8,863 research outputs found
Clarification of Disputable Amino Acid Residues in the Covalent Structure of Bovine Pancreatic Deoxyribonuclease As Derived from Chemical and X-Ray Studies
No full textMycotoxin Contamination in Grains; Papers presented at the 17th ASEAN Technical Seminar on Grain Postharvest Technology, Lumut, Malaysia, 25-27 July 1995
Full text linkCrop Production/Industries,
The Effect of Laser Chirping on Optical Subcarrier Multiplexed System with High-Bit-Rate Baseband Signal
No full textSequence changes of the infectious bronchitis virus isolates at the 3’ 7.3 kb genomes after embryonated passages.
No full text
Development and application of a multiplex reverse transcription-polymerase chain reaction for avian viral respiratory agents.
No full textClinical epidemiologic and experimental evidence for the transmission of Newcastle Disease Virus through eggs
No full textPIN trafficking factors
No full text학위논문(박사) -- 서울대학교대학원 : 자연과학대학 생명과학부, 2024. 2. 조형택.A study on the regulators for the intracellular trafficking of PIN- FORMED auxin transporters in Arabidopsis thaliana Kwang Ho Maeng Department of Biological Sciences The Graduate School Seoul National University Auxin plays as a morphogenic hormone by forming local concentration gradients. Auxin efflux carriers PIN-FORMEDs (PINs) contribute to the auxin gradient formation by asymmetrically localizing at the plasma membrane (PM) and by directionally transporting auxin in a cell-to-cell manner. The hydrophilic domain of long PINs (hereafter, PIN-HL) includes diverse molecular cues for intracellular dynamics of PINs. The characterization of these molecular cues on PIN-HL have greatly expanded our understanding of the regulatory mechanisms of polar auxin transport and auxin responses. Nevertheless, the vast spectra of auxin responses in developmental/environmental contexts imply that PIN-HL would carry even more trans-regulatory factors to modulate intracellular PIN trafficking and polarity. In order to find these additional PIN-modulating factors, our lab screened PIN-HL- interacting proteins (PIPs) by a protein pulldown method and identified some regulatory factors. In this dissertation, I introduced two PIPs, FAB1C and ROPGAP3, and their functions specifically their involvement in PIN polarity formation. In chapter 1, I presented that how FORMATION OF APLOID AND BINUCLEATE CELL 1C (FAB1C), a 1-phosphatidylinositol-3-phosphate (PI3P) 5- kinase, regulates PIN trafficking. FAB1C interacted with PIN-HLs, and this interaction was significant for PIN polarity formation. FAB1C has negative effects on PM localization of PINs through promoting the PINs degradation pathway. Among the FAB1 homologs in Arabidopsis, only FAB1C down-regulated lateral root formation. FAB1C was expressed throughout the all stages of lateral development, and negatively regulated the auxin maximum by formation weakening the periclinal PM localizations of PIN1. Moreover, FAB1C preferably interacts with phospho- defective form of PIN-HLs, and this interaction was associated with vacuolar PIN trafficking. These results suggest that FAB1C modulates the lytic trafficking of PIN upon changes in PIN phosphorylation status in response to developmental or environmental stimuli. In chapter 2, I presented that how ROP GTPase activating protein3 (ROPGAP3) maintains PIN polarity. Unlike other paralog, ROPGAP3 specifically interacts with PIN-HL, and through this interaction, it facilitates the formation of PIN clustering, thereby maintaining PIN polarity. Importantly, the formation of PIN clustering mediated by ROPGAP3 is independent of ROP signaling. Furthermore, ROPGAP3 acts as a regulator in ROP signaling, controlling PIN trafficking. This finding demonstrates that a single protein can regulate PIN polarity through two distinct mechanisms and it offers plants a significantly advantageous mechanism to respond effectively to various environmental and developmental cues. Keywords: Development, Arabidopsis, auxin, PIN-FORMED (PIN), Phosphorylation, Phosphatidylinositol 3-phosphate-5 kinase, Vacuolar trafficking, Direct interaction, Membrane, GTPase activating protein, PIN clustering. Student Number: 2017-20804옥신은 모포제닉 호르몬으로서 국소 농도 기울기가 형성된다. 옥신 유출 수송체 PIN-FORMEDs (PINs)은 세포 내 세포 막 한쪽 방향에 위치하고 옥신을 방향성 있는 세포-세포 이동을 시킴 으로서 옥신의 농도 기울기를 형성한다. PIN의 친수성 루프 (PIN-HL)는 PIN의 세포 내 활동에 다양한 분자적인 단서를 제공한다. 이러한 PIN-HL의 분자적 단서를 밝히는 것은 옥신의 극성 수송과 옥신 반응의 조절 메커니즘을 이해하는데 도움이 되었다. 그럼에도 불구하고 광범위한 발달적/환경적 옥신 반응은 PIN의 세포 내 순환과 극성 분포를 조절하는 추가적인 PIN-HL의 cis, trans 조절인자 있음을 시사한다. 추가적인 PIN 조절 인자를 찾기 위해 본 연구실에서는 단백질 pull down방법을 통해 PIN-HL 상호작용 단백질 (PIPs; PIN-HL-interacting proteins)을 찾았고 조절 인자를 밝혀 냈다. 본 논문에서는 두 PIPs인 FAB1C와 ROPGAP3가 어떻게 PIN polarity 형성에 관여하는지 소개한다.
제 1장에서는 Phosphatidylinositol 3-phosphate-5 kinase인 FORMATION OF APLOID AND BINUCLEATE CELL 1C (FAB1C)가 어떻게 PIN trafficking을 조절하는지 나타낸다. FAB1C는 PIN-HL와 상호작용하고 다시 이 상호작용은 PIN 극성 분포에 매우 중요하다. FAB1C는 또한 PIN의 분해과정을 촉진하여 PM에서 PIN의 위치에 부정적으로 작용한다. 애기장대에서 동형체 중 오직 FAB1C만 곁뿌리 발달에 관여한다. FAB1C는 곁뿌리 발달 모든 단계에서 발현하며 PIN1의 PM에서의 위치를 약화시켜 옥신 분포를 부정적으로 조절한다. 또한 FAB1C는 인산화가 덜 된 PIN-HL와 우선적으로 상호작용하고 이러한 상호작용은 PIN의 액포로의 수송에 중요하다. 이러한 결과들은 FAB1C가 발달적/환경적 자극에 반응하고 PIN의 인산화 여부를 인지하여 PIN의 분해과정을 조절함을 나타낸다.
제 2장에서는 ROPGAP3가 어떻게 PIN의 극성분포를 유지하는지 나타낸다. 다른 애기장대의 동형체와 달리 ROPGAP3는 PIN-HL와 상호작용하고 이러한 상호작용은 PIN clustering 형성을 촉진해 PIN 극성을 유지시킨다. 흥미롭게도 ROPGAP3에 의한 PIN clustering 형성은 ROP 신호전달과 무관하지만 여전히 ROPGAP3는 ROP 신호전달 조절자로 작용하여 PIN trafficking을 조절한다. 이러한 발견은 하나의 단백질이 두가지 다른 메커니즘으로 PIN의 극성분포를 조절할 수 있음을 나타내고 이는 식물이 효과적으로 다양한 발달적/환경적 단서에 반응할 수 있는 메커니즘을 제시한다.Abstract i
Contents iv
List of figures x
List of table · xiv
Abbreviations xvi
Introduction · xix
Chapter 1: FAB1C, a phosphatidylinositol 3-phosphate 5-kinase, interacts with
PIN-FORMEDs and modulates their lytic trafficking in Arabidopsis
1. Introduction 2
1.1 PIN trafficking 2
1.2 FAB1 · 4
2. Materials and methods 9
2.1 Plant materials and growth conditions 9
2.2 Transgene constructs · 9
2.3 Bacterial protein expression and in vitro protein pulldown 12
2.4 Yeast two-hybrid assay 13
2.5 FRET-FLIM analysis · 14
2.6 Sequence alignments · 15
2.7 Protein structure prediction 15
2.8 Histological observation of β-glucuronidase (GUS) Activity 15
2.9 Confocal microscopy of fluorescent proteins · 16
2.10 RNA isolation and reverse transcription (RT)- quantitative PCR analysis
16
2.11 Observation of biological parameter · 17
2.12 Secondary structure prediction 18
2.13 Statistical Analysis 18
2.14 Accession Numbers 18
3. Results · 21
3.1 FAB1C directly interacts with PIN-HL · 21
3.1.1 Screening of PINs-HL interacting proteins 21
3.1.2 Characterization of FAB1C domain 21
3.1.3 FAB1C directly interacts with PINs-HL 29
3.2 FAB1C expression and subcellular localization · 36
3.2.1 Expression patterns of FAB1C 36
3.2.2 Expression patterns and subcellular localizations of FAB1C · 36
3.3 FAB1C effects on PIN trafficking 39
3.3.1 Characterization of fab1c mutant 39
3.3.2 FAB1C is required for proper polarity of PIN1, PIN2, and PIN3
39
3.3.3 FAB1C has negative effect on PM localization of PINs 45
3.3.4 FAB1C is crucial factor for PINs PM to vacuole trafficking 48
3.3.5 FAB1C promotes vacuolar trafficking of PIN2 · 51
3.3.6 PIN-interacting domain and PIP kinase activity are required
for FAB1C-mediated vacuolar trafficking of PIN2 · 55
3.3.7 A unphosphorylated PIN2 form is preferred for dark-
induced vacuolar trafficking 62
3.4 FAB1C biological function 71
3.4.1 FAB1C regulates PIN1 trafficking during lateral root
developments. 71
3.4.2 Other auxin related phenotypes of FAB1C 79
4. Discussion 84
Chapter 2: ROPGAP3 Regulates PIN Clustering Independent of its Intrinsic
GTPase Activity
1. Introduction 88
1.1 PIN clustering 88
1.2 ROPGAP3 88
2. Materials and methods 91
2.1 Plant materials and growth conditions · 91
2.2 Transgene constructs 91
2.3 Bacterial protein expression and in vitro protein pulldown · 93
2.4 Yeast two-hybrid assay 93
2.5 FRET-FLIM analysis 93
2.6 Sequence alignments and phylogenetic analysis · 93
2.7 Protein interaction prediction 94
2.8 GTPase activity assay · 94
viii
2.9 Confocal microscopy of fluorescent proteins 94
2.10 RNA isolation and reverse transcription (RT)- quantitative PCR analysis
95
2.11 Observation of biological parameter · 95
2.12 Statistical Analysis 96
2.13 Accession Numbers 96
3. Results · 98
3.1 ROPGAP3 expression and subcellular localization 98
3.1.1 Expression patterns and subcellular localizations of ROPGAP3
98
3.2 ROPGAP3 directly interacts with PIN-HL · 100
3.2.1 ROPGAP3 interacts directly with PIN-HL in vitro 100
3.2.2 ROPGAP3 interacts directly with PIN-HL in planta 103
3.2.2 Evolutionary Analysis of ROPGAP3 107
3.3 ROPGAP3 effects on PIN polarity 112
3.3.1 Characterization of ropgap mutant 112
3.3.2 ROPGAP3 regulates PIN trafficking through ROP signaling 112
3.3.3 ROPGAP3 regulates PIN polarity by mediating PIN
clustering through interaction with PIN, rather than ROP
signaling 121
3.3.4 ROPGAP3 regulates PIN2 endocytosis through the
modulation of both PIN clustering and ROP signaling 129
3.4 ROPGAP3 biological function 132
3.4.1 ROPGAP3 is involved in gravi-tropism. 132
3.4.2 Other auxin related phenotypes of ROPGAP3 135
4. Discussion 139
5. Reference 142
국문초록 165
List of Figure
Figure 1. Topology of PIN
proteins. xx
v
Figure 2. Purpose of this
study. xx
vi
Figure 3. Subcellular Trafficking and Polarity Maintenance of PIN
Proteins. 3
Figure 4. Subcellular distribution of the PIs and PI-metabolising enzymes. 7
Figure 5. Finding new PIN interacting proteins using PIN-HL. · 22
Figure 6. Phylogenetic relationship of FAB1C orthologs. 24
Figure 7. The structure of FAB1. 28
Figure 8. FAB1C directly interacts with PIN-HL. · 30
Figure 9. PD of FAB1 interacts with PIN2-HL. · 32
Figure 10. FAB1C interacts with PIN2-HL in vivo. 34
Figure 11. Expression patterns of FAB1C. · 37
Figure 12. Expression patterns and subcellular localizations of FAB1C. 38
Figure 13. T-DNA insertion and FAB1C transcript levels in fab1c-1 and
fab1c-3 mutants. · 40
Figure 14. The loss of FAB1C alters PINs polarity. 41
Figure 15. PIN polarity is impaired in the fab1c-3 mutant. 43
Figure 16. The loss of FAB1C alters PIN2 polarity at root cortical cell. 46
Figure 17. FAB1C has negative effect on PM localization of PINs 47
Figure 18. The relationship between apical PIN2 polarity and PIN2
intensity. · 49
Figure 19. FAB1C is crucial factor for PINs PM to vacuole trafficking. 50
Figure 20. Effects of fab1c mutant on the trafficking of PIN1and PIN2. 52
Figure 21. Vacuolar trafficking of PIN is impaired in the fab1c mutant. 53
Figure 22. Trafficking of PIN2 to the vacuole is promoted by FAB1C. 54
Figure 23. The effects of mutations in FAB1C domains. 56
Figure 24. FAB1C more likely interacts with phospho-defective PIN2 than
phosphor-mimetic one. 59
Figure 25. Alignment of amino acid sequences of PIN-HLs. · 61박
An experimental investigation into pin loading effects on fatigue crack growth in Fibre Metal Laminates
No full textAbstractThis paper provides an experimental investigation into the pin loading effects on the crack growth behaviour in Fibre Metal Laminates. The pin loading effects and bypass loading effects are incorporated in two different tested joints. The analysis of the test results shows that pin loading dominates the crack growth only in the vicinity of the pin hole and the superposition method for analysing stress intensity factor in FMLs with pin loading effects can be applied
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