Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Münster
2015
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XIII, 145 Blätter Illustrationen 29 cm |
Internformat
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| 100 | 1 | |a Schürmann, Sabine |d 1986- |e Verfasser |0 (DE-588)1080977155 |4 aut | |
| 245 | 1 | 0 | |a Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells |c vorgelegt von Sabine Schürmann |
| 264 | 1 | |a Münster |c 2015 | |
| 300 | |a XIII, 145 Blätter |b Illustrationen |c 29 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Westfälische Wilhelms-Universität Münster |d 2015 | ||
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Datensatz im Suchindex
| _version_ | 1804176152042930176 |
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| adam_text | TABLE OF CONTENTS
LIST OF
FIGURES...................................................................................................................V
LIST OF
TABLES..................................................................................................................VII
LIST OF
ABBREVIATIONS......................................................................................................VIII
1
SUMMARY...................................................................................................................
1
2
INTRODUCTION................................................................................................................
4
2.1 ROLES OF THE HEDGEHOG PROTEIN FAMILY IN DEVELOPMENT AND DISEASE
.................. 4
2.2 HEDGEHOG
BIOSYNTHESIS.......................................................................................6
2.3 ROLE OF HEDGEHOG LIPID
MODIFICATIONS..................................................................7
2.4 STRUCTURAL INSIGHTS INTO THE HEDGEHOG SIGNALING DOMAIN
.....................................
9
2.5 HEDGEHOG RELEASE AND
TRANSPORT.......................................................................
13
2.5.1 DISPATCHED-MEDIATED HEDGEHOG 13
2.5.2 MICELLOUS
STRUCTURES....................................................................................
14
2.5.3 LIPOPROTEIN
PARTICLES....................................................................................
15
2.5.4
EXOSOMES...................................................................................................
16
2.5.5 HEDGEHOG TRANSPORT VIA
CYTONEMES............................................................16
2.5.6 ADAM-MEDIATED
SHEDDING..........................................................................17
2.5.7 SCUBE2-MEDIATED HEDGEHOG
SOLUBILIZATION.................................................19
2.6 ROLE OF HEPARAN SULFATE PROTEOGLYCANS IN HEDGEHOG SIGNALING
........................ 20
2.7 HEDGEHOG SIGNALING IN RECEIVING
CELLS...............................................................* *
2.8 HEDGEHOG-REGULATED DROSOPHILA WING PATTERNING
.........................................
24
3
MATERIAL....................................................................................................................
27
3.1 EQUIPMENT AND
CONSUMABLES............................................................................27
3.2
CHEMICALS..........................................................................................................
29
3.3 KITS AND
MONONUCLEOTIDES.................................................................................31
3.4
ANTIBODIES..........................................................................................................
31
3.5 MOLECULAR WEIGHT
MARKERS..................................................................................32
3.6 DNA
VECTORS.......................................................................................................
32
3.7
OLIGONUCLEOTIDES................................................................................................
33
3.8 RECOMBINANT
DNA.............................................................................................
37
3.9 ORGANISMS AND CELL
LINES...................................................................................
39
3.10 SOLUTIONS
*
BUFFERS AND
MEDIA.............................................................................41
3.10.1 MEDIA AND SOLUTIONS FOR
BACTERIA................................................................41
3.10.2 MEDIA FOR EUKARYOTIC
CELLS............................................................................42
3.10.3 SOLUTIONS AND BUFFERS FOR THE WORK WITH DNA
.............................................
42
3.10.4 SOLUTIONS FOR DENSITY GRADIENT ULTRACENTRIFUGATION AND TCA
PRECIPITATION.... 43
3.10.5 SOLUTIONS FOR
SDS-PAGE.............................................................................43
3-10.6 SOLUTIONS FOR WESTERN BLOTTING AND IMMUNODETECTION
.................................
44
3.10.7 SOLUTIONS AND BUFFERS FOR TRANSMISSION ELECTRON MICROSCOPY (***)
AND
IMMUNOGOLD
LABELING..............................................................................................
45
3.10.8 MEDIA AND SOLUTIONS FOR THE WORK ON DROSOPHILA
.......................................
46
3.11
SOFTWARE.............................................................................................................
47
4
METHODS...................................................................................................................
48
4.1 MOLECULAR BIOLOGICAL AND BIOCHEMICAL
METHODS................................................48
4.1.1 TOPO CLONING AND LR
RECOMBINATION........................................................48
4.1.2 SITE-DIRECTED
MUTAGENESIS...........................................................................49
4.1.3 SEPARATION OF DNA FRAGMENTS VIA AGAROSE GEL
ELECTROPHORESIS.................. 49
4.1.4 PURIFICATION OF PCR PRODUCTS FROM AGAROSE GEL
..........................................
49
4.1.5 TRANSFORMATION OF
*
CO *
CELLS.....................................................................50
4.1.6 PLASMID EXTRACTION FROM E.
COLI....................................................................50
4.1.7 DETERMINATION OF THE DNA
CONCENTRATION.....................................................50
4.1.8
DNASEQUENCING.........................................................................................
51
4.1.9 CULTURE OF S2
CELLS.......................................................................................
51
4.1.10 TRANSFECTION OF S2 CELLS AND RECOMBINANT PROTEIN
EXPRESSION.......... 52
4.1.11 CULTURE OF BOSC23
CELLS...............................................................................
52
4.1.12 TRANSFECTION OF BOSC23 CELLS AND RECOMBINANT PROTEIN EXPRESSION
... 53
4.1.13
CO-IMMUNOPRECIPITATION..............................................................................
54
4.1.14 PREPARATION OF DROSOPHILA LARVAL
LYSATES......................................................55
4.1.15 OPTIPREP DENSITY GRADIENT
ULTRACENTRIFUGATION..............................................55
4.1.16 GEL FILTRATION ANALYSIS
.............................................
56
4.1.17 TCA PROTEIN
PRECIPITATION.............................................................................56
4.1.18 PROTEIN AFFINITY PURIFICATION ON
HEPARIN-AGAROSE...........................................56
4.1.19 ONE-DIMENSIONAL
SDS-PAGE......................................................................57
4.1.20 SEMIDRY WESTERN BLOTTING AND IMMUNODETECTION OF
PROTEINS........*.. 57
4.1.21 STRIPPING AND REPROBING OF WESTERN
BLOTS....................................................58
4.1 *22 PREPARATION OF BOSC23 CELLS FOR TRANSMISSION ELECTRON MICROSCOPY
(***)
AND IMMUNOGOLD
LABELING.......................................................................................58
4.2 METHODS FOR THE ANALYSIS OF D 59
4.2.1 FLY STOCK MAINTENANCE AND GENETIC
CROSSES...............................................59
4.2.2 GAL4/UAS-TA
*
GETED GENE EXPRESSION IN DROSOPHILA
...................................
60
4.2.3 SITE-SPECIFIC TRANSFORMATION OF
DROSOPHILA...................................................61
4.2.4 PREPARATION AND ANALYSIS OF DROSOPHILA
WINGS............................................62
4.2.5 OBSERVATION OF DROSOPHILA INDIVIDUALS EMERGING FROM
PUPAE..................... 63
4.3 PROTEIN QUANTIFICATION AND STATISTICAL
ANALYSIS....................................................63
4.4
BIOINFORMATICS....................................................................................................
63
5
RESULTS.....................................................................................................................
64
5.1 EFFECTS OF ECTOPIC PALMITOYLATION-DEFICIENT HH EXPRESSION IN VIVO
..................... 64
5.2 IN VIVO ANALYSIS OF NTERMINALLY TRUNCATED HHNP^85S N WING
PATTERNING .*..* 69
5.3 IN VIVO OVEREXPRESSION OF NTERMINALLY TRUNCATED HHNPC85S AT
DIFFERENT
TEMPERATURES..............................................................................................................
76
5.4 ANALYSIS OF DROSOPHILA HH SHEDDING USING HA-TAGGED HHNP
VARIANTS............. 79
5.5 DROSOPHILA WING MORPHOLOGY UPON OVEREXPRESSJON OF HA-TAGGED HHNP
MUTANT
PROTEINS......................................................................................................................
83
5.6 CO-IMMUNOPRECIPITATION FOR THE IN VITRO CONFIRMATION OF
INTERMOLECULA
*
PROTEIN
INTERACTIONS BETWEEN WILD TYPE HHNP AND HHNP MUTANT
FORMS................. 88
5.7 ANALYSIS OF DROSOPHILA HH SHEDDASE CANDIDATES BY
RNAI............................... 90
5.8 ANALYSIS OF THE SHHNP MULTIMER ARCHITECTURE BY IMMUNOELECTRON
MICROSCOPY. 92
5.9 SCUBE2-FACILITATED SHH PROCESSING AND RELEASE OF SHH
CALCIUM-BINDING-
DEFICIENT MUTANT FORMS FROM MAMMALIAN
CELLS............................................................94
6
DISCUSSION................................................................................................................98
6.1 SHEDDING OF HHNP IS A PROMISING MODEL FOR HHNP MULTIMER RELEASE
COUPLED TO
MORPHOGEN
BIOACTIVATION............................................................................................
98
6.2 REPRESSION OF ENDOGENOUS HHNP ACTIVITY BY UNPALMITOYLATED HHNPC85S
CAN BE
EXPLAINED BY THE
*
CO-MULTIMERIZATION IN THE POSTERIOR WING DISC COMPARTMENT....... 101
6.3 N-TERMINALLY TRUNCATED HHNPC
*
SS LACKS BIOACTIVITY DURING DROSOPHILA WING
PATTERNING..................................................................................................................104
6.4 N-TERMINAL PROCESSING RATHER THAN N-PALMITOYLATION PER SE DETERMINES
HHNP
B
0
ACTVTY..................................................................................................................106
6.5 IMPLICATIONS OF MY FINDINGS FOR HH
TAGGING....................................................... 108
6.6 C-TERMINAL IN VITRO SHEDDING OF DROSOPHILA HHNPC
*
5S
*
HA..*............................... 109
6.7 DROSOPHILA HH SHEDDASE CANDIDATES
...............................................................
110
6.8 MECHANISMS OF SCUBE2-DEPENDENT SHH
RELEASE..............................................111
6.9 THE LINEAR HH MULTIME
*
STRUCTURE IS CONSISTENT WITH
*
AND IDEAL FOR HS-DEPENDENT
MORPHOGEN
TRANSPORT.................................................................................................113
6.10 FURTHER IMPLICATIONS FOR HH
SIGNALING...............................................................
116
6.11 THE HH MULTIMER ARRANGEMENT AS SUGGESTED BY WHALEN ET AL.
(2013)....119
7
OUTLOOK...................................................................................................................120
8
REFERENCES.............................................................................................................122
9
SUPPLEMENT............................................................................................................140
9.1 SUPPLEMENTAL
DATA...........................................................................................
140
9.2 CUITICULUM
VITAE...............................................................................................142
9.3 VERSICHERUNGEN
................................................................................................
144
9.4
DANKSAGUNG.....................................................................................................145
|
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| spelling | Schürmann, Sabine 1986- Verfasser (DE-588)1080977155 aut Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells vorgelegt von Sabine Schürmann Münster 2015 XIII, 145 Blätter Illustrationen 29 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Westfälische Wilhelms-Universität Münster 2015 Zelldifferenzierung (DE-588)4067536-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Zelldifferenzierung (DE-588)4067536-1 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028927187&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schürmann, Sabine 1986- Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells Zelldifferenzierung (DE-588)4067536-1 gnd |
| subject_GND | (DE-588)4067536-1 (DE-588)4113937-9 |
| title | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells |
| title_auth | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells |
| title_exact_search | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells |
| title_full | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells vorgelegt von Sabine Schürmann |
| title_fullStr | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells vorgelegt von Sabine Schürmann |
| title_full_unstemmed | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells vorgelegt von Sabine Schürmann |
| title_short | Structural and in vivo analysis of linear Hedgehog multimerization at the surface of producing cells |
| title_sort | structural and in vivo analysis of linear hedgehog multimerization at the surface of producing cells |
| topic | Zelldifferenzierung (DE-588)4067536-1 gnd |
| topic_facet | Zelldifferenzierung Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028927187&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schurmannsabine structuralandinvivoanalysisoflinearhedgehogmultimerizationatthesurfaceofproducingcells |