Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix: fluorescence correlation spectroscopy based single-molecule analysis in vivo
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | IX, 122 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix |b fluorescence correlation spectroscopy based single-molecule analysis in vivo |c von Gupta, Mansi |
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| adam_text | CONTENTS
SUMMARY III
LIST OF PUBLICATIONS V
1 INTRODUCTION 1
1.1 BIOLOGICAL PROCESSES MEDIATED BY DIFFUSIVE MOLECULES 1
1.2 FGF8 MORPHOGEN: ONE MOLECULE MULTIPLE FUNCTIONS 3
1.3 ESTABLISHMENT OF FGF8 MORPHOGEN GRADIENT 3
1.4 FORMATION OF AN EXTRACELLULAR MORPHOGEN GRADIENT 5
1.4.1 DIFFUSION BASED MECHANISMS OF GRADIENT FORMATION 5
1.4.2 NON-CANONICAL MECHANISMS 8
1.5 EXTRACELLULAR MATRIX AND HEPARAN SULFATE PROTEOGLYCANS (HSPG) 8
1.5.1 GLYPICAN (GPC)- HEPARAN SULFATE PROTEOGLYCANS 9
1.5.2 SYNDECAN (SDC)- HEPARAN SULFATE PROTEOGLYCANS 10
1.6 MECHANISMS BY WHICH HSPG REGULATE MORPHOGEN GRADIENTS .... 10
1.7 METHOD FOR STUDY OF MORPHOGEN TRANSPORT: FLUORESCENCE CORRELA
TION SPECTROSCOPY 12
1.8 VARIATIONS OF FCS 13
1.8.1 FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY (FCCS) ... 13
1.8.2 MODULAR SCANNING FCCS FOR MEMBRANE DIFFUSION 15
1.9 FREE DIFFUSION OR RESTRICTED DIFFUSION OF FGF8 - OPEN PROBLEM ... 16
1.9.1 DOES HEPARAN SULFATE ENHANCE OR RESTRICT FGF8 TRANSPORT? . 17
1.9.2 FCS METHOD VERSUS FRAP METHOD 18
2 AIM OF THE STUDY 19
3 RESULTS 21
3.1 IN VITRO FGF8-HEPARAN SULFATE INTERACTION MONITORED BY FCS ... 21
3.2 GLYPICAN HEPARAN SULFATE PROTEOGLYCANS 23
3.2.1 IDENTIFICATION OF GLYPICAN SEQUENCES FROM ZEBRAFISH 23
3.2.2 GLYPICAN PROTEIN SEQUENCE CONSERVATION 26
3.2.3 EXPRESSION PATTERN OF GLYPICANS 29
3.2.4 GLYPICANS ARE CLEAVED FROM THE CELL SURFACE 32
VII
HTTP://D-NB.INFO/1069089389
CONTENTS
3.3 HEPARAN SULFATE PROTEOGLYCANS INFLUENCE FGF DOWNSTREAM SIGNALING 33
3.3.1 SECRETED HSPG ENHANCE FGF SIGNALING 33
3.3.2 EXPRESSION PATTERN OF FGF8 REMAINS UNAFFECTED 35
3.3.3 OVER-EXPRESSION OF FULL LENGTH HSPG DOES NOT INFLUENCE FGF
SIGNALING 35
3.3.4 TESTING CLEAVAGE ENZYMES 36
3.4 BOBTAIL MUTANT: DEFICIENT IN HEPARAN SULFATE 38
3.5 MONITORING DIRECT INTERACTION OF FGF8 WITH EXTRACELLULAR MATRIX
MOLECULES 40
3.5.1 QUANTIFICATION OF EXTRACELLULAR BINDING OF FGF8 WITH GLYPI-
CANS AND SYNDECANS BY DUAL-COLOR FCCS 40
3.5.2 DIFFUSION OF FGF8-EGFP IS REDUCED UPON EXTRACELLULAR BINDING 46
3.5.3 REQUIREMENT OF HEPARAN SULFATE CHAINS IN EXTRACELLULAR
BINDING 48
3.5.4 DIFFERENCE IN HS SIDE CHAINS MAY ACCOUNT FOR DIFFERENT AFFINI
TIES AMONGST HSPG 49
3.6 INFLUENCE OF HSPG ON THE GRADIENT AND DISTRIBUTION OF FGF8 .... 52
3.6.1 FGF8 GRADIENT SHAPE CHANGES UPON EXTRACELLULAR GPC4 OVER-
EXPRESSION 52
3.6.2 EXTRACELLULAR SDC2 ALSO SHARPENS FGF8 GRADIENT IN THE EMBRYO 54
3.6.3 MORPHOLINO MEDIATED KNOCKDOWN OF GPC4 AFFECTS FGF8 EX
TRACELLULAR DISTRIBUTION 55
3.7 STUDY OF FGF8 BINDING WITH FGFR AND HSPG ON CELL MEMBRANE . . 58
3.7.1 FGF8 AND FGF-RECEPTOR INTERACTION IS NOT INFLUENCED BY EX
TRACELLULAR GPC4 LEVELS 58
3.7.2 GLYPICANS AND SYNDECANS INTERACT WITH FGF8 ONLY WEAKLY ON
THE CELL SURFACE 60
3.7.3 CELL SURFACE GLYPICAN OVER-EXPRESSION SHARPENS FGF8 CON
CENTRATION GRADIENT 61
4 DISCUSSION 63
4.1 FGF8 RESTRICTED DIFFUSION DURING EMBRYOGENESIS 63
4.2 HSPG STABILIZE FGF8 IN THE EXTRACELLULAR SPACE: A POSSIBLE MECHA
NISM OF SCALING THE GRADIENT 67
4.3 STRONG FGF8 AND HEPARAN-SULFATE BINDING AFFINITY OBSERVED IN VITRO
68
4.4 FGF8 AND HSPG BINDING KINETICS STUDIED IN VIVO 69
4.5 EXTRACELLULAR AND NOT CELL-SURFACE BOUND HSPG INFLUENCE FGF SIGNAL
TRANSDUCTION 70
4.6 CHARACTERIZATION OF GLYPICANS FROM ZEBRAFISH 71
4.7 MUTANTS DEFICIENT IN HEPARAN SULFATE 73
VIII
5 MATERIALS AND METHODS 75
5.1 TECHNICAL EQUIPMENT 75
5.2 COMMERCIAL KITS 75
5.3 REAGENTS AND SOLUTIONS 75
5.4 ANTIBODIES AND MORPHOLINOS 77
5.5 BIOINFORMATICS TOOLS FOR IDENTIFICATION OF GLYPICANS 77
5.6 MOLECULAR BIOLOGY METHODS 77
5.6.1 LIST OF CLONES MADE FOR THIS STUDY 77
5.6.2 REVERSE TRANSCRIPTASE - PCR 80
5.6.3 SITE-DIRECTED MUTAGENESIS 80
5.6.4 RAPID AMPLIFICATION OF CDNA ENDS 82
5.6.5 IN VITRO MRNA SYNTHESIS 84
5.6.6 ANTI-SENSE DIG-PROBE SYNTHESIS 84
5.7 FISH METHODS 84
5.7.1 FISH MAINTENANCE 84
5.7.2 GENE EXPRESSION IN FISH BY MRNA INJECTIONS 84
5.7.3 IN SITU HYBRIDIZATION 85
5.8 MICROSCOPIC METHODS 86
5.8.1 MOUNTING OF EMBRYOS FOR MICROSCOPY 86
5.8.2 FLUORESCENCE CORRELATION SPECTROSCOPY IN SOLUTION 86
5.8.3 AUTOCORRELATION IN EMBRYOS 87
5.8.4 EXPERIMENTAL SET-UP FOR FCCS 87
5.8.5 SET UP FOR SCANNING FCS 88
5.9 FGF8 GRADIENT MEASUREMENTS 89
5.10 DATA ANALYSIS AND STATISTICS 89
6 APPENDIX: THEORETICAL CONCEPTS 91
6.1 EVALUATION OF IN VITRO DISSOCIATION CONSTANT KA 91
6.2 AUTOCORRELATION ANALYSIS 92
6.3 FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY 94
6.4 BACKGROUND AND CROSS-TALK CORRECTIONS FOR FCCS 95
6.5 EVALUATION OF IN VIVO DISSOCIATION CONSTANT (KJ) 97
6.6 MODELING OF FGF8 CONCENTRATION GRADIENT IN EMBRYOS 98
BIBLIOGRAPHY 101
LIST OF FIGURES 117
ABBREVIATIONS 119
ACKNOWLEDGMENTS 121
IX
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| author | Gupta, Mansi 1985- |
| author_GND | (DE-588)1069440736 |
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| spelling | Gupta, Mansi 1985- Verfasser (DE-588)1069440736 aut Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo von Gupta, Mansi 2014 IX, 122 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2014 Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd rswk-swf Zebrabärbling (DE-588)4079468-4 gnd rswk-swf Morphogen (DE-588)4824466-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Zebrabärbling (DE-588)4079468-4 s Morphogen (DE-588)4824466-1 s Fibroblastenwachstumsfaktor (DE-588)4310092-2 s DE-188 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028117329&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Gupta, Mansi 1985- Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd Zebrabärbling (DE-588)4079468-4 gnd Morphogen (DE-588)4824466-1 gnd |
| subject_GND | (DE-588)4310092-2 (DE-588)4079468-4 (DE-588)4824466-1 (DE-588)4113937-9 |
| title | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo |
| title_auth | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo |
| title_exact_search | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo |
| title_full | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo von Gupta, Mansi |
| title_fullStr | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo von Gupta, Mansi |
| title_full_unstemmed | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single-molecule analysis in vivo von Gupta, Mansi |
| title_short | Fine-tuning of Fgf8 morphogen gradient by the extracellular matrix |
| title_sort | fine tuning of fgf8 morphogen gradient by the extracellular matrix fluorescence correlation spectroscopy based single molecule analysis in vivo |
| title_sub | fluorescence correlation spectroscopy based single-molecule analysis in vivo |
| topic | Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd Zebrabärbling (DE-588)4079468-4 gnd Morphogen (DE-588)4824466-1 gnd |
| topic_facet | Fibroblastenwachstumsfaktor Zebrabärbling Morphogen Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028117329&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT guptamansi finetuningoffgf8morphogengradientbytheextracellularmatrixfluorescencecorrelationspectroscopybasedsinglemoleculeanalysisinvivo |