| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MS 9564 Received 27 April 1999; accepted after revision 11 June 1999.
During the past few decades, unexpected similarities have emerged between neurones and endocrine cells, especially in relation to secretion and secretory mechanisms. These two cell types are, in fact, recognized as having two parallel pathways of regulated secretion, the first sustained by synaptic-like vesicles (SLV), which predominate in most neurones where they are used for the release of classical, low-molecular weight neurotransmitters, and the second sustained by dense granules (DG) employed for the release of mixtures of compounds such as amines, proteins, peptides and nucleotides. The nature of the secretory products can also be identical in the two types of cell. For example, acetylcholine and GABA, considered until recently to be typical neurotransmitters, are also released by chromaffin and pancreatic 
cells, respectively. The proteins of the chromogranin/secretogranin family, discovered in chromaffin and pituitary cells, are common to various types of neurone. Most importantly, the same integral membrane and soluble proteins known to govern the process of regulated exocytosis have been found to operate in both cell systems, including the SNAREs, the G proteins rab3A and B and the phosphoprotein rbSec1/munc18 (Söllner et al. 1993; Jacobsson et al. 1994). Thus, in spite of their well-known structural and functional differences, the definition of 'neurosecretory' appears justified for both types of cell based on the common molecular properties of their secretory programme. In the present review, the ability of the cell to express this specific programme will be referred to as neurosecretion competence.
Until recently, studies of neurosecretion competence were conducted primarily from the perspective of cellular differentiation. In various types of endocrine cells, the first traces of the specific hormone(s), accompanied by characteristic aspects of the phenotype, were observed to appear at distinct developmental stages and recognized to depend on the activity of multiple transcription factors expressed co-ordinately or in sequence during the course of differentiation (Groves et al. 1995; Anderson, 1997; Sosa-Pineda et al. 1997; St-Onge et al. 1997). However, whether the appearance of full neurosecretion competence depends exclusively on transcriptional events remains open to question. In fact (i) examples of divergence from the expected correlation between factor expression and phenotype acquisition have been reported (Schoenherr & Anderson, 1995; Scholl et al. 1996; Atouf et al. 1997), (ii) the extracellular factors and transmembrane signalling events triggering the appearance of neurosecretion remain largely unidentified, and (iii) in some cell systems the appearance of neurosecretion has been described as a complex, multistep process (Groves et al. 1995; Tiveron et al. 1996; Morin et al. 1997; Yamaoka & Itakura, 1999). For example, a protein called V1, originally identified in rat cerebellum (Taoka et al. 1994) and later localized in the cytoplasm of chromaffin cells, has recently been shown to markedly affect the phenotype of PC12 cells, increasing the expression of three enzymes participating in catecholamine biosynthesis. This action of V1 apparently takes place by the activation of an unknown mechanism independent of transcription (Yamakuni et al. 1998). In developing neurones, on the other hand, initial quantal neurotransmitter release has been shown to take place along the surface of the whole axon (Verderio et al. 1999; Zakharenko et al. 1999). As soon as synapses are established, release becomes confined to the presynaptic compartment and this is paralleled by molecular and kinetic changes (Matteoli et al. 1992; Verderio et al. 1995, 1999; Coco et al. 1998).
The overall picture that emerges from the data summarized so far confirms the importance of classical developmental mechanisms in the establishment of neurosecretion competence, but suggests the existence of additional processes that may also play a major role. Consistent with this perspective are the results obtained in our laboratory for a clone of PC12 cells which, while maintaining a variety of properties typical of its parental population (a widely employed neurosecretory cell line originating from a rat phaeochromocytoma), was found to be totally incompetent for neurosecretion, as revealed by extensive molecular, functional and morphological studies. Interestingly, the defect in the PC12 clone appears to affect only the proteins involved in neurosecretory activity, which fail to be expressed regardless of whether they are secretory, soluble or transmembrane, and regardless of their expected predominant localization in the secretory organelles (the SLVs and DGs) in the plasmalemma or cytosol. For the proteins investigated so far, the defect existing in the clone does not appear to be due to a block in transcription, but to an as yet uncharacterized post-transcriptional destabilization event, apparently taking place because of the lack of a specific protective factor. These results demonstrate the existence of a new, previously unsuspected control mechanism for neurosecretion competence which, at variance with the others, might be switched on (and possibly also off) at any time in the life of the cell.
Isolation and characterization of the neurosecretion-incompetent clone of PC12 cells
The isolation of our first neurosecretion-incompetent clone was the serendipitous result of a study aimed at obtaining the permanent overexpression of a constitutively activated form of 
s, a trimeric G protein subunit. Among the neomycin-resistant clones isolated after calcium phosphate transfection, one (designated as clone 27) attracted attention because of its inability to take up radioactive catecholamines and release them when exposed to appropriate stimuli. All the other clones (over 40) isolated during the transfection studies were able to do this. Subsequently, electron microscopy of clone 27 cells revealed a normal structure for the nucleus and many cytoplasmic organelles, such as the endoplasmic reticulum (ER) and the Golgi complex. However, the secretory organelles where catecholamines accumulate, i.e. the DGs (
0·1 µm in diameter), which in wild-type PC12 cells are primarily attached to or in the immediate proximity of the plasmalemma, were not visible in the defective clone. In addition, the vesicular and tubular superficial membrane profiles were also absent (Clementi et al. 1992). Since in other cell systems, profiles of this type have been reported to fuse with the plasmalemma during chemical fixation, the question arose as to whether such characteristics in fixed clone 27 PC12 (PC12-27) cells were an artifact or a real defect existing in the living cells. The ultrastructural study of wild-type and PC12-27 cells was therefore repeated using cell preparations processed for electron microscopy by quick freezing-freeze drying (Grohovaz et al. 1996), a physical treatment that prevents fixation artifacts as it induces the immediate block of all cellular activities, including vesicle fusion and fission. As can be seen in Fig. 1A and B, after quick freezing, the sub-plasmalemmal zone of the PC12-27 clone differed from that of the wild-type cells by the absence of DGs and SLV profiles. We concluded, therefore, that this difference was a real property of the PC12-27 clone and decided to characterize it further biochemically.
 |
View larger version [in this window] [in a new window] |
|
|
Electron microscopy of defective PC12-27 (A) and wild-type PC12 (B) cells processed by quick freezing-freeze drying (Grohovaz et al. 1996). Note that in A the cytoplasmic layer adjacent to the plasmalemma is devoid of vesicular profiles, whereas in B the same layer contains many typical dense granules (DGs), some of which are tethered to the plasmalemma, as well as some other vesicular profiles. Scale bar, 0·25 µm. | ||
The expression of various proteins in wild-type and PC12-27 cells as revealed by specific Western blotting is summarized in Table 1. For a large number of markers typical of various intracellular structures such as the ER (BiP, calreticulin, calnexin), the Golgi complex (mannosidase II, rab6) and the cytoskeleton (neurofilament H subunit; N-kinesin), the cells of the defective clone showed normal or near normal levels. Also present in PC12-27 cells were synapsin I, the protein that binds SLVs to the cytoskeleton, and tyrosine hydroxylase, the limiting enzyme in the synthesis of catecholamines. There was also another group of neurosecretory cell markers, which included the N-type Ca2+ channel (Passafaro et al. 1996), the -latrotoxin receptor and the XLs G protein (see Corradi et al. 1996). When exposed to nerve growth factor (NGF), the dendrite growth response of PC12-27 cells was limited. However, after transfection with the high-affinity receptor trkA, this response became rapid and extensive (Leoni et al. 1999). The modest effects of NGF on untransfected PC12-27 cells therefore appears not to be due to the insensitivity of the cells to the neurotrophin, but to their low expression of receptors.
Table 1. Expression of various markers in PC12 clones
The situation of regulated neurosecretion in the PC12-27 clone is, however, in marked contrast to the general neurosecretory phenotype. In fact, none of the proteins involved in the process that were investigated could be detected in PC12-27 cells. These proteins included those associated with DGs and SLVs, such as the secretory proteins chromogranin B and secretogranin II, the catecholamine-synthesizing enzyme dopamine
In conclusion, biochemical studies showed that the lack of DGs visible by microscopy in PC12-27 cells, and the lack of SLVs deducible from the vesicular profiles at the cell surface, were not due to a defect in the assembly of the specific molecular components but rather to their absence; this was accompanied by the absence of the non-granule proteins necessary for the regulated exocytosis of secretory organelles. An additional indication along the same lines was obtained when cells of the defective clone were transfected with cDNA for the human isoform of one of the missing secretory proteins, chromogranin B, which was distinguishable from the rat isoform by specific antibodies. Whereas in control PC12 cells, the protein was regularly addressed to the DGs, in PC12-27 cells the transfected chromogranin B accumulated within the Golgi complex, from which it was rapidly released to the extracellular space, but by constitutive and not regulated secretion (Corradi et al. 1996).
In addition to the protein characterization reported so far, the PC12-27 clone was analysed for the expression of a few genes considered to be of importance in neurosecretory differentiation, including the NRSF/REST/XBR product (Schoenherr & Anderson, 1995; Chong et al. 1995), a zinc finger transcription factor that has been suggested to be a negative regulator of the neuronal phenotype. In contrast to control PC12 cells, which lack the NRSF transcript, PC12-27 cells exhibited significant expression. However, when a mouse myc-tagged version of the NRSF cDNA (a generous gift from A. Paquette and D. J. Anderson) was transfected into control PC12 cells, it failed to induce the disappearance of the neurosecretory proteins. Thus, the phenotype typical of PC12-27 cells cannot simply be the consequence of NRSF expression. Additional transcription factors have been investigated by RT-PCR. In the preliminary results obtained so far, no significant differences between PC12-27 cells and controls could be observed for Phox2, which is involved in the terminal differentiation of adrenergic/noradrenergic neurons (Zellmer et al. 1995; Morin et al. 1997), whereas levels of Isl1, a widely expressed factor first recognized as participating in the differentiation of pancreatic islets (Karlsson et al. 1990), were found to be very low in the defective clone (M. L. Malosio & J. Meldolesi, unpublished observations).
In the natural history of the rat PC12 line (and possibly also of other neurosecretory cell types), the PC12-27 clone cells seem to be by no means unique. In fact, additional clones lacking DGs and other neurosecretion components have been identified over the past few years (Bitler et al. 1986; Pance et al. 1995; Leoni et al. 1999). The loss of neurosecretion competence is therefore not a rare event, and such defective clones present interesting tools that can be employed to answer questions, so far not even envisaged, concerning the expression and regulation of this differentiated cell function.
Molecular mechanism(s) of the PC12-27 cell defect: transcriptional or post-transcriptional?
The first hypothesis raised to explain the neurosecretion incompetence of PC12-27 cells (and possibly also of other, similar, PC12 clones) was that of a transcriptional defect. However, the genes of the various missing proteins are located on different chromosomes and the possibility that each of them is independently affected therefore appears unlikely. In contrast, the existence of a regulatory mechanism common to all the genes involved was conceivable. Whether such a mechanism was inductive or repressive, and whether it was selectively lost or acquired by the defective clone, was not clear at the present time. When the mRNAs of five proteins involved in neurosecretion (chromogranin B, secretogranin II, synaptophysin, VAMP2 and synaptotagmin) were analysed by Northern blotting, their levels in PC12-27 cells were found to be dramatically lower (1-10 %) than in neurosecretion-competent clones. An extension of the studies on secretogranin II by the use of RNase protection (Borgonovo et al. 1998) confirmed the Northern blot data and, in addition, led us to exclude transcriptional attenuation as the mechanism governing the observed downregulation of expression. In fact, the accumulation of unspliced or partially spliced mRNA fragments, as well as transcription from the non-coding strand of the gene, were never observed (Fig. 2). Moreover, experiments carried out with cells exposed to actinomycin D for periods of between 30 min and 24 h excluded a difference in mature mRNA turnover between mutant and control clones, while cycloheximide treatments (up to 5 h) excluded the possibility that there was a breakdown of secretion-specific mRNAs under the control of rapidly turning over proteins. Finally, Northern blots for secretogranin II and VAMP2, carried out in parallel on nuclear and cytoplasmic RNA preparations of control and PC12-27 cells, revealed identical ratios between the transcripts in both compartments, despite the huge differences in absolute values. This result excludes impaired nucleocytoplasmic mRNA transport as the defect in the PC12-27 clone.
RNase protection (left panel) shows traces of the processed (lower arrow) as well as the unprocessed (upper arrow) secretogranin II (SgII) mRNAs. Note the large quantitative difference between the two clones (control clone, lanes 3 and 4; clone PC12-27, lanes 5 and 6), without the appearance of fragments representing partially processed forms of the transcripts in either clone. No signal was observed with yeast RNA (lanes 1 and 7) or the sense SgII probe (lanes 9 and 10), which were used as specificity controls. Lanes 2 and 8 are SgII riboprobes without RNase treatment. Right panel: run-on results obtained from the nuclei of PC12-27 cells, expressed as a percentage of those obtained from the nuclei of the wild-type clone 15 (O.D., optical density). Of the three mRNAs shown, those of VAMP2 and chromogranin B (CgB) were only investigated in the full-length form, whereas that of SgII was also investigated with the 5' and 3' portions of the specific cDNAs. Reproduced with permission from Borgonovo et al. 1998.
Although all the data reported so far appeared to be compatible with a transcriptional defect, some doubt persisted based on at least two additional pieces of evidence obtained in PC12-27 cells. The first concerned the promoter activity of secretory protein genes (Fig. 3). When the full-length, 6 kb 5' flanking region of the mouse chromogranin B gene was cloned in front of an appropriate reporter (chloramphenicol acetyltransferase, CAT), considerable expression (> 50 % of control cells) was observed in transfected PC12-27 cells. Surprisingly, some expression (
Aa, schematic representation of mouse chromogranin B (mCgB) and secretogranin II (mSgII) promoter constructs, fused with CAT, used in transient transfections of wild-type PC12, PC12-27 and HeLa cells. Ab, relative activity of mCgB and mSgII promoters in PC12-27 and HeLa cells expressed as a fraction of their activity in wild-type PC12 cells. Ac, Northern blot analysis of secretogranin II (SgII) and chromogranin B (CgB) in different cell lines. Total RNA (10 µg) from NIH3T3, HeLa, PC12-27 and wild-type PC12 (PC12-15 and PC12) cells was hybridized with rat SgII and rat CgB cDNA probes. GAPDH expression was used as the internal standard. B, mapping of SgII and CgB gene transcription start sites in wild-type (PC12-15 and PC12) and PC12-27 clones. Ba, increasing amounts of RNA from wild-type clones and poly(A) RNA (2 µg) from PC12-27 cells were used in primer extension analysis. The transcription start sites are indicated by asterisks. The sequencing reaction of a 400 bp construct of the mSgII promoter was used as a molecular weight marker (C, A, T, G). Bb, genomic sequences of the rat SgII and rat CgB genes surrounding the transcription start site. The oligonucleotides used for the primer extension, spanning regions +103 to +75 in the rat CgB and +114 to +84 in the rat SgII gene, respectively, are underlined. Asterisks mark the positions of transcription start sites corresponding to the bands shown in Ba. Arrowheads indicate previously reported transcription start sites (Pohl et al. 1990; Jones et al. 1996).
Based on the results summarized so far, the hypothesis that we favour is that in PC12-27 cells, transcription of neurosecretion-specific genes may take place at a normal rate. However, this is then followed by extensive degradation of the primary transcripts which, in contrast, does not take place in the controls. Such a degradation is hypothesized to occur very rapidly, i.e. immediately after or even during synthesis, thus explaining why no differences were discerned in the standard, long-term turnover tests. Processes such as those hypothesized here in PC12 cells have been described previously in the brain, where RNA-binding proteins regulating the initial steps of gene product degradation are known to play an important role (Buckanovich & Darnell, 1997; Sakakibara & Okano, 1997).
The genetic control of the neurosecretory phenotype
The nature of the regulation of expression of neurosecretion competence was investigated by two approaches, both involving the genetic manipulation of PC12-27 cells: fusion with various other cell types and transfection of an expression library prepared from wild-type PC12 cells. Initially, the two populations to be fused were labelled with different vital dyes, and the chimerae obtained isolated by fluorescence-activated cell sorting (FACS). Results obtained with two PC12 clones, the defective clone 27 and the fully neurosecretory clone 15, are shown in Fig. 4. The fused cells were not only positive for the two markers investigated, chromogranin B and synaptophysin, but also exhibited typical DGs when analysed by electron microscopy. Moreover, the hybrids were remarkably stable, so that subclones could be isolated and characterized by their phenotype as well as their depolarization-dependent secretory activity, which was indistinguishable from wild-type PC12 (Fig. 4I). Complementation of the PC12-27 defect required fusion with a wild-type cell counterpart, and was not the simple result of the cell fusion per se, because neurosecretion did not reappear after either fusion of two PC12-27 populations or fusion between PC12-27 and another neurosecretion-incompetent clone, Trk (Fig. 4C; Leoni et al. 1999). The conclusion that can be drawn from this experiment is that the defects in PC12-27 and Trk clones may be identical or lie within the same pathway. In contrast, the results observed from hybridization studies with wild-type PC12 cells strongly indicate the existence in the wild-type of a positive neurosecretion competence regulation.
Rat chromogranin B immunolabelling (A and B) and phase-contrast microscopy (A' and B') of wild-type PC12 (A) and PC12-27 (B) cells. C-E, rat chromogranin B immunostaining of homotypic 27-27 fusion hybrids (C) and 27-wild-type hybrids, analysed 48 h (D) and 2 weeks (E) after fusion, respectively. F, hybrids from the same preparation as shown in E, but stained for synaptophysin. The scale bar in F (5 µm) also applies to A-E. The ultrastructural analysis of chemically fixed fused cells shows an abundance of DGs (G and H; scale bars, 0·1 µm). I, regulated secretion of rat chromogranin B is shown for two wild-type (PC12-15 and PC12-251) as well as for one hybrid (PC12-27/15) clone depolarized with high K+ in the presence or absence of extracellular Ca2+. In these experiments, the cells were first pulse-labelled (15 min) with [35S]sulfate, then chased (60 min), washed and exposed to the indicated treatments: -K+ = 3·6 mM; +K+ = 53·5 mM; -Ca2+ = ~0 mM; +Ca2+ = 2 mM. Radioactive rat chromogranin B released into the medium was immunoprecipitated and revealed by fluorography after SDS-PAGE. Reproduced with permission from Borgonovo et al. 1998.
An important point that could not be established with inter-PC12 clone fusion experiments was whether the neurosecretion of the hybrids was simply due to the contribution of wild-type cells whose competence was unaffected by the fusion, or to revived competence in the defective cells. In fact, there was no way of distinguishing the products coded by the genes of the two PC12 fusion partners. To answer this question, fusions were carried out between rat PC12-27 cells and the cells of two human lines, one at least potentially neurosecretive - the neuroblastoma SH-SY5Y, the other certainly incompetent - the epithelial line HeLa. In these experiments, the non-fused human cells were eliminated by exposure to G418, to which only PC12-27 cells were resistant, and the hybrids were identified by staining with human-specific antibodies, such as one against
The fusion experiments described so far offer a general outlook on neurosecretion competence, but without any information as to the nature of the competence factor(s) involved. At present this latter problem is being investigated via a phenotypic cloning approach, using as the recipient a PC12-27 subclone stably pretransfected with chromogranin B coupled to the fluorescent protein EGFP. In the defective cells, the protein is addressed to the constitutive secretion pathway, with accumulation in the Golgi area and full discharge to the extracellular space within 2 h of synthesis by constitutive secretion. In contrast, in wild-type PC12 cells the protein is sorted to DGs which, in the absence of any secretion stimulation, persist much longer within the cell. Fluorescence analysis of resting chromogranin B-EGFP PC12-27 cells transfected first with appropriate dilutions of a wild-type library and then treated for 2-4 h with the protein synthesis blocker cycloheximide can therefore provide a direct indication as to whether neurosecretion competence has been reacquired. The experiments carried out so far demonstrate unequivocally that the fluorescence does indeed persist in a significant fraction of the library-transfected cells, and that in these cells, the distribution of the secretory protein is not concentrated in the Golgi complex but appears granular and distributed through the whole cytoplasm, indistinguishable from that of wild-type cells expressing chromogranin B-EGFP (M. L. Malosio, R. Solari & J. Meldolesi, unpublished results). The search for the gene(s) responsible for this effect, and thus for the complementation of the PC12-27 defect, is at present under way.
The PC12-27 clone as a tool for cell biological studies
Because of their peculiar properties of being 'neurosecretory without neurosecretion', PC12-27 cells offer distinct advantages for the study of specific biological issues which, although of general interest, have remained poorly understood. Here we will specifically review two such issues: (1) the localization of t-SNAREs and (2) the multiplicity of regulated secretion pathways.
(1) At present, the first issue is widely recognized to be of major importance. On the one hand, all types of membrane fusion taking place not only at the surface, but also within the cell, are thought to require SNAREs. On the other hand, when tested in vitro, various isoforms of SNARE exhibit only low levels of specificity with respect to others (Yang et al. 1999). Therefore, the fusion specificity attained within the cell appears to depend, at least in part, on the specific localization of the various SNAREs. A direct indication along these lines came from our studies of syntaxin 1A (a plasmalemma t-SNARE) expressed in PC12-27 cells (Rowe et al. 1999). These results were of especial relevance, because in previous studies a marked attenuation of the discharge of a constitutively released protein, observed in a fibroblast cell line transfected with syntaxin 1A, had been interpreted as an indication of a general role of this SNARE in the establishment of regulated secretion (Bittner et al. 1996). The situation that we have identified in PC12-27 cells is profoundly different from that previously hypothesized. After single transfection, syntaxin 1A failed to reach the plasmalemma and remained trapped in the Golgi area, where it induced a progressive disruption of the cisternae, accompanied by their slow but progressive intermixing with the ER, most probably because of a direct functional interference with the Golgi SNAREs such as syntaxins 5 and 6. Due to this structural disassembly, not only secretory but also membrane proteins were delayed in their intracellular trafficking. Interestingly, however, co-transfection of rbSec1/munc18, the phosphoprotein that regulates syntaxin 1A function in regulated exocytosis, was able to prevent the trapping of the t-SNARE and re-establish its path to the plasmalemma. We conclude, therefore, that the existence of a neurosecretory programme in competent cells, based on the co-ordinated expression of a large panel of proteins, is necessary not only for their functional collaboration in the regulated neurosecretion process, but also to ensure that some of them reach their specific intracellular location (Rowe et al. 1999).
(2) The second issue concerns a new type of regulated exocytosis that had already been reported by others in neurosecretion-incompetent cells such as fibroblasts (Coorssen et al. 1996; Ninomiya et al. 1996). The mechanisms of this exocytosis, however, remained obscure. Patch-clamp studies conducted in the laboratory of Haruo Kasai demonstrated that in PC12-27 cells also, the delivery of a strong stimulation (intracellular release of Ca2+ induced by photolysis of a caged Ca2+ compound) induced large capacitance (= cell surface) increases, but with kinetics and Ca2+ dependence different from those observed for DGs and SLVs in the parental PC12 cells. Like most neurosecretory exocytosis, the secretory response in wild-type PC12 cells was sensitive to clostridial toxins, whereas that of PC12-27 was found to be totally insensitive. Thus, the PC12-27 clone appears to be a useful model for the identification of a new exocytotic system sustained by vesicles that are not pre-tethered to the plasmalemma (see 'Isolation and characterization of the neurosecretion-incompetent clone of PC12 cells', above) as those of classical neurosecretion are (Kasai et al. 1999). In the defective clone, expression of these non-conventional exocytotic vesicles appears high, and thus favourable for investigation. These vesicles may well, in fact, be ubiquitous among all cells Indeed, evidence for a toxin-insensitive exocytotic system has been reported even in a typical neurosecretory cell type, the bovine chromaffin cell (Xu et al. 1998). The isolation of these new exocytotic vesicles from PC12-27 cells and the understanding of their function represent a major aim of our laboratory.
Conclusion
Neurosecretion competence, a major property of neurones and endocrine cells, is still poorly understood with respect to its mechanisms and regulation. So far, most of the studies in this field have been conducted during development, and have focused primarily on the appearance in discrete cell populations of the mRNAs specific for particular secretion products. Whether the latter were appropriately transported within the cell, and whether the machinery was available for their regulated discharge, was often not even considered. Only in the case of cultured neurones was the evolution of regulated exocytosis and its dependence on the establishment of synapses investigated, but even in this case, the molecular mechanisms remained undefined.
Our study of PC12-27 cells has demonstrated the existence of a new, probably post-transcriptional programme of control of neurosecretion competence functioning under specific genetic control. An unexpected property of this programme is that it switches on, and possibly also off, as a whole, with the concomitant appearance (and disappearance) of all the proteins participating in the classical regulated secretion of DVs and SLVs. Whether this property of the programme becomes operative during the normal life of neurosecretory cells, or whether it primarily constitutes an emergency process to be activated only under extreme conditions, is not clear at present. Whatever its physiological significance, the programme certainly exhibits highly interesting biological properties which, in addition, offer opportunities for biotechnological intervention. Exciting developments are therefore expected in the field of neurosecretion over the next few years.
We thank Drs D. J. Anderson and A. J. Paquette (Caltech, Pasadena, CA, USA), W. Huttner (University of Heidelberg, Germany) and P. De Camilli (Yale University, New Haven, CT, USA) for the gift of cDNAs and antibodies. The contributions of Dr R. Solari (Stevenage, UK) with the phenotype cloning, P. Podini (DIBIT, Milano, Italy) with the microscopy studies, Dr E. Clementi with the original isolation of the PC12-27 clone, Drs C. Leoni and F. Valtorta for the use of the PC12-Trk clone and Dr D. Dunlap for suggestions are gratefully acknowledged. The original studies reported were supported by grants from the European Community (Biotechnology ERB B104 CT960058 to J. M. and TMR ERB4061PL95-0350 to P. R.), the Telethon Foundation (Fellowship 199/bi to M.L.M.), the Human Frontier Science Program (RG 520/95) and the Armenise Harvard Foundation.
Corresponding author
J. Meldolesi: DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy.
Email: meldolesi.jacopo{at}hsr.it
This article has been cited by other articles:
Wild-type PC12
PC12-27
ER markers
BiP 1
+
+
Calreticulin 1
+
+
Calnexin 1
+
+
Golgi complex markers
Mannosidase II 2
+
+
COP 2+
+
rab6 2
+
+
Neurosecretory markers
Cytoskeleton
Neurofilament H subunit 2
+
+
N-kinesin2
+
+
Synapsin I 1,2
+
+
Signalling
-Latrotoxin receptor 2+
+
N-type Ca2+ channel 2
+
+
XL
s G protein 2+
+
Tyrosine hydroxylase 2
+
+
Dense granules and synaptic-like vesicles
Dopamine
-hydroxylase 2+
-
vAChT 2
+
-
Chromogranin B 1
+
-
Secretogranin II 1
+
-
Synaptophysin 1
+
-
VAMP2 2
+
-
Synaptotagmin I 2
+
-
t-SNAREs
Syntaxin 1A2
+
-
SNAP25 2
+
-
SNARE regulators
rbSec1/munc18 3
+
-
rab3A 2
+
-
Membrane recycling
AP180 4
+
-
AP2 4
+
+
Dynamin4
+
±
Synaptojanin 4
+
+
Amphyphysin 4
+
+
-hydroxylase and the vesicular acetylcholine transporter (vAChT). It also included proteins directly involved in the exocytic process, synaptotagmin I, synaptophysin and the v-SNARE VAMP2, together with the t-SNAREs of the plasmalemma, syntaxin 1A and SNAP25, and the soluble proteins that regulate the functioning of the SNARE complex, rab3A and rbSec1/munc18 (Corradi et al. 1996; Rowe et al. 1999). Of the proteins participating in DG and SLV membrane recycling after exocytosis (Schmid et al. 1998), PC12-27 cells were found to lack AP180, which is found only at neuronal synapses and in neurosecretory cells, and to express those of lower specificity, i.e. AP2, synaptojanin and dynamin (G. Racchetti, B. Borgonovo & J. Meldolesi, unpublished results).
View larger version
[in this window]
[in a new window]
Figure 2. RNase protection and run-on experiments in wild-type and defective PC12 cells
25 % of control cells) was also found in a typically neurosecretion-incompetent system, i.e. the human epithelial cell line HeLa. Moreover, analogous experiments carried out with a construct that included secretogranin II revealed even smaller differences between wild-type PC12, PC12-27 and HeLa cells (Fig. 3A). The possible selection of weak transcription start sites in PC12-27 was excluded by primer extension experiments (Fig. 3B) (R. Benfante & J. Meldolesi, unpublished results). These results seem to rule out the possibility that the huge reduction in neurosecretion-specific mRNAs typical of PC12-27 cells, and the complete absence in HeLa cells (Fig. 3A), is due only to transcriptional impairment. It seems, therefore, that if regulation of the genes involved in neurosecretion occurs in competent and incompetent cells, its site of action is not the classical promoter and might include post-transcriptional events. Even more conclusive along these lines are the results obtained by run-on experiments, a technique in which mRNAs are first elongated within isolated nuclei in the presence of a radiolabelled nucleotide and then analysed specifically by hybridization. Of the three nascent transcripts investigated, only VAMP2 yielded lower levels (30 % reduction) in PC12-27 cells than in wild-type, whereas chromogranin B was similar and secretogranin II (Fig. 2) showed higher levels (200 %). These major discrepancies between Northern blot and run-on results appear to be specific to PC12-27. When, in fact, a fully non-neurosecretory cell type (Fisher rat fibroblast) was investigated, a good match was found between the three mRNAs for the very low Northern blot and run-on signals.
View larger version
[in this window]
[in a new window]
Figure 3. Analysis of chromogranin B and secretogranin II promoter activities in wild-type and defective PC12 cells
View larger version
[in this window]
[in a new window]
Figure 4. Neurosecretory phenotype of rat PC12 cells from wild-type and defective clones, before and after fusion
1 integrin. The concomitant expression of at least one of the neurosecretion-specific proteins investigated, chromogranin B, was identified by species-specific antibodies against this protein. The hybrids were found to be positive for the two neurosecretion-specific markers tested, synaptotagmin and chromogranin B, with the latter being specifically recognized by an anti-rat but not by an anti-human antibody (Borgonovo et al. 1998). We conclude therefore that complementation of the PC12-27 cell defect in the hybrids consists of the reactivation of their competence programme by factor(s) missing in the defective neurosecretory cells and provided by the fusion partners.
REFERENCES
Acknowledgements
Anderson, D. J. (1997). Cellular and molecular biology of neural crest cell lineage determination. Trends in Genetics 13, 276-280.
[Medline]
Atouf, F., Czernichow, P. & Scharfmann, R. (1997). Expression of neuronal traits in pancreatic
cells. Journal of Biological Chemistry 272, 1929-1934[Abstract/Full Text]
Bitler, C., Zhang, M. & Howard, B. D. (1986). PC12 variants deficient in catecholamine transport. Journal of Neurochemistry 47, 1286-1293
[Abstract]
Bittner, M. A., Bennett, M. K. & Holz, R. W. (1996). Evidence that syntaxin 1A is involved in storage in the secretory pathway. Journal of Biological Chemistry 271, 11214-11221
[Abstract/Full Text]
Borgonovo, B., Racchetti, G., Malosio, M. L., Benfante, R., Podini, P., Rosa, P. & Meldolesi, J. (1998). Neurosecretion competence, an independently regulated trait of the neurosecretory cell phenotype. Journal of Biological Chemistry 273, 34683-34686
[Abstract/Full Text]
Buckanovich, R. J. & Darnell, R. B. (1997). The neuronal protein Nova-1 recognizes specific RNA targets in vitro and in vivo. Molecular and Cellular Biology 17, 3194-3201
[Abstract]
Chong, J. A., Ramirez, J. T., Kim, S., Toledo-Arai, J. J., Zheng, Y., Boutros, M. C., Altshuller, Y. M., Frohman, M. A., Kraner, S. D. & Mandel, G. (1995). REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80, 949-957
[Medline]
Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C. & Meldolesi, J. (1992). Differential expression of markers and activities in a group of PC12 nerve cell clones. European Journal of Neuroscience 4, 944-953.
Coco, S., Verderio, C., De Camilli, P. & Matteoli, M. (1998). Calcium dependence of synaptic vesicle recycling before and after synaptogenesis. Journal of Neurochemistry 71, 1987-1992
[Abstract]
Coorssen, J. R., Schmitt, H. & Almers, W. (1996). Ca2+ triggers massive exocytosis in Chinese hamster ovary cells. EMBO Journal 15, 3787-3791
[Abstract]
Corradi, N., Borgonovo, B., Clementi, E., Bassetti, M., Racchetti, G., Consalez, G. G., Huttner, W. B., Meldolesi, J. & Rosa, P. (1996). Overall lack of regulated secretion in a PC12 variant cell clone. Journal of Biological Chemistry 271, 27116-27124
[Abstract/Full Text]
Grohovaz, F., Bossi, M., Pezzati, R., Meldolesi, J. & Torri Tarelli, F. (1996). High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation. Proceedings of the National Academy of Sciences of the USA 93, 4799-4803
[Abstract]
Groves, A., George, K. M., Tissier-Seta, J. P., Engel, J. D., Brunet, J. F. & Anderson, D. J. (1995). Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons. Development 121, 887-901
[Abstract]
Jacobsson, G., Bean, A. J., Scheller, R. H., Berggren, L. J., Deeney, J. T., Berggrens, P. O. & Meister, B. (1994). Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells. Proceedings of the National Academy of Sciences of the USA 91, 12487-12491
[Medline]
Jones, L. C., Day, R. N., Pittler, S. J., Valentine, D. L. & Scammell, J. G. (1996). Cell-specific expression of the rat secretogranin II promoter. Endocrinology 137, 3815-3822
[Abstract]
Karlsson, O., Thor, S., Norberg, T., Ohlsson, H. & Edlund, T. (1990). Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature 344, 879-882
[Medline]
Kasai, H., Kishimoto, T., Liu, T. T., Miyashita, Y., Podini, P., Grohovaz, F. & Meldolesi, J. (1999). Multiple and diverse forms of regulated exocytosis in wild-type and defective PC12 cells. Proceedings of the National Academy of Sciences of the USA 96, 945-949
[Abstract/Full Text]
Leoni, C., Menegon, A., Benfenati, F., Toniolo, D., Pennuto, M. & Valtorta, F. (1999). Neurite extension occurs in the absence of regulated exocyotsis in PC12 subclones. Molecular Biology of the Cell (in the Press).
Matteoli, M., Takei, K., Perin, M. S., Sudhof, T. C. & de Camilli, P. (1992). Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. Journal of Cell Biology 117, 849-861
[Abstract]
Morin, X., Cremer, H., Hirsch, M. R., Kapur, R. P., Goridis, C. & Brunet, J. F. (1997). Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a. Neuron 18, 411-423
[Medline]
Ninomiya, Y., Kishimoto, T., Miyashita, Y. & Kasai, H. (1996). Ca2+-dependent exocytotic pathways in Chinese hamster ovary fibroblasts revealed by a caged-Ca2+ compound. Journal of Biological Chemistry 271, 17751-17754
[Abstract/Full Text]
Pance, A., Guest, P., Bowers, K., Cutler, D., Dean, G. & Jackson, A. P. (1995). Protein mistargeting in a PC12 variant lacking regulated secretion. Molecular Biology of the Cell 6, suppl. 70a.
Passafaro, M., Rosa, P., Sala, C., Clementi, F. & Sher, E. (1996). N-type Ca2+ channels are present in secretory granules and are transiently translocated to the plasma membrane during regulated exocytosis. Journal of Biological Chemistry 271, 30096-30104
[Abstract/Full Text]
Pohl, T. M., Phillips, E., Song, K. Y., Gerdes, H. H., Huttner, W. B. & Ruther, U. (1990). The organization of the mouse chromogranin B (secretogranin I) gene. FEBS Letters 262, 219-224
[Medline]
Rowe, J., Corradi, N., Malosio, M. L., Taverna, E., Halban, Ph., Meldolesi, J. & Rosa, P. (1999). Blockade of membrane transport and disassembly of the Golgi complex by expresssion of syntaxin1A in neurosecretion-incompetent cells: prevention by rbSec1. Journal of Cell Science 112, 1865-1877
[Abstract]
St-Onge, L., Sosa-Pineda, B., Chowdhury, K., Mansouri, A. & Gruss, P. (1997). Pax6 is required for differentiation of glucagon-producing
-cells in mouse pancreas. Nature 387, 406-409[Medline]
Sakakibara, S. & Okano, H. (1997). Expression of neural RNA-binding proteins in the postnatal CNS: implications of their roles in neuronal and glial cell development. Journal of Neuroscience 17, 8300-8383
[Abstract/Full Text]
Schmid, S. L., McNiven, M. A. & De Camilli, P. (1998). Dynamin and its partners: a progress report. Current Opinion in Cell Biology 10, 504-512
[Medline]
Schoenherr, C. J. & Anderson, D. J. (1995). The Neuron-Restrictive Silencer Factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267, 1360-1363
[Medline]
Scholl, T., Stevens, M. B., Mahanta, S. & Strominger, J. L. (1996). A zinc finger protein that represses the transcription of the human MHC class II gene, DPA. Journal of Immunology 156, 1448-1457
[Abstract]
Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument- Bromage, H., Geromanos, S., Tempst, P. & Rothman, J. E. (1993). SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318-324
[Medline]
Sosa-Pineda, B., Chowdhury, K., Torres, M., Oliver, G. & Gruss, P. (1997). The Pax4 gene is essential for differentiation of insulin-producing
cells in the mammalian pancreas. Nature 386, 399-402[Medline]
Taoka, M., Isobe, T., Okuyama, T., Watanabe, M., Kondo, H., Yamakawa, Y., Ozawa, F., Hishinuma, F., Kubota, M., Minegishi, A., Song, S. T. & Yamakuni, T. (1994). Murine cerebellar neurons express a novel gene encoding a protein related to cell cycle control and cell fate detemination proteins. Journal of Biological Chemistry 269, 9946-9951
[Abstract]
Tiveron, M. C., Hirsch, M. R. & Brunet, J. F. (1996). The expression pattern of the transcription factor Phox2 delineates synaptic pathways of the autonomic nervous system. Journal of Neuroscience 16, 7649-7660
[Abstract/Full Text]
Verderio, C., Coco, S., Bacci, A., Rossetto, O., De Camilli, P., Montecucco, C. & Matteoli, M. (1999). Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons. Journal of Neuroscience (in the Press).
Verderio, C., Coco, S., Fumagalli, G. & Matteoli, M. (1995). Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of
-agatoxin IVA- and -conotoxin GVIA-sensitive channels. Proceedings of the National Academy of Sciences of the USA 92, 6449-6453[Abstract]
Xu, T., Binz, T., Niemann, H. & Neher, E. (1998). Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nature Neuroscience 1, 192-200
[Medline]
Yamakuni, T., Yamamoto, T., Hoshino, M., Song, S. Y., Yamamoto, H., Kunikata-Sumitomo, M., Minegishi, A., Kubota, M., Ito, M. & Konishi, S. (1998). A novel protein containing cdc10/SWI6 motifs regulates expression of mRNA encoding catecholamine biosynthesizing enzymes. Journal of Biological Chemistry 273, 27051-27054
[Abstract/Full Text]
Yamaoka, T. & Itakura, M. (1999). Development of pancreatic islets. International Journal of Molecular Medicine 3, 247-261.
[Medline]
Yang, B., Gonzalez, L. Jr, Prekeris, R., Steegmaier, M., Advani, R. J. & Scheller, R. H. (1999). SNARE interactions are not selective. Implications for membrane fusion specificity. Journal of Biological Chemistry 274, 5649-5653
[Abstract/Full Text]
Zakharenko, S., Chang, S., O'Donoghue, M. & Popov, S. V. (1999). Neurotransmitter secretion along growing nerve processes: comparison with synaptic vesicle exocytosis. Journal of Cell Biology 144, 507-518
[Abstract/Full Text]
Zellmer, E., Zhang, Z., Greco, D., Rhodes, J., Cassel, S. & Lewis, E. J. (1995). A homeodomain protein selectively expressed in noradrenergic tissue regulates transcription of neurotransmitter biosynthetic genes. Journal of Neuroscience 15, 8109-8120
[Abstract]
E. Cocucci, G. Racchetti, M. Rupnik, and J. Meldolesi
The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4
J. Cell Sci.,
September 15, 2008;
121(18):
2983 - 2991.
[Abstract]
[Full Text]
[PDF]
E. Cocucci, G. Racchetti, P. Podini, M. Rupnik, and J. Meldolesi
Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route
Mol. Biol. Cell,
December 1, 2004;
15(12):
5356 - 5368.
[Abstract]
[Full Text]
[PDF]
M. L. Malosio, T. Giordano, A. Laslop, and J. Meldolesi
Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype
J. Cell Sci.,
February 15, 2004;
117(5):
743 - 749.
[Abstract]
[Full Text]
[PDF]
C. Grundschober, M. L. Malosio, L. Astolfi, T. Giordano, P. Nef, and J. Meldolesi
Neurosecretion Competence. A COMPREHENSIVE GENE EXPRESSION PROGRAM IDENTIFIED IN PC12 CELLS
J. Biol. Chem.,
September 20, 2002;
277(39):
36715 - 36724.
[Abstract]
[Full Text]
[PDF]
This Article 
Abstract
Full Text (PDF)
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Citing Articles
Citing Articles via HighWire
Citing Articles via Google Scholar
Google Scholar
Articles by Malosio, M. L.
Articles by Meldolesi, J.
Search for Related Content
PubMed
PubMed Citation
Articles by Malosio, M. L.
Articles by Meldolesi, J.
Related Collections
Review articles
HOME
HELP
FEEDBACK
SUBSCRIPTIONS
ARCHIVE
SEARCH
TABLE OF CONTENTS