Mammalian coronin 7 and small Rho GTPases:
Gespeichert in:
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
| Veröffentlicht: |
Köln
2015
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 2 Mikrofiches (X, 100 Seiten) Illustrationen 21 cm |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
1.
INTRODUCTION..............................................................................................................
1
1.1 CORONIN FAMILY OF
PROTEINS..........................................................................................1
1.2 AN INSIGHT INTO CORONIN 7 (CRN7) - LOCALIZATION AND
SUNCTION.. *.. ** ** 3
1.3 ACTIN CYTOSKELETON REGULATION BY SMALL RHO
GTPASES..........................................4
1.4 CRIB EFFECTOR
PROTEINS................................................................................................
7
1.5 * * * . * * - STRUCTURE AND
FUNCTION............................................................................
9
1.6 AIM OF THIS
WORK.........................................................................................................12
2.
RESULTS....................................................................................................................
13
2.1 GENERATION AND CHARACTERIZATION OF A CRN7-DEFICIENT M
ICE............................ 13
2.1.1 CRN7 TARGETING
APPROACH.................................................................................13
2.1.2 VALIDATION OF THE CRN7 REPORTER INSERTION KNOCK-OUT
MOUSE**.*....... 15
2.1.3 HISTOPATHOLOGICAL SCREENING OF CRN7 KNOCK-OUT M ICE.*.***
*_...** 17
2.1.4 GOLGI MORPHOLOGY) GOLGI-CENTROSOME POSITIONING AND CENTROSOME
NUMBER
ARE AFFECTED UPON CRN7 KNOCK-OUT
..........................................................................
17
2.1.5 KNOCK-OUT OF CRN7 ALTERS GOLGI CENTROSOME POLARIZATION AND CELL
MIGRATION
OF MURINE PRIMARY FIBROBLASTS DURING WOUND HEALING OWING TO TS
POLARIZED ACTIN
CYTOSKELETON................................................................................................................
20
2.1.6 CRN7 INHIBITS FOCAL ADHESIONS AND STRESS FIBER FORMATION
......*.... 25
2.1.7 CRN7 KNOCK-OUT CELLS ATTACH AND SPREAD FASTER ON FIBRONECTIN
.*....* 28
2.1.8 CRN7 KNOCK-OUT DOES NOT ALTER THE RATE OF CELL PROLIFERATION AND
FORMATION OF
MEMBRANE
PROTRUSIONS................................................................................................29
2.1.9 RE-EXPRESSION OF GFP-TAGGED HUMAN WILDTYPE CRN7 JN CRN7 KO MURINE
FIBROBLASTS COULD RESTORE THE CELLULAR
PHENOTYPES....................................................3*
2.2 REGULATION OF MAMMALIAN CRN7 FUNCTIONS BY THE SMALL RHO GTPASES 33
2.2.1 MAMMALIAN CRN7 INTERACTS PRIMARILY WITH GDP-BOUND GTPASES AND
SHOWS A PREFERENCE FOR THE GDP-BOUND CDC42
......................................................
34
2.2.2 CRN7 EXERTS AN INHIBITORY EFFECT ON FIBROBLAST SPREADING AND ACTIN
STRESS
FIBER DENSITY VIA ITS INTERACTION WITH THE DOMINANT NEGATIVE MUTANT OF
CDC42_.._. 36
2.2.3 MUTATIONS IN THE CRN7 CRIB MOTIFS ABROGATE ITS BINDING WITH CDC42
BUT DO
NOT ALTER THE LOCALIZATION OF CR
N7............................................................................38
2.2.4 EXPRESSION OF THE CRN7 CR!B-MUT4 PARTIALLY RESTORES THE GOLGI
INTEGRITY
BUT FAILS TO RESCUE THE ACTIN PHENOTYPES IN CRN7 * *
0****..__..._....__ 40
2.2.5 CRN7 ** CELLS SHOW REDUCED GTP-CD
*
42 LEVELS BUT CRN7 DOES NOT HAVE
ANY GEF ACTIVITY TOWARDS C DC42
.............................................................................
42
2.2.6 CRN7 AND CDC42 EFFECTOR N-WASP SHOW A DIRECT
INTERACTION............... 45
2.2.7 CRN7 AND N-WASP INTERACT WITH EACH OTHER VIA THEIR
N-TERMINI.............. 46
2.2.8 CRN7 REGULATES CELLULAR PHENOTYPES THROUGH N-WASP
INHIBITION............ 48
3.
DISCUSSION...........................................................................................................**
3.1 MAMMALIAN CRN7 * ROLE IN GOLGI STRUCTURE MAINTENANCE AND BEYOND
.......... 51
3.1.1 CRN7 AND THE ACTIN CYTOSKELETON * ALTERATIONS IN THE KNOCK-OUT
MOUSE. 52
3.1.2 FOCAL ADHESION FORMATION AND CRN7
.............................................................
53
3.1.3 THE GOLGI AND CENTROSOME IN THE KNOCK-OUT MOUSE 54
3.2 CRN7 - RHO GTPASE INTERACTION IN REGULATING ACTIN-MEDIATED CELLULAR
FUNCTIONS
54
3.2.1 THE CRIB DOMAINS OF CRN7 MEDIATES ITS
FUNCTIONS................................. 56
3.2.2 CRN7 WITH MUTATIONS IN THE CRIB M
OTIFS.......................................................57
3.2.3 CRN7 BINDS SELECTIVELY TO GDP-CDC42 BUT IS NOT A GEF FOR
CDC42......... 58
3.2.4 CRN7 DYNAMICALLY CONTROLS GDP-CDC42 LEVELS N THE
CELL........................ 59
3.2.5 DOWNSTREAM EFFECTOR OF CRN7_CDC42
INTERACTION.........................................59
4.
SUMMARY...............................................................................................................
62
* .
ZUSAMMENFASSUNG...............................................................................................64
FI MATERIALS AND
METHODS.........................................................................................66
6.1
MATERIALS.......................................................................................................
66
6.1.1 MEDIA AND
BUFFERS.............................................................................................66
6.1.2
CONSTRUCTS.........................................................................................................66
6.1.3
OLIGONUCLEOTIDES...............................................................................................66
6.1.4 ENZYMES) ANTIBODIES
*
ANTIBIOTICS
*
INHIBITORS
..................................................
68
6.1.4.1
ENZYMES....................................................................................................
68
6.1.4.2
ANTIBODIES..................................................................................................
68
6.1 *4.3
ANTIBIOTICS...............................................................................................
69
6.1.4.4
INHIBITORS.................................................................................................69
6.1.5
MISCELLANEOUS...................................................................................................
69
6.1.6 BACTERIAL
STRAINS...............................................................................................70
6.1.7 MAMMALIAN CELL
LINES......................................................................................
70
6.2 M E TH O D S
..............................................................................................................
71
6.2.1 MOLECULAR BIOLOGY METHODS
............................................................................
71
6.2.1.1 PLASMID DNA PREPARATION FROM TRANSFORMED E. CO CELLS
*
MEASURING
DNA CONCENTRATION AND SEQUENCING OF DN A
.....................................................
71
6.2.1
*
2 RESTRICTION ENZYME DIGESTION
*
DNA PRECIPITATION AND AGAROSE GEL
ELECTROPHORESIS......................................................................................................71
6.2.1.3 LIGATION AND
TRANSFORMATION...................................................................72
6.2.1 _4 GENOMIC DNA
SOLATION............................................................................72
6.2.1.5 POLYMERASE CHAIN REACTION
(PCR).......................................................... 73
6.2.1.6 RNA SOLATION AND REVERSE TRANSCRIPTASE PCR
(RT-PCR)................. 73
6.2.1.7 SOUTHERN
BLOT.............................................................................................
74
6.2.1.8 NORTHERN BLOT
.............................................................................................
75
6.2.2 BIOCHEMICAL
METHODS......................................................................................75
6.2.2.1 PREPARATION OF TISSUE AND CELL HOMOGENATES AND PROTEIN
QUANTITATION 75
6.2.2.2SDS-PAG
E................................................................................................76
6.2.2.3 WESTERN
BLOT..............................................................................................77
6.2.2.4 GENERATION OF FUSION PROTEINS AND EXPRESSION/PURIFICATION OF
PROTEINS IN
BACTERIAL AND MAMMALIAN CELLS
............................................................................
78
6.225 PULL-DOWN
*
C0-MMUN0PREC*P*TAT0*N AND BLOT-OVERLAY EXPERIMENTS.* 79
6.2.2.6 NUCLEOTIDE EXCHANGE ASSAY
................................................................
80
6 .2 3 CELL BIOLOGY
METHODS.......................................................................................81
6.2.3.1 CULTURING MAMMALIAN
CELLS......................................................................
81
6.2.3.2 ISOLATION OF MOUSE PRIMARY FIBROBLASTS
...................................................
81
6.2.3.3 FREEZING AND THAWING OF
CELLS..................................................................82
6.2.3.4 TRANSFECTION OF MAMMALIAN CELLS
............................................................
82
6.2.3.5 IMMUNOFLUORESCENCE
STAINING.................................................................83
6 .2 3 6 CONFOCAL MICROSCOPY AND DATA ANALYSES
...............................................
83
6 .2 3 7 IN VITRO CELLULAR
ASSAYS..............................................................................
84
6.2.4 MOUSE
SCREENING..........................................................................................
85
7. R
EFERENCES...................................................................................................................
87
8. A BB REVIATION
S...............................................................................................................95
* .
ERKLAERUNG...............................................................................................................
98
10.
LEBENSLAUF...........................................................................................................
99
11* CURRICULUM
VITAE...............................................................................................100
|
| any_adam_object | 1 |
| author | Bhattacharya, Kurchi 1988- |
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| spelling | Bhattacharya, Kurchi 1988- Verfasser (DE-588)1084236702 aut Mammalian coronin 7 and small Rho GTPases vorgelegt von Kurchi Bhattacharya Köln 2015 2 Mikrofiches (X, 100 Seiten) Illustrationen 21 cm txt rdacontent h rdamedia he rdacarrier 24x Dissertation Universität zu Köln 2015 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1095111094/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028967537&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Bhattacharya, Kurchi 1988- Mammalian coronin 7 and small Rho GTPases |
| subject_GND | (DE-588)4113937-9 |
| title | Mammalian coronin 7 and small Rho GTPases |
| title_auth | Mammalian coronin 7 and small Rho GTPases |
| title_exact_search | Mammalian coronin 7 and small Rho GTPases |
| title_full | Mammalian coronin 7 and small Rho GTPases vorgelegt von Kurchi Bhattacharya |
| title_fullStr | Mammalian coronin 7 and small Rho GTPases vorgelegt von Kurchi Bhattacharya |
| title_full_unstemmed | Mammalian coronin 7 and small Rho GTPases vorgelegt von Kurchi Bhattacharya |
| title_short | Mammalian coronin 7 and small Rho GTPases |
| title_sort | mammalian coronin 7 and small rho gtpases |
| topic_facet | Hochschulschrift |
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