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Neurosecretory Vesicles Can Be Hybrids of Synaptic Vesicles

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Neurosecretory Vesicles Can Be Hybrids of Synaptic Vesicles Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7342-7346, August 1995 Neurobiology Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules (small dense core vesicles/sympathetic neurons) RUDOLF BAUERFEIND, RuTH JELINEK, ANDREA HELLWIG, AND WIELAND B. HUrTNER* Institute for Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany Communicated by Philip Siekevitz, The Rockefeller University, New York, NY, March 27, 1995 ABSTRACT We have investigated the relationship of the This implies that only the regulated, but not the constitutive, so-called small dense core vesicle (SDCV), the major cate- pathway of protein secretion contributes to the biogenesis of cholamine-containing neurosecretory vesicle of sympathetic SDCVs. This concept has been extended to the biogenesis of neurons, to synaptic vesicles containing classic neurotrans- synaptic vesicles in general (4, 9, 10) but has not provided a mitters and secretory granules containing neuropeptides. satisfactory explanation for the very low abundance, in secre- SDCVs contain membrane proteins characteristic of synaptic tory granules, of certain membrane proteins that are major vesicles such as synaptophysin and synaptoporin. However, components of classic synaptic vesicles. The other concept, SDCVs also contain membrane proteins characteristic of which originated from the demonstration that the classic certain secretory granules like the vesicular monoamine synaptic vesicle membrane is distinct from that ofthe secretory transporter and the membrane-bound form of dopamine granule, has viewed the SDCVs, which lack secretory proteins f3-hydroxylase. In neurites of sympathetic neurons, synapto- and morphologically resemble classic synaptic vesicles, as physin and dopamine 8-hydroxylase are found in distinct synaptic vesicles that simply contain vesicular amine transport- vesicles, consistent with their transport from the trans-Golgi ers instead of uptake systems for classic neurotransmitters (1, network to the site of SDCV formation in constitutive secre- 8). Since only the constitutive, but not the regulated, pathway tory vesicles and secretory granules, respectively. Hence, of protein secretion is thought to contribute to the biogenesis SDCVs constitute a distinct type ofneurosecretory vesicle that of classic synaptic vesicles from early endosomes (2), this is a hybrid of the synaptic vesicle and the secretory granule concept has not provided a satisfactory explanation for the membranes and that originates from the contribution of both presence, in SDCVs, of certain secretory granule membrane the constitutive and the regulated pathway of protein secre- proteins such as cytochrome b561 (11). Herein we show that tion. SDCVs are hybrids of the classic synaptic vesicle and the secretory granule membranes. Two distinct types of neurosecretory vesicles are thought to mediate the regulated release of neurotransmitters and neu- MATERIALS AND METHODS ropeptides (1). One type is the synaptic vesicles that store and release classic neurotransmitters such as glutamate, acetylcho- Electron Microscopy. Rat vas deferens was fixed with (i) 1% line, glycine, and y-aminobutyric acid but do not contain glutaraldehyde in 100 mM sodium cacodylate (pH 7.2) (caco- secretory proteins. We shall refer to these as classic synaptic dylate buffer) overnight (Fig. 1A); (ii) 2% (wt/vol) paraform- vesicles because of the data described below and the concept aldehyde in 200 mM Hepes-NaOH (pH 7.4) for 5 hr, followed derived therefrom. Classic synaptic vesicles are thought to by a postfixation with 8% paraformaldehyde in 200 mM originate from early endosomes after delivery of their mem- Hepes-NaOH (pH 7.4) for 16 hr (Fig. 1B); (iii) dichromate brane proteins via the constitutive pathway of protein secre- fixative [3% (wt/vol) potassium dichromate/4.8% paraform- tion (for review, see ref. 2). The other type is the secretory aldehyde/0.1 M sodium acetate, pH 5.8] for 1 hr (Fig. 1 C and granules (in neurons also called large dense core vesicles) that D); or (iv) 3% (wt/vol) potassium permanganate in 150 mM store and release neuropeptides, originate from the trans- sodium phosphate (pH 7.4) for 1 hr (Fig. 1E). For Epon Golgi network, and constitute the regulated pathway ofprotein embedding, fixed samples were washed in cacodylate buffer secretion (3). In certain neurons and endocrine cells, secretory and postfixed in 1% OS04 plus 1.5% (wt/vol) magnesium granules also store and release biogenic amines such as ferricyanide for 1 hr. After washes in cacodylate buffer and catecholamines (4). water, samples were incubated for 30 min in 1.5% (wt/vol) An enigma has been the nature of the so-called small dense magnesium uranyl acetate in water. Samples were then dehy- core vesicles (SDCVs) that are found in certain nerve cells, drated in ethanol and embedded in Epon. Ultrathin sections such as catecholaminergic sympathetic neurons, and store and were contrasted with lead citrate and uranyl acetate. Ultrathin release biogenic amines (e.g., catecholamines) (for review, see cryosections were prepared from samples fixed with paraform- ref. 4). SDCVs do not contain secretory proteins (5) and show aldehyde or dichromate and immunogold-labeled as described an electron dense core only after certain chemical fixations (12, 13), by using the mouse monoclonal antibody against (for example, permanganate or dichromate) that prevent the synaptophysin (14) (Boehringer Mannheim) (1 jig/ml), a loss of catecholamines (6, 7). secondary rabbit anti-mouse IgG antibody (Cappel) (50 ,ug/ The relation of SDCVs to classic synaptic vesicles and ml), and protein A-colloidal gold (9 nm), prepared as de- secretory granules has been controversial (1, 4, 8, 9). One scribed by Slot and Geuze (15), at an OD520 of 0.098. concept has been that SDCVs, because of their ability to store Subcellular Fractionation. A post-10,000 x g high-speed and take up catecholamines like certain secretory granules membrane pellet from vas deferens, prepared from 12-16 rats such as chromaffin granules, are simply formed from the as described (5, 16), was resuspended in 1 ml of in vitro buffer membrane of secretory granules after their exocytosis (4, 9). (250 mM sucrose/10 mM KCl/10 mM MgCl2/10 mM Hepes-KOH, pH 7.2) and centrifuged for 5 min at 14,000 x g,V The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: SDCV, small dense core vesicle. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7342 Downloaded by guest on September 26, 2021 Neurobiology: Bauerfeind et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7343 FIG. 1. Presence of synaptophysin on SDCVs by immunoelectron microscopy. Rat vas deferens was fixed with aldehyde (A and B), dichromate (C and D), or permanganate (E), followed by the prepara- i tion of Epon sections (A, C, and E) or cryosections with (B) or without (D) immunogold labeling for synaptophysin. The sections show axonal varicosities innervating smooth muscle. Note the presence of dense cores in SDCVs (arrowheads) after fixation with dichromate and permanganate, but not alde- hyde, whereas the dense cores in secretory granules (arrows) are observed irrespective of the type of fixation. In cryosections prepared after aldehyde fixation, gold particles indicative of synaptophysin immunoreactivity are associatedwith SDCVs but not secretory granules (B). (Inset) Immunogold-labeled vesicular profiles (50-60 nm in diameter) at higher magnifications. No synaptophysin immunoreactivity could be detected in cryosections prepared after dichromate fixation (data not shown). (Bars: A-E, 200 nm.) The pellet was resuspended in 1 ml of in vitro buffer and lected from the bottom of the gradient. Aliquots of each centrifuged for 5 min at 14,000 X gay, and the supernatants fraction were (i) analyzed for [3H]noradrenaline by liquid from the two 14,000 x g centrifugations were pooled. The scintillation counting or (ii) subjected to SDS/PAGE and pooled supernatant (1.0-1.6 ml) was supplemented with (final immunoblot analysis using the following antibodies: the rabbit concentrations) 5 mM creatine phosphate (from a 0.8 M anti-hSgIIi_2o peptide antiserum against secretogranin II (18) stock), creatine phosphokinase at 40 units/ml (from a 3200 (1:200 dilution); a rabbit antiserum (gift of T. Flatmark, units/ml stock), and 5 mM ATP (from a 100 mM stock). In Bergen, Norway) against a synthetic 21-mer peptide, corre- some experiments, the supplemented supernatant was split sponding to the cytoplasmically located portion [Gln2-Ala22 into two aliquots and 3 ,uM reserpine (from a 200 ,uM stock in (19)] of the uncleaved signal peptide (20) of rat dopamine ethanol) or the corresponding amount of ethanol only was 3-hydroxylase, specific for the membrane-bound, but not the added. The supplemented supernatant was then preincubated soluble, form of dopamine f3-hydroxylase (1:500 dilution); the for 5 min at 32°C, followed by the addition of 1-[7,8- mouse monoclonal antibody against synaptophysin (14) (0.2 3H]noradrenaline at 7.2 ,uCi/ml (180 nM) (40 Ci/mmol; 1 Ci ,g/ml); a rabbit antiserum against synaptoporin (21) (1:500 = 37 GBq; Amersham TRK.584) and a 30-min incubation. At dilution); and mouse monoclonal antibody 41.1 against syn- the end of the incubation, as shown in Fig. 2, samples were aptotagmin (22) (1 ,ug/ml). Rabbit primary antibodies were either subjected to immunoadsorption or directly applied to a detected by incubation with 125I-labeled protein A (0.12 ,uCil linear 0.6-1.6 M sucrose gradient (Beckman SW40 rotor) and ml; NEN). Monoclonal primary antibodies were detected by centrifuged at 25,000 rpm for 5 hr, with the brake off. incubation with rabbit anti-mouse IgG (Cappel) at 1 ,ug/ml Immunoadsorption was performed as described (17) by using and 1251-labeled protein A (0.12 ACi/ml; NEN). Immunore- immunobeads (Bio-Rad) precoated without or with the mouse activity was quantitated by using a Fuji BioImaging analyzer.
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