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Genes to Cells (2007) 12, 511-519. doi:10.1111/j.1365-2443.2007.01070.x
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Synaptotagmin VII modulates the kinetics of dense-core vesicle exocytosis in PC12 cells

 Takashi Tsuboi1 and Mitsunori Fukuda1,2,*

1 Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2 Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
In our previous study, we showed that PC12 cell lines stably expressing synaptotagmin (Syt) VII have greater ability to release hormones Ca2+-dependently than the original PC12 cells. However, the precise molecular mechanism of the enhancement of hormone secretion by Syt VII has never been elucidated. In this study, we established a PC12 cell line that stably expresses Syt VII-green fluorescent protein (Syt VII-GFP) or its Ca2+-binding-site-deficient mutant (D172N/D303N substitutions; Syt VII-DN-GFP), and examined the effect of Syt VII-GFP expression on the kinetics of dense-core vesicle exocytosis by total internal reflection fluorescence (TIRF) microscopy. Both Syt VII-GFP and Syt VII-DN-GFP co-localized well with dense-core vesicle markers, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y (NPY-mRFP) and cyan fluorescent protein (CFP)-tagged tissue plasminogen activator (tPA-CFP). Expression of Syt VII-GFP enhanced the number of dense-core vesicle exocytotic events, whereas expression of Syt VII-DN-GFP or knockdown of Syt VII-GFP with specific small interfering RNA (siRNA) attenuated the number of exocytotic events. Monitoring individual tPA-CFP release events revealed that "full release" events are increased in Syt VII-GFP-expressing cells, but not in Syt VII-DN-GFP-expressing or Syt VII-silenced cells. Our data indicate that Syt VII modulates the kinetics of Ca2+-dependent dense-core vesicle exocytosis in neuroendocrine PC12 cells, possibly by modulating fusion pore opening.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Hormones accumulate in the dense-core vesicles of neuroendocrine cells, and the cells exocytose their dense-core vesicle cargo in a Ca2+-dependent manner. Synaptotagmins (Syts) are a family of transmembrane proteins with C-terminal tandem C2 domains and are thought to regulate Ca2+-dependent vesicle exocytosis in a variety of cell types (see reviews; Chapman 2002; Südhof 2002; Koh & Bellen 2003; Fukuda 2006). SytI is an abundant synaptic vesicle protein and is thought to function as the major Ca2+ sensor for neurotransmitter release (Fernandez-Chacon et al. 2001; Mackler et al. 2002; Yoshihara & Littleton 2002; Nishiki & Augustine 2004) and to regulate synaptic vesicle docking, fusion and recycling via its C2 domains (Mikoshiba et al. 1995; Fukuda et al. 2000; Llinás et al. 2004). SytI has been the most extensively studied member of the Syt family, but there are at least 15 different Syt isoforms, and they have different affinities for Ca2+ and phospholipids, and different subcellular localizations (Fukuda 2003, 2006; Craxton 2004). Syt VII is an evolutionarily conserved Syt isoform that is abundantly expressed in non-neuronal tissues, and it is thought to function as a Ca2+ sensor for dense-core vesicle exocytosis in neuroendocrine PC12 cells (Sugita et al. 2001; Fukuda et al. 2002c, 2004; Wang et al. 2005) and for lysosomal exocytosis in fibroblasts (Martinez et al. 2000 and reviewed in Andrews & Chakrabarti 2005). Others and we previously showed that Syt VII is localized on dense-core vesicles, together with SytI and IX, and enhances dense-core vesicle exocytosis by PC12 cells (Fukuda et al. 2002c, 2004; Wang et al. 2005), suggesting that Syt VII functions as a vesicular Ca2+ sensor in neuroendocrine cells. However, it has never been elucidated whether Syt VII molecules are involved in the control of the dense-core vesicle docking step, or whether they modulate the kinetics of hormone secretion or the number of exocytotic events.

In this study, we established a PC12 cell line that stably expresses Syt VII-green fluorescent protein (Syt VII-GFP) or the Ca2+-binding-site-deficient mutant Syt VII-DN-GFP molecule (D172N/D303N substitutions; see Fig. 1A) (Fukuda et al. 2002a), and we used vesicle cargo-targeted fluorescent protein to investigate the effect of the wild-type and mutant Syt VII molecules on the motion of a single dense-core vesicle during exocytosis in the stable PC12 cells by total internal reflection fluorescence (TIRF) microscopy, also called evanescent wave or evanescence microscopy (Axelrod 1981; Tsuboi & Fukuda 2005, 2006a,b). The number of dense-core vesicle exocytotic events was significantly increased in the PC12 cells stably expressing Syt VII-GFP, whereas expression of Syt VII-DN-GFP or silencing of Syt VII-GFP with small interfering RNA (siRNA) decreased the number of exocytotic events. Interestingly, the majority of Syt VII-GFP-containing dense-core vesicles undergo exocytosis in the form of "full release" of the vesicle cargo, rather than "partial release". We discuss the possible role of Syt VII in dense-core vesicle exocytosis in PC12 cells based on our findings.


Figure 1
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  		Figure 1  Co-localization of Syt VII-GFP and Syt VII-DN-GFP with dense-core vesicle cargo marker NPY-CFP. (A) Schematic representation of Syt VII-GFP constructs used in this study. Syt VII and GFP are separated by a short Gly linker. TM, transmembrane domain. (B) Confocal image of fixed Syt VII-GFP stable PC12 cells showing the distribution of NPY-CFP (a); image of Syt VII-GFP fluorescence in the same cells (b); and overlay (c) of (a) and (b). Confocal image of fixed Syt VII-DN-GFP stable PC12 cells showing the distribution of NPY-CFP (d); image of Syt VII-DN-GFP fluorescence in the same cells (e); and overlay (f) of (d) and (e). Note that NPY-CFP co-localized well with Syt VII-GFP and Syt VII-DN-GFP (yellow in c and f, respectively). Bar = 5 µm. (C) Typical TIRF images of plasma membrane-docked NPY-CFP-containing vesicles before high-KCl stimulation of control (control), Syt VII-GFP stable (Syt VII), Syt VII-DN-GFP stable (Syt VII-DN) and Syt VII-siRNA-expressing cells (Syt VII-GFP + siRNA). Bar = 5 µm. (D) The density of plasma membrane-docked NPY-CFP-containing vesicles was determined by counting the vesicles in each image (n = 8 cells in each). Note that expression of Syt VII, Syt VII-DN or Syt VII siRNA had no effect on the number of plasma membrane-docked NPY-CFP-containing vesicles (D).

 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Dense-core vesicle cargos are localized in synaptotagmin VII-GFP-containing vesicles in PC12 cells

In our previous study, we showed that Syt VII-GFP molecules are mainly localized on dense-core vesicles and dramatically enhance Ca2+-dependent hormone secretion (Fukuda et al. 2004). We first investigated whether dense-core vesicle cargo proteins are targeted to the Syt VII-GFP-expressing vesicles in PC12 cells by labeling the dense-core vesicle cargo by expressing a 4-kDa-cargo protein neuropeptide Y fused to cyan fluorescent protein (NPY-CFP). Confocal microscopy showed that both Syt VII-GFP-positive vesicles (Fig. 1B, a–c; 74 ± 8%, n = 3 cells) and Syt VII-DN-GFP-positive vesicles (Fig. 1B, d–f and 71 ± 6%, n = 3 cells) co-localized well with NPY-CFP. To explore the effect of Syt VII-GFP and Syt VII-DN-GFP protein on the docking step of dense-core vesicle exocytosis in PC12 cells, we used TIRF microscopy to count the number of plasma membrane-docked NPY-CFP-containing vesicles before stimulation (Tsuboi & Fukuda 2005, 2006a,b ). As shown in Fig. 1C,D, expression of neither Syt VII-GFP nor Syt VII-DN-GFP protein altered the number of plasma membrane-docked NPY-CFP-containing vesicles in comparison with the control cells. We also assessed the effect of the specific siRNA against Syt VII in the stable PC12 cells on the number of plasma membrane-docked NPY-CFP-containing vesicles, but the Syt VII-siRNA had no effect on the number of plasma membrane-docked NPY-CFP-containing vesicles either (Fig. 1D), presumably because the endogenous Syt VII expression level in PC12 cells is very low (Zhang et al. 2002; Fukuda et al. 2004).

Effect of Syt VII-GFP and Syt VII-DN-GFP protein on monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y (NPY-mRFP) release

To investigate whether expression of Syt VII or Syt VII-DN protein modulates the kinetics of dense-core vesicle exocytosis, we analyzed the dynamics of single vesicle fusion events in a single NPY-mRFP-expressing vesicle near the plasma membrane by TIRF microscopy. After application of the high-KCl (70 mM) buffer, most of the NPY-mRFP-containing vesicles in the control, Syt VII-GFP, Syt VII-DN-GFP and Syt VII-siRNA-expressing cells briefly exhibited a diffuse cloud of fluorescent peptides (200–400 ms) immediately before their disappearance, consistent with the previous studies (Tsuboi et al. 2000, 2004; Tsuboi & Fukuda 2005, 2006a,b ), and the momentary cloud was associated with a marked increase in brightness (Fig. 2A and 5.2 s column). We next measured fluorescence intensity in the sequential images by digitizing the pixel values in the center of NPY-mRFP-containing vesicles; however, the kinetics or time course of individual NPY-mRFP release events was identical in all cell types (Fig. 2B).


Figure 2
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  		Figure 2  Effect of Syt VII-GFP, Syt VII-DN-GFP and Syt VII-siRNA expression on the kinetics of NPY-mRFP release. (A) Typical sequential images of a single NPY-mRFP vesicle observed after 70 mM KCl stimulation of control cells (control) and cells expressing Syt VII-GFP (Syt VII), Syt VII-DN-GFP (Syt VII-DN) or Syt VII-siRNA (Syt VII-GFP + siRNA), acquired at 200-ms intervals, through a TIRF microscope. The third column of images (5.2 s) shows a diffuse cloud of NPY-mRFP fluorescence, and the fourth column of images (5.6 s) shows disappearance of the cloud. (B) Time course of the fluorescence changes measured in the center of NPY-mRFP vesicles in the control (closed squares), Syt VII-GFP- (closed circles), Syt VII-DN-GFP- (closed triangles) and Syt VII siRNA-expressing (closed inverted triangles) cells. Mean fluorescence intensity before fusion was set equal to 100% (n = 18 vesicles in each experiment). Bar = 2 µm. Note that expression of Syt VII, Syt VII-DN or Syt VII siRNA had no effect on the kinetics of vesicle fusion of the dense-core vesicles, when dense-core vesicle exocytosis was monitored by NPY-mRFP. (C) The number of NPY-mRFP release events (R) during a 5-min stimulation period as a percentage of the number of plasma membrane-docked vesicles (D) before stimulation in the control (black bar), Syt VII-GFP stable (open bar), Syt VII-DN-GFP stable (hatched bar) and Syt VII-siRNA-expressing cells (shaded bar) observed by TIRF microscopy (n = 6 cells in each). Data are shown as means ± SEM. *P < 0.05 in comparison with the control.

 
Next, we counted the number of NPY-mRFP release events (R) during the 5-min stimulation period and the number of plasma membrane-docked vesicles (D) before stimulation and then calculated the ratio between R and D and expressed it as a percentage, that is, as normalized NPY-mRFP release events (Tsuboi & Fukuda 2006b). R/D (%) was significantly reduced in the Syt VII-DN-GFP stable cells in comparison with the control cells (hatched bar in Fig. 2C). By contrast, R/D (%) was significantly increased in the Syt VII-GFP stable cells (open bar), but it was reduced to the control cell level by Syt VII-siRNA (shaded bar in Fig. 2C), consistent with the results of our previous biochemical analysis (Fukuda et al. 2004).

Effect of Syt VII-GFP and Syt VII-DN-GFP protein on cyan fluorescent protein (CFP)-tagged tissue plasminogen activator (tPA-CFP) release

Although Syt VII expression had no effect on the kinetics of mRFP-NPY release (Fig. 2B), because the release kinetics of fluorescent peptides must be affected by their size, Syt VII may affect the kinetics of release of vesicle cargos larger than NPY-mRFP (4 kDa + 26 kDa). To investigate this possibility, we used a 63 kDa serine protease tPA (tissue plasminogen activator) found in endothelial, PC12 and chromaffin cell granules (Parmer et al. 1997) as a marker to monitor dense-core vesicle exocytosis, because previous experiments in PC12 cells and pancreatic clonal MIN6 ß-cells showed that some portions of tPA-GFP molecules are often retained in secretory vesicles after exocytosis (Taraska et al. 2003; Tsuboi et al. 2004). First, we confirmed the co-localization between tPA-CFP and Syt VII-GFP (or Syt VII-DN-GFP) in the stable PC12 cells by confocal microscopy. Most of the tPA-CFP-positive vesicles co-localized with Syt VII-GFP-positive vesicles (Fig. 3A, a–c; 78 ± 9%, n = 3 cells) or Syt VII-DN-GFP-positive vesicles (Fig. 3A, d–f; 72 ± 7%, n = 3 cells). We also confirmed that tPA-CFP and NPY-mRFP were mostly targeted to the same dense-core vesicles by confocal microscopy (Supplementary Fig. S1). We then counted the number of plasma membrane-associated tPA-CFP-containing vesicles by TIRF microscopy (Fig. 3B), but consistent with the results of NPY-mRFP, expression of Syt VII-GFP, Syt VII-DN-GFP or the Syt VII-siRNA had no effect on the number of plasma membrane-docked tPA-CFP-containing vesicles in comparison with the control cells (Fig. 3C).


Figure 3
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  		Figure 3  Co-localization of Syt VII-GFP and Syt VII-DN-GFP with dense-core vesicle cargo marker tPA-CFP. (A) Confocal image of fixed Syt VII-GFP stable PC12 cells showing the distribution of tPA-CFP (a); image of Syt VII-GFP fluorescence in the same cells (b); and overlay (c) of (a) and (b). Confocal image of fixed Syt VII-DN-GFP stable PC12 cells showing the distribution of tPA-CFP (d); image of Syt VII-DN-GFP fluorescence in the same cells (e); and overlay (f) of (d) and (e). Note that tPA-CFP co-localized well with Syt VII-GFP and Syt VII-DN-GFP (yellow in c and f, respectively). Bar = 5 µm. (B) Typical TIRF images of plasma membrane-docked tPA-CFP-containing vesicles before high-KCl stimulation of control (control), Syt VII-GFP stable (Syt VII), Syt VII-DN-GFP stable (Syt VII-DN) and Syt VII-siRNA-expressing cells (Syt VII-GFP + siRNA). Bar = 5 µm. (C) The density of plasma membrane-docked tPA-CFP-containing vesicles was determined by counting the vesicles in each image (n = 7 cells in each). Note that expression of Syt VII-GFP, Syt VII-DN-GFP or Syt VII siRNA had no effect on the number of plasma membrane-docked tPA-CFP-containing vesicles (C), the same as on the NPY-CFP-containing vesicles (Fig. 1D).

 
We also analyzed the dynamics of a single tPA-CFP-expressing vesicle movement just beneath the plasma membrane in the wild-type PC12 cells by TIRF microscopy (Fig. 4A). Depolarization of cells with high-KCl caused an increase in tPA-CFP fluorescence initially, which then slowly dimmed, presumably reflecting either partial release of tPA or slow vesicle resealing (referred to as "partial release" below; Fig. 4B). This observation is consistent with the previous data obtained in MIN6 cells (Tsuboi et al. 2004) and in PC12 cells (Taraska et al. 2003). Interestingly, we observed complete release of tPA-CFP by some exocytosing tPA-CFP vesicles in the form of a transient increase in fluorescence followed by diffusion away from the site of exocytosis (referred to as "full release" below; Fig. 4C).


Figure 4
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  		Figure 4  TIRF analysis of the kinetics of tPA-CFP release during high-KCl stimulation. (A) Typical TIRF image of plasma membrane-docked tPA-CFP vesicles observed before stimulation. The circles represent vesicles that have fused with the plasma membrane after stimulation. Bar = 10 µm. (B) Sequence images (400-ms intervals) of a single vesicle showed "partial release" of tPA-CFP (top panels; the number corresponds to the circle 1 in A). The changes in fluorescence intensity within circle 1 were plotted against time (bottom graph). Average fluorescence intensity prior to fusion was set equal to 100%. Bar = 1 µm. (C) Sequence images of a single vesicle showed "full release" of tPA-CFP (top panels). The changes in fluorescence intensity in circle 2 were plotted against time (bottom graph). Bar = 1 µm.

 
Syt VII modulates tPA-CFP release kinetics

Finally, to determine whether expression of Syt VII-GFP alters the ratio between partial and full tPA-CFP release events, we counted the number of partial and full tPA-CFP release events during the 5-min stimulation period. As shown in Fig. 5A,B, 42.3 ± 4.4% of the release events in the control cells (n = 4 cells; black bars) showed the tPA-CFP partial release, whereas the percentage of partial tPA-CFP release events was significantly decreased in the Syt VII-GFP stable cells (31.8 ± 2.8%, n = 4 cells; open bars). The increased fraction of full-release events in the Syt VII-GFP stable cells must be attributable to the Ca2+-binding function of Syt VII, because no change was observed in the Syt VII-DN-GFP stable cells or the Syt VII siRNA-expressing cells in comparison with the control cells (hatched and shaded bars, respectively). The same as the result for NPY-mRFP release (Fig. 2C), the probability of tPA-CFP release (i.e., R/D-value) by the Syt VII-GFP stable cells was significantly increased (Fig. 5C, open bar), whereas the probability of release by the Syt VII-DN-GFP stable cells was significantly decreased (hatched bar). Taken together, these findings indicate that the Ca2+-binding sites of Syt VII are required for both promotion of Ca2+-dependent hormone secretion and modulation of the release kinetics of tPA-CFP in PC12 cells.


Figure 5
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  		Figure 5  Effect of Syt VII-GFP, Syt VII-DN-GFP and Syt VII-siRNA expression on the kinetics of tPA-CFP release. (A and B) The percentage of partial release (A) and full release (B) events during high-KCl stimulation was calculated in control (closed bars), Syt VII-GFP stable (open bars), Syt-VII-DN stable (hatched bars) and Syt VII-siRNA-expressing cells (shaded bars). (C) The number of tPA-CFP release events (R) during 5-min stimulation as a percentage of the number of plasma membrane-docked vesicles (D) before stimulation in Syt VII-GFP-expressing cells observed by TIRF microscopy (n = 6 cells in each). Data are shown as means ± SEM. *P < 0.05 in comparison with the control.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In a previous study, we showed that stably expressing Syt VII-GFP molecules in PC12 cells dramatically enhanced high-KCl-induced NPY release from dense-core vesicles (Fukuda et al. 2004). In the present study, we explored the molecular mechanism of the facilitation of dense-core vesicle exocytosis by Syt VII by monitoring two fluorescently labeled dense-core vesicle cargos of different molecular size (i.e., NPY-mRFP and tPA-CFP). Although involvement of Syt VII in the docking step of dense-core vesicles to the plasma membrane was suggested by the previous in vitro biochemical study (Chieregatti et al. 2004), the direct observations of the number of plasma membrane-docked NPY-CFP- or tPA-CFP-containing vesicles by TIRF microscopy in the present study clearly indicated that Syt VII does not promote vesicle docking or alter vesicle distribution (Figs. 1 and 3), and instead increases the probability of both NPY-mRFP and tPA-CFP release events. A more detailed analysis indicated that Syt VII enhances tPA-CFP full-release events, rather than partial-release events (Fig. 5A,B), suggesting that Syt VII modulates expansion of the fusion pore and/or the duration of fusion pore opening.

How does Syt VII modulate the release kinetics of tPA-CFP in PC12 cells? Since the Ca2+-binding-site-deficient Syt VII-DN mutant failed to modulate the release kinetics (Fig. 5A,B), Ca2+-binding activities of the tandem C2 domains must be required for modulation of the release kinetics by Syt VII. We and others previously showed that the Syt VII C2 domains exhibit Ca2+/phospholipid-, t-SNARE- (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and self-oligomerization activity (Fukuda & Mikoshiba 2000, 2001; Shin et al. 2002; Sugita et al. 2002; Fukuda et al. 2002a, 2004; Rao et al. 2004; Rickman et al. 2004), the same as the C2 domains of Syts I and IX. In contrast to SytI, however, phospholipid binding to the C2 domains of Syt VII has been shown to be activated by 5–10 times lower concentrations of Ca2+ than required to activate SytI or IX (Sugita et al. 2001, 2002; Wang et al. 2005), suggesting that such high-affinity Ca2+-sensing ability of Syt VII may stabilize the fusion pore (or prolong fusion pore opening) during exocytosis. Alternatively, Syt VII may increase fusion pore size (Fukuda et al. 2002a; Wu et al. 2003) by formation of a homo-oligomer or hetero-oligomer with endogenous Syts I and IX (Fukuda et al. 2004).

Although the Ca2+-binding-site-deficient Syt VII-DN-GFP mutant did not alter the ratio between partial and full tPA-CFP release events, the mutant has a dominant negative effect and reduces the probability of exocytosis (Figs. 2C and 5C). Since the Syt VII-DN mutant is unable to bind Ca2+ (and thus Ca2+-dependent phospholipid binding and oligomerization do not occur), the mutant may trap t-SNARE hetero-dimers (i.e., SNAP-25 and syntaxin-1a), which are required for dense-core vesicle exocytosis (Rickman et al. 2004), Ca2+-independently. Alternatively, Syt VII-DN-GFP Ca2+-independently binds endogenous Syts I and IX via the N-terminal domain (Fukuda et al. 2004) and may inhibit their function in dense-core vesicle exocytosis.

In summary, we have demonstrated by live cell TIRF imaging that the Syt VII molecules on dense-core vesicles in the stable PC12 cells enhance Ca2+-dependent hormone secretion by increasing the total number of exocytotic events and the number of full fusion events (i.e., modulation of release kinetics). Since the endogenous expression level of Syt VII molecules in PC12 cells is much lower than that of Syts I and IX (Fukuda et al. 2002b, 2004; Zhang et al. 2002), in the future it will be important to determine whether endogenous Syt VII molecules in other neuroendocrine cells also modulate the kinetics of hormone secretion. A more recent analysis of endogenous Syt VII molecules in pancreatic ß-cells, where Syt I is not endogenously expressed (Gao et al. 2000; Gut et al. 2001), has indicated that Syt VII plays a critical role in insulin secretion by ß-cells (B.R. Gauthier & C.B. Wollheim, personal communications), consistent with our findings. We therefore speculate that Syt VII actually functions as a Ca2+-sensor for dense-core vesicle exocyotosis by certain neuroendocrine cells.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Site-directed mutagenesis and plasmid construction

A mutant Syt VII containing Asp-to-Asn mutations at amino acid positions 172 (within the C2A domain) and 303 (within the C2B domain) was prepared as previously described (Fukuda et al. 2002a) (Fig. 1A). The mutant Syt VII-DN fragment was subcloned into the pEF-T7 expression vector modified from pEF-BOS (Mizushima & Nagata 1990; Fukuda et al. 1994, 1999), and the mutant Syt VII fragment was subcloned into the pShooter-FLAG-GFP vector as previously described (Fukuda et al. 2002c; Saegusa et al. 2002). The sequence of all plasmid inserts was verified by automated sequencing. A plasmid encoding CFP-tagged tissue plasminogen activator (tPA) were generously provided by Dr Bethe A. Scalettar (Lochner et al. 1998). Other expression vectors, including tPA-mRFP, NPY-CFP, NPY-mRFP and pSilencer-Syt VII, were prepared as previously described (Fukuda et al. 2004; Tsuboi et al. 2004).

Screening of PC12 cell lines stably expressing FLAG-Syt VII-DN-GFP protein

PC12 cells (2 x 106 cells, the day before transfection) were cultured on 10-cm dishes in Dulbecco's modified Eagle's medium containing 10% horse serum and 10% fetal bovine serum at 37 °C under 5% CO2. The expression vector encoding the Syt VII-DN-GFP fusion protein was transfected into PC12 cells with LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The transfected cells expressing a neomycin-resistance gene were selected by using Geneticin (Invitrogen) at a concentration of 400 µg/mL. Expression of the Syt VII-DN-GFP fusion protein in established cell lines was verified by immunocytochemical analysis.

Immunocytochemisty and confocal microscopy

For microscopic analysis, the cloned PC12 cells stably expressing Syt VII-GFP or Syt VII-DN-GFP fusion proteins were transfected with either NPY-CFP or tPA-CFP. Two days after transfection the cells were fixed with 4% paraformaldehyde (Wako Pure Chemicals, Osaka, Japan) for 20 min (Saegusa et al. 2002) and then examined with a confocal microscope (Fluoview500, Olympus, Tokyo, Japan). The bottom of the cell was located by inspection in the confocal mode, and the focal plane was then maintained at that level throughout the experiments. The images were processed with METAMORPH software (version 6.3, Universal Imaging Corporation, Downingtown, PA).

TIRF microscopy

For TIRF imaging, original PC12 cells, Syt VII-GFP stable cells or Syt VII-DN-GFP stable cells were plated on to poly L-lysine-coated coverslips, and the cells were transfected with 4 µg of NPY-CFP, NPY-mRFP, tPA-CFP, tPA-mRFP or pSilencer-Syt VII by using LipofectAMINE 2000 as described above. The imaging was performed in a modified Ringer buffer at 37 °C (RB: 130 mM NaCl, 3 mM KCl, 5 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose and 10 mM HEPES, pH 7.4). Stimulation with high-KCl was achieved by perfusion of 70 mM KCl containing RB (NaCl was reduced to maintain the osmolarity). We monitored exocytosis of NPY-mRFP or tPA-CFP at the single vesicle level by using a TIRF microscope similar to that previously described (Tsuboi et al. 2000; Tsuboi & Fukuda 2005). In brief, a high numerical aperture objective lens (Plan Apochromatic, 100 x, NA = 1.45, infinity corrected, Olympus) was mounted on an inverted microscope (IX81, Olympus), and incident light for total internal reflection illumination was introduced from the high numerical aperture objective lens through a single mode optical fiber and two illumination lenses (IX2-RFAEVA-2, Olympus). Images were acquired every 200 ms. To analyze the TIRF imaging data, single exocytotic events were selected manually, and the average fluorescence intensity of individual vesicles in a 0.7 µm x 0.7 µm square placed over the vesicle center was calculated. The number of fusion events during a 5-min period was counted manually. Data are reported as means ± SEM of at least four individual experiments. Statistical significance and differences between means were compared by one-way ANOVA followed by a Newman–Keuls multiple comparison test with GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego, CA).


   Acknowledgements
 
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants 17657067, 18022048, 18050038, 18057026 and 18207015 to M.F.; Grant-in-Aid for Young Scientists (A) 18689008 to T.T.), by the Kato Memorial Bioscience Foundation, by the Brain Science Foundation (to M.F.), by the Astellas Foundation for Research on Metabolic Disorders (to M.F.), by the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.T.), by the Uehara Memorial Foundation (to T.T.), and by the FY2005 DRI Research Grant (to T.T.). T.T. was also supported by Special Postdoctoral Researchers Program of RIKEN. We thank Dr Bethe A. Scalettar (Lewis & Clark College, Oregon, USA) for kindly donating tPA-CFP, and Eiko Kanno and Megumi Satoh for technical assistance.


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: nori{at}mail.tains.tohoku.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Andrews, N.W. & Chakrabarti, S. (2005) There's more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII. Trends Cell Biol. 15, 626–631.[CrossRef][Medline]

Axelrod, D. (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89, 141–145.[Abstract/Free Full Text]

Chapman, E.R. (2002) Synaptotagmin: a Ca2+ sensor that triggers exocytosis? Nat. Rev. Mol. Cell Biol. 3, 498–508.[CrossRef][Medline]

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Received: 30 October 2006
Accepted: 15 January 2007




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