Localization of Goα in the Developing and Adult Mouse Brain
발생 중인 신경세포와 성체 생쥐 뇌에서의 Goα 단백질 발현 위치
- 주제(키워드) Go protein , cerebellum , Purkinje cell , plasma membrane
- 발행기관 아주대학교
- 지도교수 서해영
- 발행년도 2013
- 학위수여년월 2013. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000013539
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
-Abstracts- Localization of Goα in the Developing and Adult Mouse Brain Heterotrimeric G proteins mediate signal transduction generated by hormone and neurotransmitters. Among G proteins, Go, a member of the Gi/o family, is the most abundant heterotrimeric G protein in the brain but the roles and effectors for the Go have not been clearly defined. As previously reported, the expression of Go protein is found in various brain regions, including cerebrum, hippocampus, thalamus, and cerebellum. Although Go is already known to be present on both plasma- and endo-membranes, the precise subcellular location of Go protein has been a controversial issue because diverse detection methods such as in situ hybridization versus and immunohistochemistry yielded apparently contradictory results. We previously found that Go functions as a signaling scaffolding molecule that determines the subcellular localization of cAMP-dependent protein kinase A (Ghil et al., 2006). In this study, we showed that Go is expressed in Purkinje cell dendrites of developing and adult cerebellum by comparing the Go expression in wild type and Go knockout mice and confirm the subcellular location of Go protein through the hippocampal neuron in vitro culture. Consequently, this study provides more accurate information to speculate the role of Go protein on the basis of its correct localization as a character of the Go.
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TABLE OF CONTENS
ABSTRACTS ⅰ
TABLE OF CONTENTS ⅱ
LIST OF FIGURES ⅳ
LIST OF TABLE ⅴ
Ⅰ. INTRODUCTION 1
1. Heterotrimeric Guanine nucleotide-binding protein alpha subunit (Gnαo)
1
2. Localization of Goα subunit in the brain 2
3. Cerebellar cortex 2
Ⅱ. MATERIALS AND METHODS 6
1. Animals 6
2. Tissue preparation (for immunohistochemistry) 6
3. Immunohistochemistry 6
4. Cell cultures 7
4.1. Cerebellar neuron/Purkinje cell culture 7
4.2. Hippocampal neuron culture 7
5. EGFP-IRES3-Goα-CL, GFP-Akt-PH, GFP-PLCδ-PH Construct 8
6. Transfection 8
6.1. 293T cell transfection 8
6.2. F11/COS cell transfection and Forskolin treatment 8
6.3. Hippocampal neuron transfection 9
7. Western blot 9
8. Immunocytochemistry 10
9. In situ hybridization 11
9.1 In vitro RNA transcription 11
9.2 Tissue preparation (for in situ hybridization) 11
9.3 In situ hybridization 12
Ⅲ. RESULTS 15
Ⅳ. DISCUSSION 29
Ⅴ. CONCLUSION 33
REFERENCES 34
국문 요약 38
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LIST OF FIGURES
Figure. 1. Structure of the cerebellar cortex 5
Figure. 2. Construct map of EGFP-IRES3- Goα-CL 13
Figure. 3. In situ hybridization probe for Goα 14
Figure. 4. Goα is abundant in the molecular and granule cell layers. 19
Figure. 5. Expression of Goα is neuron specific 20
Figure. 6. Neuron-specific expression of Goα in in vitro cultured cerebellar
neurons. 21
Figure. 7-1. Subcellular distribution of Goα in Purkinje cells 22
Figure. 7-2. Subcellular compartmentalization of Goα and calbindin
in Purkinje dendrites. 23
Figure. 8. Expression of Goα mRNA in Purkinje cells 24
Figure. 9. Goα does not affect the cytogenesis of cerebellar cortex. 25
Figure. 10. Expression of Goα in transfected 293T is plasma membrane
targeting. 26
Figure. 11. Expression of Goα is plasma membrane dominant 27
Figure. 12. Distribution of Goα merges with GFP-Akt-PH, GFP-PLCδ-PH
28
