2004 Fiscal Year Final Research Report Summary
Analysis of intracellular translocation of choline acetyltransferase by bioimaging
| Project/Area Number |
15500257
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
MATSUO Akinori Shiga University of Medical Science, Molecular Neuroscience Research Center, Assistant Professor, 分子神経科学研究センター, 助手 (20324585)
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| Co-Investigator(Kenkyū-buntansha) |
SAITO Naoaki Kobe University, Biosignal Center, Professor, バイオシグナル研究センター, 教授 (60178499)
AIMI Yoshinari Shiga University of Medical Science, Molecular Neuroscience Research Center, Assistant Professor, 分子神経科学研究センター, 助手 (20231756)
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| Project Period (FY) |
2003 – 2004
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| Keywords | pChAT / cChAT / GFP / translocation / HEK293 / PC12 |
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| Research Abstract |
Choline acetyltransferase(ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type(pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type(cChAT) using green fluorescent protein(GFP) in living human embryonic kidney cells (HEK293 cells) and PC-12 cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.
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