Synaptotagmin IV Is Present at the Golgi and Distal Parts of Neurites

Abstract

Synaptotagmin IV (SytIV) is an immediate early gene induced by membrane depolarization in PC12 cells and in rat brain. However, little is known about the function of SytIV or the functional relationship between SytIV and SytI, because SytIV has yet to be localized. Here we show that SytIV was localized at the Golgi and distal part of neurites in nerve growth factor-differentiated PC12 cells and cultured hippocampal neurons by immunocytochemistry using an isoform-specific antibody (antiSytIV). These SytIV signals were not colocalized well with SytI signals. Upon membrane depolarization, SytIV signals were increased at both the Golgi and distal part of neurites within several hours in both types of cells. We further show that the increase of SytIV protein levels results from protein kinase A-dependent gene up-regulation. In hippocampal neurons, SytIV was developmentally regulated. These results suggest that SytIV may play a role at the Golgi and tips of neurites during development and synaptic plasticity. Key Words: Synaptotagmin-Depolarization-Forskolin-Immediate early genesNeuronal development-Synaptic plasticity. J. Neurochem. 74, 518 -526 (2000).The synaptotagmin family consists of 11 isoforms [synaptotagmins (Syt) I-XI] in rat or mouse and shares a short amino terminus, one transmembrane region, and two C2 domains homologous to the C2 regulatory region of protein kinase C (known as the C2A and C2B domains) (Perin et al

Synaptotagmin IX Regulates Ca2+-dependent Secretion in PC12 Cells

“…The Abs were affinity-purified by exposure to antigenic peptide bound to FMP-activated Cellulofine (Seikagaku Co.) as described previously (21). Specificity of these antibodies was checked by immunoblotting using recombinant T7-tagged Syts I-XIII expressed in COS-7 cells (22)(23)(24). Under our experimental conditions, we could not observe cross-reactivity of anti-Syt IX-C2A and anti-Syt IX-N Abs with Syt I in immunoblotting.…”

“…After washing twice with phosphate-buffered saline, the cells were stimulated for 10 min at 37°C with either low KCl buffer (5.6 mM KCl, 145 mM NaCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES-KOH, pH 7.4) or high KCl buffer (56 mM KCl, 95 mM NaCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM glucose, and 15 mM HEPES-KOH, pH 7.4) containing rhodamine-labeled anti-Syt I-N Ab (1 g/ml) and fluorescein-labeled anti-Syt I Ab (10 g/ml). The cells were immediately washed twice with phosphate-buffered saline and then fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer for 20 min at room temperature as described previously (20,22). Incorporated antibodies were analyzed with a fluorescence microscope (TE300, Nikon) attached to a laser confocal scanner unit CSU 10 (Yokogawa Electric Corp.) and HiSCA CCD camera (C6790, Hamamatsu Photonics).…”

“…Incorporated antibodies were analyzed with a fluorescence microscope (TE300, Nikon) attached to a laser confocal scanner unit CSU 10 (Yokogawa Electric Corp.) and HiSCA CCD camera (C6790, Hamamatsu Photonics). Images were pseudo-colored and superimposed with Adobe Photoshop software (Version 4.0) (20,22).…”

Synaptotagmin IV regulates glial glutamate release

“…Rabbit polyclonal antibodies against Syts I-XIII were used in Western blots (27,28). Specificity of reactivity was confirmed by testing each antibody against recombinant Syts I-XIII (Fig.…”

Formation of Crystalloid Endoplasmic Reticulum Induced by Expression of Synaptotagmin Lacking the Conserved WHXL Motif in the C Terminus

“…Transfection was achieved by using the LipofectAMINE Plus reagent according to the manufacturer's instructions (Life Technologies Inc., Gaithersburg, MD). Three days after transfection, PC12 cells were fixed, permeabilized, and stained with both rabbit anti-FLAG antibody (1/1000 dilution) and anti-TGN38 mouse monoclonal antibody (1/500 dilution) or anti-BiP mouse monoclonal antibody (1/200 dilution), as described previously (43,44). Immunoreactivity was analyzed with a fluorescence microscope (TE300, Nikon, Tokyo, Japan) attached to a laser confocal scanner unit CSU 10 (Yokogawa Electric Corp., Tokyo, Japan) and HiSCA charge-coupled device camera (C6790, Hamamatsu Photonics, Hamamatsu, Japan).…”