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Development of genetically encoded fluorescent sensors to study cellular neurophysiology : 세포신경생리학 연구를 위한 유전자부호화된 형광센서의 개발

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dc.contributor.advisor송윤규-
dc.contributor.author이성무-
dc.date.accessioned2019-05-07T06:33:17Z-
dc.date.available2021-04-13T05:10:38Z-
dc.date.issued2019-02-
dc.identifier.other000000154492-
dc.identifier.urihttps://hdl.handle.net/10371/152564-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 융합과학기술대학원 융합과학부(나노융합전공), 2019. 2. 송윤규.-
dc.description.abstractOptical sensors of cellular neurophysiology have thrived to a level where they can enable probing the voltage change of dendritic spines or neuronal activities from electrophysiologically indistinguishable cell types in a small region. The on-going success of optical recording was achieved by a combination of genetic engineering, optimization of optics for biological imaging, and the development of advanced fluorescent sensors. The emergence of genetically encoded fluorescent bio-sensors facilitated cell-type specific targeting of the optical sensors by employing the cell of interests own protein production machinery.

This dissertation will discuss rationally designed strategies to develop genetically encoded voltage indicators, to optimize membrane trafficking of the sensor molecules, and to modify fluorescent proteins property to confer photoactivatability.

Recording voltage is a direct measurement of neuronal activity. Genetically encoded voltage indicators were first reported in 1997 and had shown considerable improvements in the last seven years. The first part of this thesis paper will introduce the basic concepts and a brief history in genetically encoded voltage indicator development.

One of the main advantages of using a genetically encoded optical sensor is the ability to send the proteins to specific regions, even subcellular. Therefore, means to better locate the sensor proteins were empirically studied and will be discussed in the second part.

Finally, a set of rationally designed mutations to make a sensor protein optically highlightable will be shown. Previous results from the report of the first photoactivatable green fluorescent protein were applied to develop photoactivatable genetically encoded voltage indicators and pH indicators.
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dc.description.abstract광학 센서들의 눈부신 발전으로 인해 세포신경생리학 연구에 있어 전기생리학적으로는 밝혀내기 어려운 정보의 습득에도 이러한 광센서들이 사용되고 있다. 예를 들면, 신경 세포 dendritic spine의 전기적인 신호 변화나 혹은 한 지역에 있으나 전기적으로는 구별해 내기 힘든 서로 다른 세포들을 구분해 내어 광학적으로 그 활성을 측정하는데 활용되고 있다. 이러한 광학 센서의 성공은 생명공학 기술의 발전, 생물 실험에 최적화된 광학 장비 그리고 향상된 수준의 형광 센서 개발에 기인한다. 특히, 유전적으로 부호화된 형광 바이오센서들의 출현은 조사하려는 세포 자체의 단백질 생산 메커니즘을 사용함으로써 광학 센서의 세포 유형별 표적화를 가능하게 하였다.

본 학위 논문은 유전자 부호화 전압 표시장치를 개발하고, 센서 분자의 세포막 발현을 최적화하며, 또한 형광 단백질의 광활성화를 가능케 하는 방법을 다루고 있다.

신경세포의 전압변화를 측정하는 것은 곧 신경 활동을 직접 측정한다는 것을 의미한다. 유전자 부호화된 전압 표시장치는 1997년에 처음 보고되었는데, 특히 지난 7년간 눈부신 발전을 이뤄왔다. 이 학위 논문의 첫 번째 부분에서는 유전자 부호화된 전압 표시장치의 기본 개념과 그간의 개발 이력을 간략히 소개함과 동시에 이번 연구에서 새롭게 개발한 전압 표시장치를 소개하고 있다.

유전적으로 암호화된 광학 센서의 주요 장점 중 하나는 해당 단백질을 특정 지역, 더 나아가 세포 소기관으로 보내는 표적 능력이다. 따라서, 센서 단백질을 특정한 위치에 더 잘 발현 시키기 위한 수단들을 경험적으로 연구하였고, 이 또한 이 논문의 두 번째 부분에서 논의될 것이다.

마지막으로, 센서 단백질을 광학적으로 활성화하기 위해 설계된 논리적 돌연변이 전략을 소개한다. 이 부분에서는 2002년에 처음 보고된 광활성이 가능한 녹색 형광 단백질의 개발에 사용된 전략을 적극적으로 활용하였다. 이를 통하여 광활성화가 가능한 유전자 부호화된 전압 표시장치 및 산성도 표시장치를 개발한 과정과 결과를 소개한다.
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dc.description.tableofcontentsContents

Abstract i

Contents v

List of figures xi

List of tables xvi

Chapter 1. Introduction 1

1.1 Methodologies to study neuronal activity 2

1.1.1 Electrophysiological recordings 2

1.1.2 Alternatives to electrophysiological techniques 8

1.2 Genetically encoded voltage indicator 14

1.2.1 Voltage imaging 14

1.2.2 Early versions of genetically encoded voltage indicator 18

1.2.3 GEVIs using voltage-sensing domains from non-conducting voltage-sensitive phosphatase 21

1.2.4 GEVIs using microbial rhodopsin 26

1.3 The importance of membrane trafficking and means to improve it 29

1.3.1 Consequence of bad membrane expression 29

1.3.2 Optogenetic actuators with optimized membrane expression 33

1.3.3 Voltage indicators with the membrane targeting motifs 35

1.4 Photoactivation and its application in cellular neurophysiology 39

1.4.1 Photoactivatable fluorescent proteins 39

1.4.2 Photoactivatable calcium indicators 42

1.4.3 Genetically encoded pH indicators 45

Chapter 2. Rational modification of an interdomain linker region enabled single pixel voltage imaging of action potentials 49

2.1 Introduction 50

2.2 Materials and methods 52

2.2.1 Plasmid DNA construction 52

2.2.2 Cell culture and transfection 55

2.2.3 Animals for brain slice voltage imaging 56

2.2.4 Stereotaxic surgery for viral injection in CA1 area 56

2.2.5 Hippocampal brain slice preparation 57

2.2.6 Electrophysiology and fluorescence imaging for HEK 293 cells and primary neurons 58

2.2.7 Electrophysiology and fluorescence microscopy for brain slice 60

2.2.8 Data acquisition and analysis 61

2.2.9 Photobleaching experiment of HEK 293 cells 62

2.2.10 Baseline change analysis of neurons 62

2.2.11 Statistics 63

2.3 Results 64

2.3.1 Insertion of positively charged amino acid residues in linker regions increased the ΔF/F signal size for depolarization of the plasma membrane, while the negatively charged linkers reduced the size 64

2.3.2 Effects of different charge, size, and polarity of the linker amino acid residues on voltage imaging of membrane potential 69

2.3.3 Introducing voltage-sensing domain mutations to CC1-Pos6 indicated that the positively charged linker shifted the voltage range to more negative membrane potentials 73

2.3.4 A single arginine residue inserted at the third position of Bongwooris linker region improved the signal size and maintained the voltage range for an efficient measurement of action potentials 78

2.3.5 The improved fluorescence response and the near zero millivolt V1/2 of Bongwoori-R3 resolved action potentials with better contrast 82

2.3.6 Single pixel resolution of action potentials with Bongwoori-R3 87

2.3.7 The baseline fluorescence change of Bongwoori and Bongwoori variants is due to the intracellular pH change 92

2.3.8 Slice recording 96

2.4 Conclusion 101

Chapter 3. Deciphering consequences of introducing membrane targeting motifs into genetically encoded voltage indicators 105

3.1 Introduction 106

3.2 Materials and methods 108

3.2.1 Plasmid DNA construction 108

3.2.2 Cell culture and transfection 111

3.2.3 Electrophysiology and voltage imaging 111

3.2.4 Data acquisition and analyses 111

3.3 Results 112

3.3.1 Determining the location of Golgi to membrane trafficking signal and/or ER export signal sequences in Bongwoori linker variants 112

3.3.2 Bongwoori-Pos6 with Golgi TS and / or ER export signal did not improve fluorescence signal for membrane depolarization 112

3.3.3 Bongwoori-R3 with Golgi TS and ER export motifs showed increased ΔF/F signal size for both hyperpolarization and depolarization 118

3.3.4 Bongwoori-R3 variants with different ER export signal locations resulted in diminished ΔF/F signal size 123

3.3.5 Placing the ER motif at the inter-domain linker region also decreased voltage induced fluorescence response 126

3.3.6 Inserting a short 6 amino acid long spacer in between the FP and ER motif improved the kinetics 128

3.3.7 Bongwoori-R3_Golgi & ER with longer amino acids spacers recovered the kinetics while maintaining the large ΔF/F 131

3.3.8 Inserting membrane targeting motifs affected V1/2 value 136

3.4 Conclusion 139

Chapter 4. Development of photoactivatable optical bio-sensors of physiological voltage and pH changes 141

4.1 Introduction 142

4.2 Materials and methods 145

4.2.1 Gene constructs design and cloning 145

4.2.2 Cell culture and transfection 149

4.2.3 Photoactivation, voltage and pH imaging 149

4.2.4 Analyses 151

4.3 Results 152

4.3.1 Schematics of designing photoactivatable voltage indicator 152

4.3.2 ASAP1-ssPA did not express well and ASAP1-PATM was bright already before photoactivation 156

4.3.3 The photoactivatable FRET donor in PA-Nabi 2.242 was photoactivated for more than 2 - fold but the FRET acceptor showed a mild increase its fluorescence 158

4.3.4 Photoactivatable-Bongwoori-R3 was well expressed in membrane region and photoactivated by 385 nm LED 163

4.3.5 Photoactivatable ecliptic pHluorin with T203H mutation 169

4.4 Conclusion 173

Chapter 5. Conclusion 175

References 177

국 문 초 록 189
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dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subject.ddc620.5-
dc.titleDevelopment of genetically encoded fluorescent sensors to study cellular neurophysiology-
dc.title.alternative세포신경생리학 연구를 위한 유전자부호화된 형광센서의 개발-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorSungmoo Lee-
dc.description.degreeDoctor-
dc.contributor.affiliation융합과학기술대학원 융합과학부(나노융합전공)-
dc.date.awarded2019-02-
dc.identifier.uciI804:11032-000000154492-
dc.identifier.holdings000000000026▲000000000039▲000000154492▲-
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