axon cylinder is about 7 mm. 61 The axons form voluminous ramifications within the electric organ requiring an intense exchange of material between the soma and the axon. The accumulation of immunoreactive material at the site of ligation was investigated between 0 min and 72 h after ligation. Immunofluorescence in frozen sections was analysed qualitatively and quantitatively using a confocal laser microscope and dual channel scanning. Crushing the nerve alone did not cause an accumulation of immunoreactive axonal material (MVP100, Fig. 1A; SV2, Fig. 1A 0 ). No significant accumulation of MVP100 immunoreactivity was detected up to 6 h following the crush operation (1h, Fig. 1B; 3 h, Fig. 5A), but weakly fluorescent traces of accumulated MVP100 were detected in the confocal microscope at 3 h. From 12 h onwards vault immunoreactivity was accumulated significantly on either side of the crush (Fig. 1C) and the accumulation increased markedly with time (48 h, Fig. 1D). In fact, the cytofluorimetric scanning curve indicated an acceleration in accumulating amounts with time after 24 h. In contrast, an accumulation of the synaptic vesicle protein SV2 could first be detected 1 h after the crush operation both, proximal and distal to the crush (Fig. 1B 0 ). The accumulation was increased during the following hours (12 h, Fig. 1C 0 ) and days (48 h, Fig. 1D 0 ), but appeared to decelerate during the time after the initial 24 h. For both, SV2 and MVP100 the accumulation at the proximal side of the crush was always larger than at the distal side (Fig. 1). After 48 h about twice as much material had accumulated at the proximal side for either protein. Figure 2 reveals the distribution of the two proteins at higher magnification. Generally, all MVP100-containing axons also revealed SV2, except that the immunostaining for SV2 was more intense throughout the axon. This is also the case for axon profiles proximal and distal to the crush at time-point zero (Fig. 2A). The fast transport of SV2 is reflected by a significant increase in immunoreactivity of axon profiles close to the crush already after 1 h (Fig. 2B). After 12 h axonal profiles
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proximal and distal to the crush are clearly double labeled for MVP100 and SV2 even though the immunofluorescence was more intense for SV2 (Fig. 2C). The two proteins were essentially colocalized. After more than 24 h the immunofluorescence for MVP100 had also become very intense and axonal profiles were equally labeled (Fig. 2D, 48 h). The antibody against MVP100 stained also some non-axonal structures that presumably represent connective tissue surrounding axon bundles (Fig. 2D). This labeling was also observed on some occasions in uncrushed nerves. In contrast to SV2, expression of MVP100 is not neuron-specific. An analysis of the immunoreactive structures at high magnification revealed an association of MVP100 with granular organellar structures, indicating that MVP100 is associated with particulate structures. The subcellular distribution was identical proximal (Fig. 3A) and distal (Fig. 3B) to the crush. In contrast, immunoreactivity of SV2 at the sites of the crush was rather evenly distributed revealing a very fine punctuate pattern. The immunolabeling of SV2 was different at sites distal to the crush or in non-crushed axons. There, distinct organellar structures of round or elongated shape were labeled (Fig. 3C, D). MPV100, however, was not detected in the normal, non-accumulated part of the axons. Cytofluorimetric scanning studies showed that the increase in SV2 gradually decelerated between 24 and 48 h, and further between 48 and 72 h (Fig. 4), in agreement with early observations in longterm crush-operated rat sciatic nerves. 6,7 In contrast, the accumulations of MVP100-immunoreactive material accelerated (Fig. 4). Thus, while during the early phase after crushing the SV2-carrying vesicles accumulated at a rapid rate and the vault protein appeared to increase very slowly, the reverse was apparent during the interval between 48 and 72 h. We also analysed the crush-induced accumulation of mitochondria as an additional organelle, using an antibody directed against the b-subunit of the mitochondrial ATPase. An accumulation of immunoreactivity could be observed as early as 3 h after application of the crush (Fig. 5B). The immunoreactivity for mitochondria was thereafter increased on either side of the crush. The accumulation of immunoreactive material increased further with time at an apparently steady rate, and was always more pronounced than that of MVP100. The specificity of the antibodies applied for immunocytochemistry was investigated using total tissue homogenates of electric nerves (Fig. 6). All antibodies proved to be monospecific. The antibody against MVP100 recognized a single band at 100,000 mol. wt that against the proteoglycan SV2 revealed the known broad band between 90 and 110,000 mol. wt, and the antibody against the b-subunit bound to the expected band at 52,000 mol. wt.
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Fig. 1. Nerve sections and their cytofluorimetric scanning curves after varying times following the crush operation. Longitudinal sections were double labeled for MVP100 (FITC; A, B, C, D) and SV2 (Texas Red; A 0 , B 0 , C 0 , D 0 ). Nerves were analysed after 0 h (A, A 0 ), 1 h (B, B 0 ), 12 h (C, C 0 ) and 48 h (D, D 0 ) following the crush. Arrowheads indicate sites of the crush. The proximal is to the left. The peaks of the upper scanning curves can be identified in the microscope photographs. The pixel numbers under the curves, representing the amounts of accumulation, are shown in the left boxes, below the photographs; the upper right boxes show the percentage between the distal and proximal accumulation.
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Fig. 2. CLSM photographs of bundles of electromotor axons double labeled for MVP100 and SV2 0 h (A), 1 h (B), 12 h (C) and 48 h (D) after the crush operation. M, labeling for MVP (FITC); S, labeling for SV2 (Texas Red). The site of the crush is indicated by large arrows with the proximal side above and the distal side below. The vertical line in the middle indicates the border between the two channels. Proximal accumulation of SV2 becomes apparent after 1 h and is then further increased. Accumulation of MVP100 is distinct at 12 h and 48 h. At these time-points the two proteins are co-localized within the axons proximal and distal to the crush. Small arrows indicate immunostaining for MVP100 of connective tissue. Scale bars 100 mm.
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Fig. 3. CLSM photographs of axons 48 h after the crush operation at high resolution. Photographs were taken closely proximal (A) or distal (B) to the crush. M, labeling for MVP (FITC); S, labeling for SV2 (Texas Red). The distribution of SV2 immunoreactivity is shown in addition at large distance either proximal (C) or distal (D) to the crush. The immunoreactivity for SV2 in close proximity to the crush reveals largely a very fine punctuate pattern, however, at larger distance away from the crush it is associated with large organelles of varying shape. Scale bars 5 mm. DISCUSSION
Vaults represent granular structures High resolution immunofluorescence analysis revealed that MVP100 is associated with distinct granular structures in the axon. In principle, a granular structure is in agreement with previous biochemical findings. MVP100 is associated with a granular structure on sucrose density gradient or glycerol velocity gradient centrifugation. 22 It cosediments with synaptic vesicles on sucrose gradients but can be separated from vesicles by gel filtration where it elutes with a smaller particle size. Negative staining of the MVP100 containing subcellular fractions derived from Torpedo electric organ revealed the presence of barrel-shaped particles with 45 nm width and 65 nm length. For comparison, synaptic vesicles in the Torpedo electric organ have a diameter of about 90 nm. 21 As revealed by immunoelectron microscopy, vaults are highly enriched within the nerve terminal, and appear to be closely associated with the SV2containing synaptic vesicles. 22 Although it is difficult to derive the size of a particle from immunofluorescence data the size of many of the MVP100-immunopositive structures is
presumably larger than that expected for the size of vaults. This suggests that vaults are clustered together in the axon or are associated with larger organelles driven by axonal transport. Little is known concerning the subcellular distribution of vaults in other tissues. A punctuate distribution of the major vault protein has been observed in fibroblasts where the protein can colocalize also with the tips of actin filaments at the edge of the cultured cells. 28 Synaptic vesicle protein 2 can be associated with a variety of axonal organelles In previous immunoelectron microscopic studies of electromotor axons the synaptic vesicle protein SV2 was found in association with a variety of organelles including multivesicular bodies, vesiculotubular structures, lamellar bodies, electron-dense granules and also electron-lucent vesicles of the size of synaptic vesicles. Some of these structures may represent organelles moving in anterograde direction, others organelles directed to the cell body. 26 The association of SV2 with distinct organellar structures of round or elongated shape as revealed by immunofluorescence corresponds to
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Fig. 4. Cytofluorimetric scanning curves of crush-operated Torpedo nerves, showing the accumulation of immunoreactive MVP100 and SV2 up to 72 h after the operation. Note that with time after the initial 24 h the accumulations of MVP100 increases at an accelerated speed, while the accumulation of SV2 appears to decelerate. The intensity of immunoreactive material cannot directly be compared between the two antigens, since the settings for the confocal scanning equipment was different for SV2 and MVP100. Values represent means ^ S.E.M. from two independent experiments. Acc, accumulation.
the electron microscopic observations. Close to the crush the subcellular distribution of SV2 was different from that of MVP100 and revealed a much more intense and fine punctuate immunofluorescence. This could represent the crush-induced formation of very small vesicular organelles. Alternatively, it may be due to the pile-up of large numbers of small organelles in the short segment proximal and distal to the crush. This could preclude the identification of individual organelles. In unligated electromotor axons, SV2-containing organelles are accumulated proximally and distally of each individual node of Ranvier suggesting that this structure represents a natural site of organelle accumulation. 26,60,61 The accumulation of immunofluorescent material in individual axons near the crush observed in the present study is discontinuous. It presumably reflects the intense accumulation of organelles at individual nodes. The crush-induced accumulation of SV2 in the electromotor nerves corresponds to that previously observed with the cytofluorimetric scanning method in mammalian peripheral axons. Similar results were obtained also for the synaptic vesicle membrane proteins synaptophysin and synaptobrevin that reveal significant anterograde and retrograde
transport. 12,38 In contrast, proteins such as the synapsins, rabphilin 3A or the small GTP-binding protein rab3A that are loosely associated with the synaptic vesicle membrane reveal a fast anterograde but only a marginal retrograde component. 12,37,40 Dynamics of axonal transport of vaults may differ from those of vesicle proteins and mitochondria Vault particles can be transported in either direction but about half of the material transported to the synapse appears to be degraded there. Significant accumulation of vaults can be observed first 6–9 h after application of the crush, but traces were observed in the confocal microscope already 3–6 h after crush. Transport appeared initially considerably slower than that of the organelles carrying the synaptic vesicle protein SV2 (significant after 1 h) and also slower than that of mitochondria (significant after 3 h). However, the accumulation was faster than would be expected for material susceptible to slow axonal transport. 13 It was interesting to observe that the increase in accumulation proximal to a crush of vault protein appeared to accelerate with time after crush, while the increase in SV2 protein decelerated (Fig. 4). This behaviour of
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Fig. 6. Analysis of the antibodies by immunoblotting using homogenates of electric nerve. Equal amounts of protein (40 mg) were loaded per lane. 1, MVP100 antibody; 2, SV2 antibody; 3, antibody against F1-subunit of Fl-ATPase. KD, relative molecular masses of standard proteins.
Fig. 5. Comparison of the localization of MVP100 (A) and the F1-subunit of mitochondrial Fl-ATPase (B) 3 h after the crush operation. The site of the crush is indicated by arrows with the proximal side above and the distal side below. Labelling for MVP100 (M) and FlATPase (Fl) (both FITC) was performed on different sections derived from the same nerve. Whereas MVP100 reveals very little accumulation Fl-ATPase is significantly accumulated on either side of the crush. Scale bars 100 mm.
synaptic vesicle protein-immunoreactive material has been observed earlier in rat sciatic nerve; Bo¨o¨j and colleagues 6,7 observed that the accumulation of RASVA immunoreactivity (rabbit antisynaptic vesicle antibody, 6 the first antibody that recognized SV2 in cholinergic vesicles 51) and of 10H-immunoreactive material, 7 decelerated during periods later than 12 h after the crush. Both these antibody preparations recognize the proteoglycan in synaptic vesicle that is labeled by the SV2 antibody. This indicates that the soma production of synaptic vesicle proteins, as a reaction to the axonal trauma, reduce the synthesis of material involved in transmission. In contrast, an axotomized neuron increases the production of reparative material. Thus, the differences in initial accumulation rate in the Torpedo nerves demonstrated here during the first 24 h after crush need not necessarily indicate dramatic differences in normal rate of anterograde axonal transport, but may be due to differences in the normal levels of vault protein on the one hand, and SV2-containing organelles on the other. At later intervals, beyond the 48 h period, the situation is altered, in that the amount of vault protein in transport is increased while the SV2 production and
amount in transport is down-regulated. For this reason it is difficult to estimate the rate of anterograde transport of the vault protein. One may speculate, based on the observations here, that it is slower than that of the SV2-carrying organelles, but, at least during the late phase (48–72 h) as fast as, or possibly faster, than that of the large mitochondria. It is interesting to consider the mechanisms of fast axonal transport of different components in the axon. An in vitro association of sea urchin vaults with microtubules has recently been observed. 20 If vaults were linked to molecular motors for transport these may be different from motors associated with synaptic vesicle precursors or mitochondria. In extracts of axonal organelles from adult rat cauda equina KIFlA, a monomeric motor for anterograde axonal transport, is associated with organelles containing the synaptic vesicle proteins synaptotagmin, synaptophysin and rab3A, but not SV2. This suggests that synaptic vesicle proteins may be transported into the axon terminal by differing organellar carriers. 16,48 Mitochondria are transported considerably more slowly than vesicular organelles (up to 70 mm/ d). 16,58 Purified KIFlB can transport mitochondria along microtubules in vitro. 47 Cross-bridges have been observed between mitochondria and microtubules. 24,50 But filamentous actin has also been implicated in axonal transport of mitochondria in vivo. 45 Little is known concerning the mechanisms of RNA-particle transport. Messenger RNA anchoring to actin filaments (fibroblasts) 2,52 and also to microtubules (neurons) 3 has been demonstrated. Using the membrane-permeable nucleic acid stain SYTO 14 the translocation of endogenous RNA could be visualized in neurites of cultured nerve cells. RNA
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was found to be associated with granules. Anti-kinesin antisense-oligonucleotides inhibit the transport of myelin basic protein mRNA in processes of cultured oligodendrocytes. 10 In cultured neurons RNA granules were found to be transported in anterograde and also in a retrograde direction. The motility had characteristics of an actively transported organelle that might be transported along cytoskeletal tracks. 34 Our observations demonstrate the anterograde and retrograde axonal transport of ribonucleoprotein particles in situ. At present the functional role of vaults is unknown. The only functional implication supported by experimental evidence so far is a potential involvement of vaults in multi drug-resistance. It is well documented that the lung resistance-related protein (LRP), the human homologue of MVP100, correlates with a poor response to chemotherapy. Multi-drug-resistant cancer cells frequently overexpress the LRP protein and LRP over-expression has been found to predict a multi-drug-resistance phenotype in acute leukemia and ovarian carcinoma. 25,41,52 Furthermore, vault particles interact with estrogen receptor in MCF-7 breast cancer cells. The functional role of the association with nuclear receptors is not known but it could relate to the intracellular traffic. 1 Whatever its function, major vault protein may be modulated by protein
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kinases. MVP100 is phosphorylated in vivo by protein tyrosine kinase and it can be phosphorylated in vitro by protein kinase C and casein kinase II. 21,22 CONCLUSIONS
Our studies demonstrate that vault particles are transported in the cholinergic electromotor axons of the electric ray. Transport occurs both in an anterograde and retrograde direction and transport velocity appears slower than that of the synaptic vesicle membrane protein SV2. An up-regulation of the vault protein appears to take place late after crushing and a simultaneous down-regulation of the SV2 protein. High resolution immunofluorescence reveals a granular distribution of the protein distinct from that of SV2. At present the functional role of vaults is unknown. Studies of the transport of this organelle could reveal insights into the transport mechanisms of ribonucleoprotein particles in general whether in neurons or in other cells.
Acknowledgements—This work was supported by an EUproject No. BMH4-CT 96-1586, by the Swedish MRC (2207) (A. D.), by the Swedish Medical Society (J.Y.L) and by a grant from the Deutsche Forschungsgemeinschaft to W.V. We are grateful to Andrea Winter for expert technical support.
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