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Journal of Physiology (2001), 532.3, pp. 649-659
© Copyright 2001 The Physiological Society
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ABSTRACT |
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rat glioma hybrid NG108-15 cells in culture.
-COP, an individual coat subunit of the coatomer complex present on Golgi-derived vesicles.
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INTRODUCTION |
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Neuronal calcium sensor-1 (NCS-1) is a protein bearing four EF-hand binding motifs (McFerran et al. 1998) and functions as a calcium sensor, because it co-operatively binds two Ca2+ with affinity constants of 8.9 10-4 and 1.4 10-8 M, respectively (Cox et al. 1994). NCS-1, originally termed frequenin, exhibited an enhanced frequency-dependent facilitation of neurotransmitter release, and was first described in Drosophila and Xenopus (Pongs et al. 1993; Angaut-Petit et al. 1993; Poulain et al. 1994; Olafsson et al. 1995) and more recently in Crustacea (Jeromin et al. 1999). The protein sequence of Drosophila frequenin contains 187 amino acids. NCS-1 (frequenin) is a member of a superfamily of related Ca2+-binding proteins. Within this family are two subclasses of proteins; type A includes proteins such as visinin, recoverin and s-modulin, which are expressed in photoreceptors; type B includes proteins expressed in neurons such as neurocalcin, hippocalcin, frequenin/NCS-1 and VILIP (Paterlini et al. 2000). Frequenin has been shown to be localized at the frog neuromuscular junction, muscle spindle and nerve (Werle et al. 2000) and to enhance synaptic efficacy at the Drosophila and Xenopus neuromuscular junction (Pongs et al. 1993; Rivosecchi et al. 1994; Angaut-Petit et al. 1998). Overexpression of frequenin in transgenic flies only increased evoked transmitter release, while acute infusion of Xenopus frequenin as a recombinant protein increased both spontaneous and evoked release. The basis for this discrepancy is unclear at present.
Though frequenin was shown to co-localize with synaptophysin in vestibular efferent endings in mouse inner ear (Sage et al. 2000) and in rat brain and neuroendocrine (phaeochromocytoma PC12) cells (authors' unpublished results), it is not known whether it is related to synaptotagmin, a vesicle-associated protein that also binds Ca2+. In rat PC12 cells, overexpression of NCS-1 (Nef et al. 1995: Olafsson et al. 1997; McFerran et al. 1998, 1999; Martone et al. 1999) led to an increase in dense-core granule exocytosis (McFerran et al. 1998) only in intact, but not in permeabilized cells. The authors concluded that NCS-1/frequenin may play an indirect modulatory role in the secretion of dense-core vesicles. Whether or not NCS-1 plays a more direct role in neurotransmitter release, especially from clear vesicles containing small molecules such as acetylcholine or glutamate, has not been elucidated.
To address this question, we have chosen the NG108- myocyte co-culture model (Nirenberg et al. 1983a,b). It has been reported that mouse neuroblastoma rat glioma hybrid NG108-15 cells do not form functional autaptic synapses (Nelson et al. 1976). In contrast, co-culturing of NG108-15 cells with muscle cells leads to the formation of cholinergic synapses, involving intracellular cAMP-dependent signalling pathways (Nelson et al. 1976; Puro & Nirenberg, 1976; Higashida et al. 1981; Yano et al. 1984; Ogura et al. 1990; Kimura & Higashida, 1992). In the present study, we examined the role of NCS-1. First, we analysed the subcellular localization of endogenous NCS-1 in NG108-15 cells. Second, we established an overexpression method for rat NCS-1 by transient and stable transfection of the wild-type rat NCS-1 or rat NCS-1 co-expressed with green fluorescent protein (GFP) in NG108-15 cells. We demonstrate here an increase in functional synapse formation with myotubes and an increased acetylcholine release in transfected NG108-15 cells, by recording muscle endplate potentials arising from spontaneous or evoked release of acetylcholine.
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METHODS |
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Cell culture of control and transfected NG108-15 cells
Wild-type mouse neuroblastoma rat glioma hybrid NG108-15 cells (Nirenberg et al. 1983a; Hamprecht et al. 1985) and NG108-15 cells transfected with expression vector alone or NCS-1 cDNA were maintained as described previously (Morikawa et al. 1995; Zhong et al. 1995a,b, 1997). NG108-15 cells were differentiated with 0.25 mM dibutyryl cAMP as described previously (Higashida & Brown, 1986; Higashida, 1988).
Preparation of NCS-1 cDNA constructs
DNA encoding full-length rat NCS-1 (GenBank accession no. L27421) was described previously (McFerran et al. 1998). The NCS-1 cDNA with or without an engineered Kozak consensus sequence was inserted into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA, USA). Two cDNAs with a Kozak sequence, designated as pcDNA3RFrqF12 and pcDNA3RFrqF21, were constructed. The pcDNA3 vector was used for mock transfection. pCINeoRFrq8 without a Kozak sequence and pCINeoGFPRFrq19B1 with a Kozak sequence are based on a bi-cistronic vector co-expressing GFP. The DNA constructs were amplified in E. coli and purified with a plasmid DNA purification kit (Qiagen, Hilden, Germany).
Stable NG108-15 clones overexpressing NCS-1 were generated by transfecting the cells with pcDNA3RFrqF12 and subsequent selection of the transfectants in 300 µg ml-1 G418 (Gibco-BRL Life Technologies, Grand Island, NY, USA). Individual clones, designated as NG108-FA1, NG108-FHB1 and NG108-FC1 cells, were characterized by Western blotting.
Transient transfection of NCS-1 cDNA constructs into NG108-15 cells
NG108-15 cells were plated in 60 mm dishes (5 105 cells per dish). Twenty-four hours later, the cells were transfected with 10 ng each of the above five pcDNA3 constructs mixed with 25 µl Lipofectamine or Lipofectamine plus (Gibco-BRL). The transfection efficiency was about 15-35 % (n = 5) for NG108-15 cells transfected with pcDNA3RFrqF12-lipofectamine judging from the number of fluorescence-positive cells. Cells were harvested 1 day after transfection and further cultured in fresh 60 mm dishes for 4 days for immunoblot analysis, or co-cultured with myotubes in 35 mm dishes for 1-7 days for electrophysiological measurements.
Antibodies
Antibodies against NCS-1 were raised in New Zealand White rabbits and are described in detail elsewhere (Werle et al. 2000). For Western blotting and immunocytochemistry, affinity-purified antibodies were used. A different antibody against the full-length protein was also generated in chicken, and is described in further detail elsewhere (Weisz et al. 2000). This antibody was used for some of the co-localization experiments. The anti--COP antibody was kindly provided by Y. Ikehara (Fukuoka University, Japan) and the anti-SNAP-25 monoclonal antibody was purchased from Sternberger Monoclonals (Baltimore, MD, USA).
Secondary antibodies used for immunocytochemistry were Texas Red goat anti-mouse IgG (H+L) conjugate (Molecular Probes, Eugene, OR, USA), fluorescein anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA, USA), and Cy3-conjugated donkey anti-chicken IgG (Jackson Immunoresearch, West Grove, PA, USA).
Immunoblot analysis
The different types of NG108-15 cells were cultured for 4 days with 0.25 mM dibutyryl cAMP. Cells were solubilized in 5 SDS-sample buffer and the protein concentration was determined for each protein extract by a Bio-Rad protein assay method (Hercules, CA, USA). A 200 µg sample of each protein was separated by 8 % SDS-PAGE (Laemmli, 1970) and then transferred electrophoretically to nylon membranes (Nihon Millipore, Yonezawa, Japan). The membranes were first incubated with 5 % non-fat milk in PBS (blocking buffer) for 60 min at room temperature, according to the method described previously (Zhong et al. 1997). The membranes were then incubated overnight at 4 °C with the rabbit polyclonal antibodies that recognize mouse and rat NCS-1, and afterwards washed 3 times for 10 min with the blocking buffer. The membranes were then incubated for 1 h at room temperature in blocking buffer containing horseradish peroxidase-conjugated goat anti-rabbit antibody (EY Laboratories Inc., San Mateo, CA, USA). The membranes were washed 3 times for 10 min with the blocking buffer and reactive proteins were visualized with a Western blotting analysis system (Amersham, Bucks, UK).
Immunostaining
For immunocytochemical analysis, NG108-15 cells were plated on collagen-coated coverslips, differentiated with 0.25 or 0.5 mM dibutyryl cAMP for 3 days, then fixed with 4 % paraformaldehyde and permeabilized in 0.4 % saponin as described elsewhere (Andersson et al. 2000). Primary and secondary antibodies were applied for 30 min each and the mounted cells were examined in a Radiance Plus laser-scanning confocal imaging system (Bio-Rad, Hercules, CA, USA). The pictures were processed using Adobe Photoshop software and printed using a Fuji Pictrography 3000 printer.
Neurite outgrowth analysis
Various types of NG108-15 cells were cultured on collagen-coated coverslips in the presence or absence of 0.25 or 0.5 mM dibutyryl cAMP for 2-4 days. The cells were photographed using a phase contrast microscope for quantitative analysis and in some experiments images were processed on a computer. A neurite was defined as a process extending at least one cell diameter from the cell body.
Co-culture with myotubes and electrophysiological measurements
New-born rats were chilled on ice, decapitated and then rinsed in 70 % ethanol. Rat hindlimb muscle cells isolated by trypsinization were cultured for 7 days and grown to form myotubes 20-30 µm in diameter and > 100 µm long as described previously (Puro & Nirenberg, 1976; Higashida et al. 1981). Non-transfected and mock- or NCS-1-transfected NG108-15 cells were overlaid at a density of 2 104 cells per 35 mm dish on the already fused and contracting muscle cells (Yano et al. 1984; Zhong et al. 1997). The co-culture was maintained in DMEM supplemented with 10 % horse serum, 100 µM hypoxanthine, 16 µM thymidine and 0.25 mM dibutyryl cyclic AMP for 1-7 days. The co-culture medium was replaced with a recording medium (10 mM Hepes-buffered DMEM supplemented with 2 mM CaCl2 and 0.1 mM choline chloride), as described previously (Nelson et al. 1976). Pre- and postsynaptic activities were electrophysiologically studied by a conventional intracellular recording method with sharp microelectrodes filled with 1 M potassium citrate (7-15 M
) (Yano et al. 1984). Membrane potentials of myotube and hybrid cells were amplified via an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA, USA). Membrane potentials of DC-coupled or high gain RC-coupled recordings were continuously monitored on a Nihon Koden thermal array recorder (model RTA-1100, Tokyo, Japan) with frequency characteristics of DC to 10 kHz. The noise level was usually less than 0.2 mV. The presence of miniature endplate potentials (MEPPs) in a given myotube that showed synaptic connection to NG108-15 cells morphologically was judged by eye from the potential waveform on an oscilloscope and from a rapid upwards deflection (with a time constant of 1-2 s) above the noise level (usually > 0.3 mV) on a chart record. To avoid contamination of other responses such as muscle contraction, we recognized a pair of cells as synaptically connected if their frequency was greater than four events during a stable recording for a minimum of 2 min. These criteria are the same as in previous studies (Nirenberg et al. 1983b; Higashida, 1988).
In such pairs, endplate potentials (EPPs) elicited 1-50 ms after the peak of the action potentials of NG108-15 cells were considered to be evoked EPPs. Action potentials were usually evoked by injecting 0.1-3 nA of depolarizing current for 0.2-1 s into NG108-15 cells through the bridge circuit in Axoclamp-2A. The failure rate indicates the mean number of quanta released by an impulse (m) given by the formula: m = lnT/F, where T represents the total number of action potentials for a hybrid cell and F represents the number of action potentials that failed to evoke a response in a muscle cell (Katz, 1966).
Statistical analysis
All values are represented as means ± S.E.M. Homogeneity of variances was tested with Fisher's F test followed by Student's t test using two-way analysis of homogeneous variance.
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RESULTS |
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Endogenous NCS-1 in NG108-15 cells
The presence of NCS-1 at the mRNA level in wild-type NG108-15 cells was confirmed by Northern blotting (data not shown). NCS-1 protein in NG108-15 cells was detected by immunoblot analysis using rabbit polyclonal antibodies against mouse/rat NCS-1, as shown in Fig. 1. These antibodies detected a single band at the predicted molecular mass of mouse/rat NCS-1 of 21 kDa.
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Figure 1. Immunoblot analysis showing NCS-1 overexpression in NG108-15 cells using rabbit anti-mouse/rat NCS-1 antibodies Non-transfected cells (lane 1), mock-transfected cells (lane 2), stably transfected clones of NG108-FHB1 (lane 3) or NG108-FA1 (lane 4) cells, and cells transiently transfected with pcDNA3RFrqF12 (lane 5) were cultured for 4 days with 0.25 mM dibutyryl cAMP. The cells were solubilized, and 200 µg of protein for each cell type was used for electrophoresis. Note that the three types of transfected cells show a strong immunoreactive band at around 21 kDa, unlike with the non-transfected or mock-transfected cells.
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Immunochemical localization of NCS-1 in dibutyryl cAMP-differentiated NG108-15 cells
NCS-1 immunoreactivity was punctate in the cytoplasm of individual NG108-15 cells. There was also prominent NCS-1 immunoreactivity in the plasma membrane (Fig. 2A, B and E) and in large granules distributed in the cytoplasm as demonstrated by both anti-NCS-1 antibodies. The chicken anti-NCS-1 antibody gave a more punctate staining pattern as compared to the rabbit NCS-1 antiserum. NCS-1 and the Golgi marker -COP, a coat subunit of the coatomer complex of Golgi-derived vesicles (Oprins et al. 1993; Torii et al. 1995), had distinct subcellular localization patterns; punctate NCS-1 immunoreactivity was present in the cytoplasm and in the plasma membrane (Fig. 2D), whereas -COP was present in the perinuclear region representing Golgi-derived vesicles (Oprins et al. 1993; Fig. 2C). Therefore, there is no evidence for co-localization of NCS-1 and -COP. Double labelling of NCS-1 and the exocytotic protein SNAP-25 (Bark et al. 1995; Andersson et al. 2000; Fig. 2F) showed that the two immunoreactivities were partially co-localized (yellow) in the plasma membrane (Fig. 2G).
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Figure 2. Confocal images of NCS-1 immunoreactivity in NG108-15 cells differentiated with dibutyryl cAMP Images of endogenous NCS-1 were obtained by confocal microscopy after incubation with chicken (A and B) or rabbit (E) affinity-purified polyclonal antibody to NCS-1 and after direct double labelling with rabbit antiserum to
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NCS-1 immunoreactivity was investigated in NG108-15 cells overexpressing NCS-1 and co-cultured with rat myotubes (Fig. 3). In neurites and growth cones, NCS-1 was mainly present at the plasma membrane and partially co-localized with SNAP-25 (Fig. 3A-C). In areas of cell-cell contact between processes of NG108-15 cells and rat myotubes, NCS-1 immunoreactivity showed a different and more punctate staining pattern than in growth cones (Fig. 3D). There was only a partial co-localization between NCS-1 and SNAP-25 at synaptic contacts between NG108-15 cells and myotubes (Fig. 3D-F). Noteworthy is that the large NCS-1-immunoreactive cytoplasmic granules observed in mono-cultures of NG108-15 cells (Fig. 2) were absent in the co-cultures (data not shown).
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Figure 3. NCS-1 immunoreactivity in growth cones and at synaptic contacts Images obtained with confocal microscopy of NG108-15 cells overexpressing NCS-1 and co-cultured with rat myotubes. The cells were double labelled with antibodies against NCS-1 (A and D; green) and SNAP-25 (B and E; red). In C and F the images showing NCS-1 and SNAP-25 labelling are combined and the yellow colour indicates the co-localization of the two proteins. Note the co-localization of NCS-1 and SNAP-25 in the plasma membrane of a growth cone (see A-C) and in a cell associated with myotubes (see D-F). Asterisks indicate muscle cells and arrows indicate the co-localization of NCS-1 with SNAP-25 in NG108-15 cells. Bars = 10 µm.
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Overexpression of NCS-1 in NG108-15 cells
The overexpression of rat NCS-1 in transiently and stably transfected NG108-15 cells was confirmed by Northern blotting (data not shown) and Western blotting (Fig. 1). We did not attempt to quantify the relative level of overexpression of NCS-1, as the value is likely to represent an underestimation in the transiently transfected cells.
Microfluometric analysis revealed that 20-30 % of individual NG108-15 cells exhibited strong fluorescence 3 days after transfection of the bi-cistronic vector (data not shown).
Neurite outgrowth induced by dibutyryl cAMP in NG108-15 cells transiently or stably transfected with GFP-NCS-1
The effects of NCS-1 overexpression on neurite outgrowth were first examined in transiently transfected NG108-15 cells that were photographed under transmitted light. NCS-1 expression was identified by GFP fluorescence. NG108-15 cells that had been cultured in the presence of 0.25 mM dibutyryl cAMP for 4 days were used. With cAMP treatment, long and branched neurites emerged in both GFP-negative and -positive NG108-15 cells as reported previously (Nirenberg et al. 1983a,b). However, the number of neurites per cell and branches per neurite were slightly different between the two types of cells.
The mean number of neurites per cell in three identical experiments was 3.60 ± 0.87 in 47 GFP fluorescence-negative cells and 2.02 ± 0.94 in 42 GFP-positive cells (P < 0.001). The mean length of neurites in GFP-negative and -positive cells was 19.1 ± 5.8 µm (n = 47) and 18.4 ± 6.3 µm (n = 42), respectively. On average each neurite had 2.5 ± 0.57 (n = 47) and 1.4 ± 0.38 (n = 42) branches in GFP-negative and -positive cells, respectively (P < 0.001).
Next, this tendency was re-examined in clonal NG108-15 cells transformed to stably express NCS-1. As shown in Fig. 4, values of the mean number of neurites per cell, mean length of neurites and mean number of branches of neurites in three NCS-1-overexpressing cells were noticeably smaller than those in non-transfected and mock-transfected control cells. The results obtained in transiently transfected and stably transformed NG108-15 cells suggest that NCS-1 overexpression does not promote but rather slightly suppresses the neurite outgrowth initiated by dibutyryl cAMP.
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Figure 4. Plots of the mean number of neurites per cell (A), mean length of neurites (B) and number of branches (C) in different clonal cells Results presented in the graphs were obtained from computer images of photographs of two sets of control NG108-15 cells (48 non-transfected cells, 16 mock-transfected cells) and three sets of cells stably transformed with NCS-1 (36 NG108-FA1, 50 NG108-FHB1 and 73 NG108-FC1 cells) in three independent experiments. Cells were cultured for 4 days in the presence of 0.25 mM dibutryl cAMP. Significant differences from control values in non-transfected and mock-transfected cells are indicated (* P < 0.01; ** P < 0.001).
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Enhancement of functional synapse formation induced by dibutyryl cAMP in NCS-1-transfected NG108-15 cells
We next determined whether the expression of NCS-1 in NG108-15 cells alters functional synapse formation, using the presence or absence of MEPPs in myotubes as an electrophysiological indicator. The number of myotubes with MEPPs increased with time when co-cultures were treated with 0.25 mM dibutyryl cAMP for 1-7 days (Fig. 5). The rate of synapse formation by NCS-1-transfected cells was not significantly different from that of non-transfected or mock-transfected cells during the early phase of co-culture (days 1-3), but was significantly higher during the late phase (days 4-7). The mean level of functional synapse formation at days 5-7 was 40.5 ± 5.8 % (n = 93) for non-transfected cells, 39.2 ± 6.9 % (n = 104) for mock-transfected cells and 70.3 ± 6.3 % (n = 104) for NCS-1-transfected cells, respectively (P < 0.01).
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Figure 5. Effect of NCS-1 transfection in NG108-15 cells on functional synapse formation with myotubes Functional synapse formation was examined in non-transfected (
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The frequency of spontaneous transmitter release was estimated by MEPP frequency, which varied between each synapse pair (data not shown). The mean MEPP frequency observed during days 4-7 was 3.3 ± 1.5 events min-1 (n = 19) in non-transfected cells, 4.4 ± 3.5 events min-1 (n = 16) in mock-transfected cells and 5.4 ± 3.8 events min-1 (n = 29) in NCS-1-transfected cells, respectively, with no significant difference between the three types of cells.
The effect of overexpression of NCS-1 on synapse formation was confirmed by the use of different constructs of NCS-1 cDNA and another transfection reagent (Lipofectamine plus). Though we used four different constructs with or without the Kozak sequence and also with or without GFP in the expression vector, all DNAs tested increased synapse formation by up to 60-85 % without exception (data not shown). Transfection of these cDNAs using Lipofectamine plus also slightly and equally elevated the synapse formation rate. One possible explanation for the lack of a significant difference between different cDNA constructs and transfection reagents might be that the efficiency of synapse formation is saturated at high levels.
Evoked synaptic potentials between stably transfected cells and myotubes
To verify the role of NCS-1 in evoked acetylcholine release and to overcome the uncertainty arising from transient transfection, we established NG108-15 cell clones stably expressing rat NCS-1, designated NG108-FA1 (Fig. 1), NG108-FHB1 (Fig. 1) and NG108-FC1 cells. By monitoring the number of action potentials evoked by a series of depolarizing current steps in a current-clamp mode (see Fig. 2 in the report by Takahashi et al. 1999), we found the membrane excitability as an overall electrical neuronal feature of these clones to be similar to that of control cells (data not shown). After co-culturing these stable clones with myotubes under conditions identical to those for transiently transfected cells, the membrane potentials of NG108-15 cells and myotubes were simultaneously recorded. Figure 6 shows two examples in which action potentials elicited in two NG108-FHB1 cells evoked EPPs in a paired myotube. The latency in the generation of EPPs after action potentials varied from a few milliseconds to about 100 ms, as demonstrated for wild-type NG108-15 cells (Nelson et al. 1976, 1978). The proportion of action potentials that evoked EPPs varied with different cell types and was higher in stably transfected clones compared with wild-type NG108-15 cells. The mean number of quanta (m) liberated by an action potential was calculated in each clone from the percentage of action potentials that failed to generate EPPs. The mean m value for wild-type and mock-transfected NG108-15 cells ranged from 0.01 to 0.36, with mean values for each of 0.06 ± 0.02 (n = 19) and 0.09 ± 0.02 (n = 17), respectively, which was very low. In NCS-1-transformed clones, the m value was significantly increased (P < 0.001), with a mean of 0.63 ± 0.12 (n = 7), 0.68 ± 0.14 (n = 4) and 0.47 (n = 2) for NG108-FA1, NG108-FHB1 and NG108-FC1 cells, respectively (Fig. 7). The maximal quantal content, however, did not exceed 1.14, 0.92 and 0.58 for the three clones, respectively.
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Figure 6. Synaptic interaction between NG108-FHB1 cells and myotubes Records in A and B are shown on different time scales. Single action potentials (Vh, membrane potential of hybrid cells) were elicited in two NG108-FHB1 cells by each depolarizing stimulation (I) of 0.6 nA (A) or 0.9 nA (B) for 1 s. Five or four traces of muscle activity are high gain AC-coupled records (Vm, membrane potential of muscle cells). Endplate potentials evoked soon after the peak of the action potentials are indicated by arrows or arrow heads. Some stimuli (1st and 5th stimuli) evoked double end-plate potentials, as observed in control NG108-15 cells (see Fig. 2A in the report by Nelson et al. 1978). Membrane potentials of NG108-FHB1 and muscle cells are, respectively, -40 and -59 mV in A and -43 and -70 mV in B.
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Figure 7. Plot of mean quantum value (m) in different clonal cells The number of quanta liberated by an action potential (m) for each clone was obtained from the number of cells in parentheses. * Significant difference from control values in wild-type and mock-transfected NG108-15 cells (P < 0.001).
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NG108-15 cells sometimes exhibited two to five repetitive action potentials elicited with stimuli of 0.2-0.5 nA, with inter-spike intervals of > 100 ms. Double pulse protocols are usually very difficult to apply to NG108-15 cells, as the stimulation is usually required to last 100-200 ms for the whole time course of the action potential. Therefore, facilitation of EPPs and frequencies as evoked by the double pulse protocol (Pongs et al. 1993) could not be appropriately examined.
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DISCUSSION |
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In the present study, we have shown that overexpression of rat NCS-1 in NG108-15 cells increased functional synapse formation with myotubes and potentiated the release of acetylcholine. However, overexpression of NCS-1 did not stimulate additional neurite outgrowth in response to dibutyryl cAMP-induced differentiation compared to control cells, but rather reduced neurite outgrowth. This is, to our knowledge, the first report of the role of NCS-1 in synaptic physiology in mammalian cells. In accordance with our findings, overexpression of frequenin in Xenopus also enhanced basal and evoked synaptic transmission (Olafsson et al. 1995), while in Drosophila only evoked release was enhanced (Rivosecchi et al. 1994).
In our previous overexpression experiments in NG108-15 cells with cDNAs coding for human L1 (Zhong et al. 1997) and rat synapsins (Zhong et al. 1999), we observed an enhancement of functional synapse formation under a similar protocol. In the case of the L1 extracellular matrix protein, however, the promotion of synapse formation was largely accompanied by elongation or maturation of neurites. In other words, it was due to an increased number of encounters between the two cells of the pre- and postsynaptic elements. In the second case with synapsins, the enhancement of synapse formation was due to morphological changes (Han et al. 1991; Fried et al. 1995) followed by an increase of acetylcholine release (Zhong et al. 1999). In sharp contrast, the reduction of neurite outgrowth after NCS-1 overexpression, a result similar to that reported previously in Drosophila (Angaut-Petit et al. 1998), suggests that the enhancement acetylcholine release results from synapse maturation and formation stimulated by NCS-1 expression.
The mechanisms by which NCS-1 upregulates acetylcholine secretion are not known. Is it conceivable that NCS-1 might modulate cAMP-dependent intracellular signalling pathways and thereby affect synapse formation and ion channel activities (Nirenberg et al. 1983b). The effect of NCS-1 appeared in the late phase of culture after treatment with dibutyryl cAMP. This delay might reflect the structural changes underlying the remodelling of the release machinery.
It is interesting to note that NCS-1 has been shown to interact with phosphatidylinositol 4-kinase (PI4K) in yeast (Hendricks et al. 1999). These studies in yeast also show that NCS-1, called FRQ1 in yeast, is an essential gene, as the mutant is lethal (Hendricks et al. 1999). Transgenic strategies in mice will also be helpful to address the physiological role of NCS-1.
Phosphatidylinositols (Janmey, 1998) and frequenin (Weisz et al. 2000) play a variety of roles in cellular signalling, in particular in cellular trafficking. Whether NCS-1 influences PI4K-dependent signalling pathways underlying the remodelling of the secretion machinery in NG108-15 cells remains to be investigated.
In this study we obtained evidence for the enhancement of acetylcholine release from NCS-1-expressing cells. Overexpression of NCS-1 in stably transfected NG108-15 clones also increased evoked release associated with increased quantal content (m) estimated from the failure rate. It is important to further validate this by measuring endplate currents and finding discrete classes of current size.
In our findings, upregulation of acetylcholine release by NCS-1 might be a reflection of a direct modulation of vesicle recycling. Immunolocalization of NCS-1 in differentiated NG108-15 cells showed partial co-localization with the exocytotic protein SNAP-25, an observation that is in line with its presence in the synaptic vesicle fraction in neurons (Andersson et al. 2000). Phosphatidylinositol, in particular phosphatidylinositol 4,5-bisphosphate, has been shown to play an important role in exo- and endocytosis (McFerran et al. 1999). Whether NCS-1 and PI4K are directly involved in vesicle recycling is under investigation.
In summary, NCS-1 localizes to the plasma membrane, including a synapse-like compartment in dibutyryl cAMP-differentiated NG108-15 cells as shown by the partial co-localization with the exocytotic protein SNAP-25. We have also shown that overexpression of NCS-1 in NG108-15 cells enhances the evoked release of acetylcholine in co-cultures with myotubes without further elongation and maturation of neurites. The availability of these cells will allow us to further dissect the signalling pathways modulated by NCS-1; for instance, the relationship between release and neuronal outgrowth, which were affected differentially in our study.
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Acknowledgements
This study was supported by grants from the Japanese Ministry of Education, Sciences, Sports and Culture to H.H., the Medical Research Council of Canada to J. R. and A.J., the Swedish Medical Research Council to P.-O.B., the Swedish Natural Science Research Council to C.B. and the Swedish Medical Research Council to B.M.
Corresponding authors
H. Higashida: Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan.
Email: haruhiro{at}med.kanazawa-u.ac.jp
A. Jeromin: Mount Sinai Hospital, SLRI-860, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5.
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, con), mock-transfected (
) and NCS-1 (pcDNA3RFrqF12)-transfected NG108-15 cells (
). The three different types of NG108-15 cells were co-cultured with myotubes for the indicated number of days in the presence of 0.25 mM dibutyryl cAMP. MEPPs were recorded from postsynaptic myotubes. Each value is the mean of 30-33 muscle cells collected in three experiments. Significant differences from the control values in non-transfected and mock-transfected cells at each culture day are indicated (* P < 0.02; ** P < 0.001).