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Expression and localisation of synaptotagmin isoforms in endocrine beta-cells: their function in insulin exocytosis
GUT, A, et al.
Abstract
Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic beta-cells and derived cell lines is mainly controlled by Ca(2+). Several lines of evidence have demonstrated a role of the Ca(2+)- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca(2+) determined by their two C(2) domains (C(2)A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca(2+) sensors in LDCV exocytosis, syt III was not expressed in primary beta-cells, whereas syt IV was only found adjacent to the TGN.
However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary beta-cells the introduction of recombinant peptides (100 nM) corresponding to the C(2) domains of syt V, VII and VIII, but not of syt III, IV [...]
GUT, A, et al . Expression and localisation of synaptotagmin isoforms in endocrine beta-cells:
their function in insulin exocytosis. Journal of Cell Science , 2001, vol. 114, no. Pt 9, p. 1709-16
PMID : 11309201
Available at:
http://archive-ouverte.unige.ch/unige:34827
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INTRODUCTION
The release of neurotransmitters and peptide hormones proceeds by exocytosis, that is, fusion between the membranes of the corresponding vesicles and the plasma membrane (Jahn and Sudhof, 1999). The release of potent substances must be tightly controlled and in most systems an increase in cytosolic Ca2+ constitutes the major stimulatory signal for exocytosis (Burgoyne and Morgan, 1998). During the last decade a number of proteins have been identified which are required for this process. A heterotrimeric complex of proteins, the so- called SNARE proteins, exerts a major role (Rothman and Sollner, 1997). The complex encompasses synaptobrevin/
VAMP, which is located on exocytotic vesicles, as well as SNAP-25 and syntaxin, mainly present on the plasma membrane. Assembly and disassembly of this complex seems to be necessary for exocytosis.
Ca2+-dependent regulation may be imposed through a class of vesicle proteins, the synaptotagmins (syt), which interact with both the SNARE proteins syntaxin and SNAP-25, and with membrane phospholipids (Fernandez-Chacon and Sudhof, 1999). 13 isoforms of synaptotagmin are presently known and are predicted to exhibit several common structural features (Marquèze et al., 2000). The best-characterised
isoform, syt I, contains a short intravesicular N terminus followed by a single transmembrane domain and a region of variable length connecting to two C2domains, C2A and C2B.
These C2 domains were first characterised in protein kinase C, where they mediate phospholipid binding at micromolar concentrations of free Ca2+. In addition, binding of Ca2+to the C2A domain alters its electrostatic potential and thus permits binding to the SNARE protein syntaxin at Ca2+concentrations above 100 µM (Rizo and Sudhof, 1998). These concentrations of the cation are in accordance with levels of cytosolic Ca2+ reported for exocytosis from bipolar retinal neurones (Heidelberger et al., 1994).
The implication of synaptotagmin as a Ca2+ sensor in exocytosis has been strengthened by the observations that cytosolic applications of recombinant peptides corresponding to the C2 domains or of antibodies inhibit Ca2+-dependent exocytosis in neuroendocrine cells (Elferink et al., 1993; Ohara Imaizumi et al., 1997; Lang et al., 1997a). More importantly, transgenic deletion mutations of synaptotagmin I in mice and Drosophila selectively abolish fast neurotransmission (Geppert et al., 1994; Littleton et al., 1994).
In the synaptotagmin family several distinct features are apparent. Northern blot analysis has indicated that some isoforms are mainly expressed in neuronal cells (I-V, X), Exocytosis of insulin containing Large Dense Core Vesicles
(LDCVs) from pancreatic β-cells and derived cell lines is mainly controlled by Ca2+. Several lines of evidence have demonstrated a role of the Ca2+- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca2+
determined by their two C2domains (C2A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca2+ sensors in LDCV exocytosis, syt III was not expressed in primary β-cells, whereas syt IV was only found adjacent to the TGN.
However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary β-cells the introduction of recombinant peptides (100 nM) corresponding to the C2domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca2+-evoked insulin exocytosis by 30% without altering GTPγS-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary β-cells whereas V, VII and VIII may mediate Ca2+- regulation of exocytosis.
Key words: Exocytosis, Large dense core vesicle (LDCV), Ca2+, Endocrine cell, Insulin
SUMMARY
Expression and localisation of synaptotagmin
isoforms in endocrine β -cells: their function in insulin exocytosis
Anne Gut1, Catherine Eva Kiraly1, Mitsunori Fukuda2, Katsuhiko Mikoshiba2,3, Claes B. Wollheim1and Jochen Lang1,4,*
1Division de Biochimie Clinique, Département de Médecine, Université de Genève, CH-1211 Genève 4, Switzerland
2Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Hirosawa, Wako, Saitama 351-0198, Japan
3Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan
4Institut Européen de Chimie et Biologie, Bordeaux, France
*Author for correspondence at address 4 (e-mail: j.lang@iecb-polytechnique.u-bordeaux.fr) Accepted 11 February 2001
Journal of Cell Science 114, 1709-1716 © The Company of Biologists Ltd
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whereas others such as syt VI-IX and XI are seemingly more widely distributed (Marqueze et al., 2000). Moreover, only the isoforms I-V and VII bind phospholipids at low micromolar Ca2+. The interaction with syntaxin is apparent either at low (III and VII) or high (I, II and V) micromolar levels of Ca2+or is absent (Li et al., 1995). Their differential distribution and biochemical characteristics have contributed to the notion that synaptotagmins may function in a variety of distinct membrane fusion events (Marqueze et al., 2000), although their precise role remains a matter of debate. Indeed, conflicting results on the localisation and function of the isoforms III (Butz et al., 1999; Brown et al., 2000) and IV (Ferguson et al., 1999; Berton et al., 2000; Ibata et al., 2000) have been reported
To study the putative role of synaptotagmin isoforms in exocytosis of peptide-containing large dense core vesicles (LDCVs) we have used pancreatic β-cells and derived cell lines (Lang, 1999). These preparations represent a valid model as the use of permeabilised cells permits the study of exocytosis (Lang et al., 1997b). Under these conditions the release of insulin is stimulated upon an increase in intracellular Ca2+or the addition of the slowly hydrolyzable GTP analogue GTPγS (Vallar et al., 1987). Ca2+-stimulated exocytosis of insulin requires functional SNARE proteins as shown by the use of clostridial neurotoxins (Lang et al., 1997c; Martin et al., 1995;
Regazzi et al., 1996; Sadoul et al., 1995). We have previously demonstrated a role for the two synaptotagmin isoforms syt I and II, in two β-cell derived cell lines in exocytosis evoked by Ca2+, but not by GTPγS (Lang et al., 1997a). In view of the different isoforms known we have now set out to characterise the localisation and involvement of syt III and IV in this model system of LDCV exocytosis as well as the putative role of syt V-VIII.
MATERIALS AND METHODS
Materials
Streptolysin-O was produced and purified as described (Weller et al., 1996). The generation and testing of antibodies against the C2A domains of syt III or syt IV have been described previously (Fukuda et al., 1999; Ibata et al., 2000). Plasmids encoding GST-tagged synaptotagmin I-VIII were generously donated by Dr T. E. Südhof (Dallas, USA). GST-tagged recombinant proteins purified according to the instructions of the manufacturer and in the absence of detergents were >95% pure, as judged by Coomassie Blue stained SDS-PAGE gels.
Cell culture and release studies
Culture of the insulin-secreting cell lines HIT-T15 or MIN6 and preparation of primary islet cells were from male Wistar rats as described previously (Lang et al., 1998). Preparation of intracellular buffers and permeabilisation with streptolysin-O were as described (Lang et al., 1997a,b). 100 nM GST-syt-C2A/B recombinant peptides were added to the permeabilised cells in intracellular buffer at 0.1 µM free Ca2+. After 10 minutes preincubation, this solution was exchanged for intracellular buffer containing 0.1 µM free Ca2+, 10µM free Ca2+or 100 µM GTPγS (Boehringer, Rotkreuz, Switzerland).
After 7 minutes of stimulation, supernatants were removed, centrifuged and their insulin content determined by radioimmunoassay (Lang et al., 1997b).
Northern blot analysis
Total RNA was extracted from different insulin-secreting cell lines and pancreatic islets using guanidine thiocyanate (Gibco, Basel,
Switzerland). For RNA transfer blots, 30 µg of total RNA from the various cells were denatured with glyoxal, separated by electrophoresis in 1% agarose gels and transferred to nylon membranes. The blots were probed with synaptotagmin cytoplasmic fragments encompassing both C2 and the C-terminal domains of synaptotagmins I-VIII, kindly provided by Dr T. E. Südhof (Li et al., 1995). Hybridisations were carried out in 1 M NaCl/1% SDS/10%
dextran sulfate for 16 hours at 55°C. The nylon membranes were washed in 1×SSC/1% SDS for 15 minutes at 50-60°C before autoradiography.
Western blotting and immunofluorescence
Preparation of homogenates from insulin-secreting cell lines and SDS/PAGE immunoblots were performed as published (Zhang et al., 1999). For immunofluorescence, cells were grown on native (HIT- T15, MIN6) or extracellular matrix-coated glass coverslips (primary cells) at 105cells/well. They were either cultured as such or transiently transfected using Fugene (Roche, Germany) at a concentration of 1 µl/250 ng cDNA/well. Experiments were performed 48 hours after cotransfection. Coverslips with cells were washed twice with phosphate-buffered saline (PBS), fixed for 15 minutes in PBS-4%
paraformaldehyde, washed twice again for 5 minutes in PBS and permeabilised for 50 minutes with PBS containing 0.1% saponin (Sigma, Buchs, Switzerland) and 5% BSA. Primary antibodies were added for 1-4 hours at room temperature or overnight at 4°C. Cells were then washed three times for 5 minutes with PBS-1% BSA, and incubated with secondary antibodies in PBS containing 0.1% saponin and 5% BSA for 1 hour. After three washes with PBS for 5 minutes, cells were mounted on glass slides using a mounting medium (Vector Lab., Burlingame, USA). Confocal laser microscopy was performed with a Zeiss LSM 410 inverse laser scan microscope equipped with an argon and helium-neon laser (Zürich, Switzerland). Primary antibodies were diluted as indicated in the figure legends.
Statistical analysis
Data are presented as mean ± s.e.m. from experiments performed independently on at least three different cell preparations. Statistical analysis was performed by Student’s two-tailed t-test for unpaired data (2P).
RESULTS
The synaptotagmin protein family contains a number of isoforms and some of them have been implicated in the regulation of insulin exocytosis (Brown et al., 2000; Lang et al., 1997a; Mizuta et al., 1994; Mizuta et al., 1997). Syt III has previously been reported to play a role as a Ca2+sensor in the exocytosis of secretory granules (Brown et al., 2000; Mizuta et al., 1997), whereas syt IV was found to be important in the exocytosis of LDCVs and synaptic vesicles (Ferguson et al., 1999; Littleton et al., 1999; Thomas et al., 1999). Therefore we studied their subcellular distribution to determine whether their localisation permits them to intervene in exocytosis.
First we examined the specificity of the antibodies directed against syt III or IV as used here. Whereas the anti- synaptotagmin III antibody was highly specific for the syt III- C2A/B peptide (Fig. 1A), the anti-synaptotagmin IV antibody demonstrated some minor crossreaction with other recombinant proteins, mainly syt II. We further determined the specificity by testing whether the binding could be blocked by the corresponding recombinant peptide. As shown in Fig. 1B, the syt III peptide, but not the syt IV peptide, inhibited the binding of the anti-syt III antibody on western blots from rat brain homogenates. Similarly, the reactivity of the anti-syt IV JOURNAL OF CELL SCIENCE 114 (9)
antibody was blocked by the syt IV peptide, but not by the syt III. Using these antibodies in western blots from cell homogenates (Fig. 1C) we found immunoreactive bands of the corresponding molecular mass for syt III mainly in MIN6 cells and to a lesser extent in RINm5F and HIT-T15 cells, whereas no staining was detectable in INS-1 or primary islet cells. A 45 kDa band corresponding to syt IV was found in all cell lines tested except for HIT-T15 cells.
To further establish the distribution of these two synaptotagmin isoforms we resorted to confocal laser immunofluorescence (Fig. 2). Insulin-secreting β-cells represent the majority of primary islet cells; however, such a preparation also contains somatostatin-secreting δ-cells and glucagon-secreting α-cells. It is therefore important to identify precisely the cell type involved. Within the primary islet cells the anti-synaptotagmin III antibody stained a few cells which were not, however, β-cells as they were not recognized by an antibody against insulin (Fig. 2a). In contrast, these cells were stained with an antibody against somatostatin, suggesting that syt III is only expressed in pancreatic δ-cells (data not shown).
The presence of syt III on secretory vesicles has been reported for the insulin-secreting cell line MIN6 (Mizuta et al., 1997).
Our antibody did indeed detect immunoreactive material with a granular distribution in this cell line (Fig. 2c). However, close inspection revealed only minor colocalisation between the immunoreactivity for syt III and for insulin (Fig. 2d). Taking into account the resolution obtained by confocal microscopy, these data suggest that syt III is most unlikely to be expressed on insulin-containing granules.
To obtain information on the targeting of syt III we performed transient expression in both primary islet cells and in HIT-T15 cells, as in the latter we could not reliably detect syt III by immunofluorescence. Primary β-cells transiently expressed syt III upon transfection. However, the immunoreactivity was mainly present at the level of the plasma membrane (Fig. 2b, green) and did not coincide with insulin (Fig. 2b, red). To identify transiently transfected HIT- T15 cells, they were cotransfected with a plasmid encoding eGFP. As shown in Fig. 2e, synaptotagmin III was again solely found at the plasma membrane and not on intracellular structures.
In the case of syt IV, a strong immunostaining was found in a few cells in preparations of primary islet cells (Fig. 3a, green). This strong signal was confined to glucagon-secreting α-cells (Fig. 3c) and associated with structures distinct from secretory granules. A fainter signal was observed in all cells (Fig. 3b). Costaining with an antibody against TGN38, a marker for the trans-Golgi network, revealed that syt IV was mainly found within or adjacent to the TGN (Fig. 3b). A similar perinuclear distribution was apparent in MIN6 cells and again no colocalisation with secretory granules was apparent.
In HIT-T15 cells only a very faint signal was observed at a location compatible with the TGN (data not shown).
As syt III and syt IV are not expressed on secretory vesicles we also investigated the relative distribution of the synaptotagmin isoforms V-VIII in these various cell lines. To this end northern blots were hybridised with specific probes and compared to the expression of actin. As shown in Fig. 4, all isoforms assayed are expressed in insulin-secreting cells, although at different levels. The results suggest that synaptotagmins V-VIII are widely expressed in these cells and
may eventually be involved in the exocytosis of secretory granules.
We employed next a functional approach to gain insight into the potential role of synaptotagmin isoforms. The C2A/B domains are known to be important in the function of synaptotagmins and their binding to other components of the exocytotic machinery (Fernandez-Chacon and Sudhof, 1999;
Rizo and Sudhof, 1998). Consequently, it has been shown that Fig. 1. Characterisation of anti-syt III and anti-syt IV antibodies.
(A) Western blots of 50 ng of recombinant GST-syt-C2A/C2B peptides (syt I-VIII) were incubated with anti-syt III or anti-syt IV antibodies (1:2000). (B) Western blots of rat brain homogenate (25 µg/lane) were incubated with the indicated antibodies, which had been preincubated with buffer alone (−) or with buffer containing GST-syt III- or GST-syt IV-C2A/C2B recombinant protein (10 µg/100 µl). Specific bands are indicated by arrows. (C) Western blots of crude membranes from rat brain, HIT-T15, INS-1, RINm5F, MIN6 or islet cells (50 µg/lane) were incubated with antibodies against syt III or syt IV. The positions of molecular mass markers are shown.
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the cytosolic application of the corresponding domains of syt I blocks the exocytosis of LDCVs in PC12 cells (Elferink et al., 1993). We therefore examined the effect of these domains of recombinant syt I-VIII on insulin exocytosis in streptolysin- O permeabilized primary islet cells (Fig. 5). We also compared
the stimulation of insulin exocytosis by Ca2+ with the stimulation by the slowly hydrolysable GTP analogue GTPγS, as the guanine nucleotide exerts its effect independent of Ca2+
(Vallar et al., 1987). In primary islet cells, only syt V, VII and VIII were effective and as expected, the GTPγS-induced effects JOURNAL OF CELL SCIENCE 114 (9)
Fig. 2. Subcellular localization of endogenous and transiently expressed synaptotagmin III in primary islet, MIN6 and HIT-T15 cells. Islet cells:
cells grown on coverslips were fixed, permeabilised and incubated with the anti-syt III polyclonal antibody (1:200) and monoclonal anti-insulin antibody (1:200). Anti-syt III binding was revealed with an FITC-coupled second antibody (green), anti-insulin binding with a TRITC-coupled second antibody (red). (a) Native cells; (b) cells transiently expressing synaptotagmin III. MIN6 cells: primary antibodies bound to native syt III or insulin were visualized with fluorophore-coupled secondary antibodies; (c) TRITC, anti-syt III; (d) FITC anti-syt III, TRITC, anti-insulin).
HIT-T15: cells were transiently cotransfected with plasmids expressing syt III and plasmids expressing eGFP. Primary antibodies bound to native syt III were visualized with TRITC-coupled secondary antibodies. Immunofluorescence and phase-contrast images are given in the upper and lower panels, respectively. Bars, 5 µm.
Fig. 3. Subcellular localization of synaptotagmin IV in pancreatic islets, MIN6 and HIT-T15 cells. Islet cells: cells were permeabilised and fixed. Staining for syt IV (a-c) was revealed with an FITC-coupled second antibody (green), costaining for insulin (a), anti-TGN (b) or anti- glucagon (c) was revealed with a TRITC-coupled second antibody (red). MIN6: staining for syt IV (d) was revealed with a FITC-coupled second antibody (green), costaining for insulin was revealed with a TRITC coupled second antibody (red).
were not altered. We also performed experiments in the hamster insulinoma cell line HIT- T15: the peptides corresponding to syt I or II inhibited Ca2+-induced insulin release by 35.7±4.1% and 28±6.2%, respectively (2P<0.05) without altering GTPγS-evoked release (data not shown). These observations are in line with our previous results in HIT-T15 cells obtained by the use of site-specific antibodies or point mutations (Lang et al., 1997a). In addition, the recombinant C2A/B domains of syt VI, VII and VIII specifically inhibited Ca2+ induced exocytosis (28.4±4.7%, 29.8±5.9% and 23.4±4.3%, respectively; 2P<0.05) but did not significantly alter GTPγS mediated release. This suggests that syt VII and VIII may have a general function in insulin exocytosis, whereas the effect of the isoforms I, II, V and VI are cell-type dependent.
DISCUSSION
Within the family of the Ca2+- binding protein synaptotagmin, the two isoforms syt I and syt II play a role in the exocytosis of LDCVs and SVs (Elferink et al., 1993;
Geppert et al., 1994; Littleton et al., 1994; Lang et al., 1997a). We have previously shown that syt I and II are operational in endocrine exocytosis probably involving their capacity to bind phospholipids.
However, in contrast to derived cell lines, primary β-cells do not express syt I or II, raising the question about the nature of the relevant isoform present in primary
cells (Lang et al., 1997a). Moreover, a considerable number of additional isoforms has been identified during recent years and their respective roles still remain unclear (reviewed in Marqueze et al., 2000). Our study has therefore addressed the expression of synaptotagmin isoforms in a model system of LDCV exocytosis, namely pancreatic β-cells or derived cell lines, to investigate their potential implication in Ca2+evoked exocytosis.
Syt III has been proposed to play an essential role in regulated exocytosis from endocrine β-cells (Brown et al., 2000; Mizuta et al., 1994; Mizuta et al., 1997), whereas syt IV regulates exocytosis in Drosphila neurons and neuroendocrine PC12 cells (Ferguson et al., 1999; Littleton et al., 1999; Thomas et al., 1999). In our system we could not find any evidence for their expression on relevant organelles
or a role in the exocytosis of peptide hormone containing LDCVs. Clearly the investigation of isoforms always implies the problem of crossreactivity. Phylogenic analysis reveals a close relationship between syt III and syt V, VI or X as well as between syt IV and syt XI (Marqueze et al., 2000). The C2A domain of syt III used as an antigen exhibits 74%
identity to the corresponding domains of syt V, syt VI or syt X and less than 65% identity to other synaptotagmin isoforms. It should be noted that the antibodies used in our study had been preabsorbed to syt VI to remove crossreactivity and the specificity has been tested against syt I-VIII (our results) and syt IX-XI (Fukuda et al., 1999; Ibata et al., 2000). We therefore assume that any crossreactivity with these isoforms is unlikely.
Although high expression levels of syt III mRNA were Fig. 4. Northern blot analysis of synaptotagmin
III-VIII expression in insulin-secreting cells and rat brain. Total RNA (30 µg per lane) of whole rat brain, or the indicated cell lines and primary islet cells, were separated on agarose gels, transferred to nylon membranes and hybridized with specific probes as indicated in Materials and Methods. Upper panels:
expression of synaptotagmin III-VIII mRNAs.
Lower panels: actin mRNA, for comparison.
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evident in the rather differentiated cell line INS-1 and in concordance with a previous study in MIN6 cells (Mizuta et al., 1994), only low levels were found in pancreatic islets.
Indeed, the level of expression in islets was comparable to or even lower than RINm5F cells. The latter are poorly granulated and contain 100-fold less insulin than islet cells (Wollheim et al., 1990). A scarcely expressed protein may, of course, fulfill an important function. However, immunohistochemistry demonstrated that in primary islet cells syt III is only expressed in somatostatin-secreting δ-cells, but not in β-cells. Therefore, the low level of expression in islets is most likely to reflect its expression in somatostatin-secreting cells, which represent less than 10% of islet cells. It is noteworthy that the distribution in δ-cells was granular, but did not match with somatostatin- containing secretory granules (not shown). Similar to a previous report (Mizuta et al., 1997) we found a granular distribution of syt III in MIN6 cells, from which it was originally been cloned. However, these syt III-containing structures overlapped only partially with insulin-containing vesicles. Finally transiently expressed syt III in pancreatic β- cells or HIT-T15 cells was almost exclusively localised at the plasma membrane. In nerve terminals the majority of syt III is also not expressed on synaptic vesicles but on the plasma membrane or, alternatively, on a still undefined compartment (Butz et al., 1999). Although overexpression may eventually result in a saturation of the sorting machinery, such an artifact is not responsible for the observed localisation as staining of the correct compartement should still be observed. However, we could not detect any evidence for localisation outside the plasma membrane or any evidence for intracellular aggregation. Hence, syt III seems in general to be directed to the plasma membrane. The different expression pattern in MIN6 and δ-cells may be explained by the presence of a cell- specific cofactor, which routes syt III to an organelle with a granular distribution.
We were also unable to localise syt IV on any exocytotic compartment, in contrast to reports of pheochromocytoma PC12 cells and Drosophila brain (Ferguson et al., 1999;
Littleton et al., 1999; Thomas et al., 1999). In accordance with two other reports on localisation of syt IV in hippocampal neurons and PC12 cells (Berton et al., 2000; Ibata et al., 2000), a structure adjacent to the TGN was immunostained in all cells examined. Interestingly the expression of syt IV has recently been demonstrated on immature granules with subsequent retrieval during maturation (Eaton et al., 2000). In contrast to β-cells, syt IV was strongly expressed in glucagon-secreting primary α-cells, but again on a compartment distinct from peptide hormone-containing LDCVs. Immunopurification and in vitro binding studies demonstrated an interaction between syt I and syt IV, which may be of functional importance (Littleton et al., 1999). In view of the distinct distribution of syt I in derived β-cells, such an interaction may eventually only occur at the level of the TGN, and certainly not at the sites of exocytosis. Finally, we were unable to observe any prominent staining in somatostatin-containing δ-cells, in contrast to a previous report (Brown et al., 2000). As syt IV presents an immediate early gene product (Vician et al., 1995), we cannot exclude the possibility that the discrepancy between its expression in δ- and α-cells may reside in the use of fresh versus cultured islets.
The functional assay used also argues against a role for syt
III or IV in insulin exocytosis. The assay is based on an early observation that cytosolic application of C2A/B domains of syt I inhibits Ca2+ stimulated exocytosis from PC12 cells (Elferink et al., 1993). The same approach has also been used recently to investigate the putative function of syt VII in the exocytosis of lysosomes (Martinez et al., 2000). The use of specific peptides or antibodies offers the advantage of an acute intervention, thus avoiding problems inherent in the selection of stable clones. The mechanisms of how these peptides inhibit are not completely clear but may involve two distinct mechanisms. First, synaptotagmins undergo homo- and heterodimerisation, which may be required for their function (Fukuda et al., 1999; Fukuda and Mikoshiba, 2000), and may even occur at low micromolar concentrations of Ca2+ (Osborne et al., 1999). Binding of endogenous synaptotagmins to recombinant C2A/B domains lacking the transmembrane domain may result in non-productive interactions in terms of exocytosis. Second, several other proteins required for exocytosis, such as syntaxin or SNAP, interact with the C2A/B domain (Li et al., 1995; Gerona et al., 2000). Their binding to recombinant C2A/B domains may prevent their interaction with endogenous synaptotagmins required for exocytosis. In essence, this assay does not directly measure whether a given isoform is implicated in exocytosis, but whether this isoform is capable of interacting in a productive manner with the different cell-specific components of the exocytotic machinery under the conditions present in permeabilised cells.
The specificity of the approach used here is underlined by several findings. First, the effect of recombinant peptides was JOURNAL OF CELL SCIENCE 114 (9)
GST syt I syt II syt IIIsyt I V
syt V
syt VI
syt VII
syt VIII 0
10 20 30 40 50
* *
*
% inhibition of insulin release
10 µM Ca2+
100 µM GTP γS
recombinant peptides
Fig. 5. Differential effects of synaptotagmin C2A/B peptides on insulin release from streptolysin-O permeabilized primary rat islet cells. Primary islet cells were permeabilized with streptolysin-O and subsequently exposed to basal or stimulatory concentrations of Ca2+
(0.1 µM or 10 µM) or 100 µM GTPγS (at 0.1 µM Ca2+) in the presence of GST fusion peptides corresponding to the C2A/C2B domains of synaptotagmin isoforms I-VIII (syt I-VIII; GST, GST alone). Insulin secretion was measured by radioimmunoassay and results are expressed as a percentage of inhibition of net release as compared to the absence of peptides. *2P<0.05 as compared to GST alone.
restricted to Ca2+-induced exocytosis, whereas GTPγS evoked release remained unchanged. Indeed, GTPγS-evoked release is known to proceed independently of Ca2+(Vallar et al., 1987) or synaptotagmin, as indicated by the use of site-specific antibodies and mutants (Lang et al., 1997a). Second, in concordance with previous experiments using site-specific antibodies and mutants (Lang et al., 1997a), the peptides corresponding to syt I and II inhibited exocytosis in HIT-T15 cells. In contrast, these peptides did not alter exocytosis in primary β-cells that do not express these proteins.
Syt I and II are known to interact in vitro with the SNARE protein syntaxin (Li et al., 1995). This interaction exhibits an EC50 for Ca2+ between 200 and 500 µM, in accordance with subplasmalemmal Ca2+ concentrations occuring in neuroexocytosis (Heidelberger et al., 1994). Both HIT-T15 cells and primary β-cells express syntaxin 1 and require its function for exocytosis (Lang et al., 1997c; Martin et al., 1995).
The failure of the recombinant C2A/B domains of syt I and II to inhibit exocytosis from primary β-cells suggests that Ca2+- dependant interactions with syntaxin may not be the major mechanism probed for with the recombinant peptides.
Although the true Ca2+dependency of the interaction between syntaxin and synaptotagmin in-vivo is unknown, exocytosis from permeabilised cells was tested under conditions providing the maximal stimulation in our system as measured by biochemical means, that is 10 µM Ca2+. At these levels of Ca2+, syt III, V and VII bind to phospholipids (Li et al., 1995).
Disturbance of this interaction of synaptotagmin dimerisation or of binding to still unknown factors may form the basis for the effect of recombinant C2A/B domains corresponding to specific isoforms.
Our functional data argue strongly against the possibility that syt III or IV play a role in Ca2+stimulated exocytosis, at least in primary β-cells and clonal HIT-T15 cells. In contrast, we observed significant effects in the case of syt V, VII and VIII in primary β-cells, whereas syt VI-VIII were active in HIT-T15 cells. The differential activities of the recombinant peptides syt V and VI again underscore the cell-type specificity, as also observed for syt I and II. Syt V-VIII are expressed in primary islet cells and clonal cell lines; we still do not know whether they are present in β-cells and on which organelle they reside. Syt V and VII both exhibit Ca2+
sensitivity in their binding to phospholipids and syntaxin 1 (Li et al., 1995). Whereas the subcellular localisation and precise function of syt V, VI and VIII are still unknown, syt VII has been shown to reside on lysosomal compartments in fibroblasts and is involved in the Ca2+-dependent exocytosis from this compartment (Martinez et al., 2000). It is therefore likely that the Ca2+-dependent exocytosis from lysosomes and LDCV exocytosis involve common mechanisms of binding to syt VII.
β-cells provide a tested model to study the exocytosis of peptide-hormone containing LDCVs (Lang, 1999). The role of syt in endocrine exocytosis has been established and specifically linked to Ca2+-stimulated release. As far as primary cells are concerned, syt I-IV are not involved in this event. Our data indicate that syt VII and VIII, which are both Ca2+sensitive, may constitute promising candidates for further investigation.
We are grateful to Dr T. Sudhof for providing us with the plasmids encoding GST-tagged synaptotagmine I-VIII. We are indebted to
Danielle Nappey, Franca Minafra and Dominique Duhamel for skilled technical assistance and to Jean Gunn for her patience in correcting the manuscript. This study was supported by the Swiss National Science Foundation (FN 32-49755.96 to C. B. W. and J. L., FN 31- 54023.98 to J. L.) and the Human Frontier Science Program.
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