Actin organization at intracellular membranes: targeting and regulatory interactions of the spir formin actin nucleator complex
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VII, 119 S. Ill., graph. Darst. |
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| adam_text | TABLE OF CONTENTS
J
ABSTRACT I
ZUSAMMENFASSUNG III
1 INTRODUCTION 1
1.1 ACTIN 2
1.2 ACTIN NUCLEATION FACTORS 3
1.2.1 ARP 2/3 COMPLEX 4
1.2.2 FORMINS 4
1.2.3 WH2 DOMAIN CONTAINING ACTIN NUCLEATION FACTORS 6
1.3 THE ACTIN NUCLEATION FACTOR .SPIR 6
1.3.1 DOMAIN STRUCTURES OF THE SPIR-FAMILY 7
1.3.2 SPIR/FORMIN COOPERATION 9
1.3.3 SPIR FUNCTION 10
1.3.3.1 SPIR FUNCTION IN VESICLE PROCESSES 11
1.4 REGULATION OF VESICLE TRANSPORT PROCESSES 11
1.4.1 THE RAS SUPERFAMILY 11
1.4.1.1 THE RAB GTPASE FAMILY 12
1.4.1.2 THE ARF GTPASE FAMILY 12
1.4.2 MYOSIN SUPERFAMILY 13
1.4.3 RABLLA/MYOSIN VB COOPERATION 14
1.5 BIOMEMBRANES 15
1.5.1 FUNCTIONS AND STRUCTURE OF CELLULAR MEMBRANES 15
1.5.2 MEMBRANE-BINDING DOMAINS 16
1.5.3 MODEL MEMBRANE SYSTEMS 17
1.6 FLUORESCENCE-BASED TECHNIQUES 18
1.6.1 PRINCIPLE OF FLUORESCENCE 18
1.6.2 FLUORESCENCE CORRELATION SPECTROSCOPY (FCS) 19
1.6.3 FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY (FCCS) 22
1.6.4 FLUORESCENCE LIFETIME IMAGING/ FORSTER RESONANCE ENERGY TRANSFER
(FLIM/FRET) 23
2 AIM OF THE
WORK 25
V
HTTP://D-NB.INFO/1064639585
TABLE OF CONTENTS
3 MATERIAL AND METHODS 27
3.1 REAGENTS 27
3.2 DNA CONSTRUCTS 27
3.3 PROTEIN EXPRESSION AND PURIFICATION 27
3.3.1 EXPRESSION AND PURIFICATION OF SPIR-2 VARIANTS 27
3.3.2 EXPRESSION AND PURIFICATION OF MYRISTOYLATED ARFL 28
3.4 LABELING OF THE PROTEINS 29
3.5 GEL ELECTROPHORESIS AND WESTERN BLOT 30
3.6 GST-PULLDOWN ASSAY 31
3.7 PREPARATION OF MODEL MEMBRANES 32
3.7.1 LIPIDS USED IN THIS STUDY 32
3.7.2 GIANT UNILAMELLAR VESICLES (GUVS) 33
3.7.3 LARGE UNILAMELLAR VESICLES (LUVS) 33
3.8 LUV FLOATATION ASSAY 33
3.9 ELECTROCHEMILUMINESCENCE IMMUNOASSAY (EIA) 34
3.10 CELL CULTURE 35
3.11 CELL SEEDING AND TRANSFECTION 35
3.12 CONFOCAL LASER SCANNING MICROSCOPY 36
3.13 FCS AND FCCS DATA ACQUISITION 36
3.14 FCS AND FCCS DATA ANALYSIS 37
3.14.1 IN VITRO FCS AND FCCS 37
3.14.2 IN VIVO FCS AND FCCS 38
3.14.3 TITRATION OF HISACGFP-SPIR-2-CT AND SPIR-2-KIND*ALEXA
647
39
3.15 FLIM-FRET 40
3.16 STATISTICAL ANALYSIS 40
4 RESULTS 41
4.1 CHARACTERIZATION OF MEMBRANE BINDING OF SPIR-2 41
4.1.1 PREFACE 41
4.1.2 CHARACTERIZATION OF THE MEMBRANE BINDING DOMAINS OF SPIR-2 43
4.1.3 CHARACTERIZATION OF THE PHOSPHOLIPID INTERACTIONS OF SPIR-2 45
4.1.4 SPIR-2 IS A MONOMER IN THE CYTOSOL 49
4.2 CIS-REGULATORY INTERACTIONS OF SPIR-2 51
4.2.1 PREFACE C. 51
4.2.2 IDENTIFICATION OF AN INTRAMOLECULAR SPIR-2-INTERACTION 53
4.2.3 QUANTIFICATION OF THE INTRAMOLECULAR SPIR-2-INTERACTION 55
VI
TABLE OF CONTENTS
4.2.4 MEMBRANE BINDING REGULATES THE INTRAMOLECULAR SPIR-2 INTERACTION
57
4.2.5 FMN-2-EFSI REGULATES THE INTRAMOLECULAR SPIR-2 INTERACTION 59
4.3 TRANS-REGULATORY INTERACTIONS OF SPIR-2 61
4.3.1 SPIR INTERACTS WITH ARFL PROTEIN 61
4.3.2 SPIR-ARFL INTERACTION DOES NOT INFLUENCE THEIR BINDING PROPERTIES
63
4.3.3 SPIR-2 INTERACTS WITH MYOSIN VB 66
4.3.4 SPIR-2 LINKER REGION MEDIATES THE INTERACTION WITH MYOSIN VB-TAIL
72
5 DISCUSSION 75
5.1 SPIR-2 POSSESSES A FYVE-RELATED DOMAIN 75
5.2 SPIR-2-FYVE DOMAIN IS A PROTEIN-PROTEIN INTERACTION MODULE 77
5.3 SPIR-2 IS REGULATED BY AN INTRAMOLECULAR COMPLEX 78
5.4 SPIR-2 IS AN UNCONVENTIONAL EFFECTOR PROTEIN OF ARFL.. . 84
5.5 SPIR-2/MYOSIN VB INTERACTION REGULATES VESICLE PROCESSES 86
6 FUTURE DIRECTIONS 93
7 BIBLIOGRAPHY 95
8 APPENDIX 109
8.1 LIST OF CONSTRUCTS 109
9 ABBREVIATIONS . 113
10 SYMBOLS 116
11 ACKNOWLEDGEMENTS 117
12 DECLARATION 119
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| spelling | Tittel, Janine 1984- Verfasser (DE-588)1061017249 aut Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex von Janine Tittel 2014 VII, 119 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2014 Actin (DE-588)4201876-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Actin (DE-588)4201876-6 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027951170&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Tittel, Janine 1984- Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex Actin (DE-588)4201876-6 gnd |
| subject_GND | (DE-588)4201876-6 (DE-588)4113937-9 |
| title | Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex |
| title_auth | Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex |
| title_exact_search | Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex |
| title_full | Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex von Janine Tittel |
| title_fullStr | Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex von Janine Tittel |
| title_full_unstemmed | Actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex von Janine Tittel |
| title_short | Actin organization at intracellular membranes |
| title_sort | actin organization at intracellular membranes targeting and regulatory interactions of the spir formin actin nucleator complex |
| title_sub | targeting and regulatory interactions of the spir formin actin nucleator complex |
| topic | Actin (DE-588)4201876-6 gnd |
| topic_facet | Actin Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027951170&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT titteljanine actinorganizationatintracellularmembranestargetingandregulatoryinteractionsofthespirforminactinnucleatorcomplex |