Nuclear actin polymerization by formins:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VII, 88 S. Ill., graph. Darst. 21 cm |
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| adam_text | AUS DEM INSTITUT FUR PHARMAKOLOGIE
GESCHAFTSFLIHRENDER DIREKTOR: PROF. DR. ROBERT GROSSE
DES FACHBEREICHS MEDIZIN DER PHILIPPS-UNIVERSITAT MARBURG
NUCLEAR ACTIN POLYMERIZATION BY FORMINS
INAUGURAL-DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER NATURWISSENSCHAFTEN
DEM FACHBEREICH MEDIZIN DER PHILIPPS-UNIVERSITAT MARBURG
VORGELEGT VON
HAICUI WANG
AUS SHANDONG, CHINA
MARBURG, 2014
HTTP://D-NB.INFO/1052352375
TABLE OF CONTENTS
ABBREVIATIONS................................................................................................
IV
1 INTRODUCTION... 1
1.1 ACTIN ASSEMBLY AND DISASSEMBLY 2
1J NUCLEATION OF ACTIN FOR SPONTANEOUS ASSEMBLY IN CELLS
.....................4
1.2.1 MECHANISM OF ARP2/3 COMPLEX ON ACTIN ASSEMBLY 4
1.2.2 MECHANISM OF FORMINS ON ACTIN ASSEMBLY S
1J REGULATION OF FORMIN ACTIVITY ON CYTOSKELETON REMODELING IN CELLS 8
1.4 ACTIN AND ACTIN BINDING PROTEINS IN THE NUCLEUS..- 9
1.5 ACTIN AND ACTIN BINDING PROTEINS ON TRANSCRIPTIONS IN THE NUCLEUS
.11
1.6 FORMINS IN THE NUCLEUS 13
1.7 AIM OF THIS
STUDY 14
2. MATERIALS AND METHODS _ - 15
2.1
MATERIALS...............................................................................................
15
2.2 CELL
CULTURE...........................................................................................
26
2.2.1 GENERAL CELL CULTURE 26
2.2.2 CELL TRANSFECTION 26
2 J BIOCHEMICAL
METHODS...........................................................................27
2.3.1 PURIFICATION OF RECOMBINANT PROTEINS 27
2.3.2 IMMUNOPRECIPITATION 28
2.3.3 NUCLEAR AND CYTOPLASMIC FRACTIONATION 28
2.3.4 SDS-PAGE AND WESTERN BLOTTING 28
2.3.5 COOMASSIE STAINING AND DESTAINING 30
2.3.6 PYRENYL-ACTIN ASSEMBLY ASSAY 30
2.4 MOLECULAR BIOLOGICAL METHODS 30
2.4.1 DNA CLONING 30
2.4.2 AGAROSE GEL ELECTROPHORESIS 32
2.4.3 RNA ISOLATION FROM CELLS 32
V
TABLE OF CONTENTS
2.4.4 REVERSE TRANSCRIPTION OF TOTAL RNA 33
2.4.5 QUANTITATIVE REAL TIME PCR (QPCR) 33
2.5 IMMUNOFLUORESCENCE AND MICROSCOPY... .......... *.34
2.5.1 IMMUNOFLUORESCENCE 34
2.5.2 MICROSCOPY 34
3.1 NUCLEAR FORMINS ON RNA POL TRANSCRIPTION .... ..... 35
3.1.1 GENERAL TOOL FOR SCREENING OF FORMINS INVOLVED IN RNA POLYMERASE
II TRANSCRIPTION ACTIVITY 35
3.1.2 SCREENING FOR FORMINS REQUIRED FOR RNA POLYMERASE II DEPENDENT
TRANSCRIPTION 37
3.1.3 EFFECTS OF FORMINS ON BASAL HISTONE H3 ACETYLATION 38
3.2 POTENT ACTIN ASSEMBLY ACTIVITY OF HUMAN FORMIN DIAPH3. ..41
3.2.1 PURIFICATION OF HUMAN DLAPH3 IN E.COLI 42
3.2.2 ACTIVITY OF HUMAN DIAPH3 ON ACTIN ASSEMBLY 43
3 J FORMIN-MEDIATED ACTIN ASSEMBLY IN THE NUCLEUS..... ....... 44
3.3.1 DETECTION OF NUCLEAR ACTIN AND FORMINS 44
3.3.2 ASSOCIATION OF ENDOGENOUS MDIAL AND ACTIN IN THE NUCLEUS 45
3.3.3 NUCLEAR ACTIVE MDIA (DIALCT) PROMOTES ACTIN ASSEMBLY 46
3.3.4 MDIAL/2 DEPENDENT BASAL NUCLEAR ACTIN TURNOVER 47
3.3.5 ARP2/3 REGULATES CYTOPLASMIC BUT NOT NUCLEAR ACTIN ASSEMBLY 50
3.3.6 IMPAIRMENT OF ACTIN POLYMERIZATION BY NUCLEAR EXTRACTS RESULTING
FROM DEPLETION OF ENDOGENOUS FORMINS 54
3.4 ACTIVATION OF FORMINS IN THE NUCLEUS ....... ......... 56
3.4.1 ANALYZATION OF SUBCELLULAR DISTRIBUTIONS OF ENDOGENOUS RHO-
GTPASES 56
3.4.2 ROLE OF RHO-GTPASES IN ACTIN ASSEMBLY OF NUCLEAR EXTRACTS 56
3.4.3 EFFECTS OF SERUM STIMULATION ON NUCLEAR ACTIN ASSEMBLY 59
3.4.4 ROLE OF MDIA FORMINS IN SERUM-INDUCED NUCLEAR ACTIN ASSEMBLY ...60
4 DISCUSSION ... 62
VI
TABLE OF CONTENTS
5
SUMMARY..................*.................................................................................68
6 ZUSAMMENFASSUNG 69
7 REFERENCES 70
8 PUBLICATIONS... ... ....... ....... ... 83
8.1 ORIGINAL PUBLICATIONS ........................................ 83
.8.2 COMMUNICATIONS AND POSTERS 83
9 CURRICULUM VITAE 84
10 ACKNOWLEDGMENTS...
....................*....................*..._...*.......................86
11 ACADEMIC TEACHERS............. 87
12 DECLARATION
................................................................................................88
12 EHRENWORTIICHE ERKLSRUNG 88
VII
|
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| language | English |
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| physical | VII, 88 S. Ill., graph. Darst. 21 cm |
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| spelling | Wang, Haicui 1983- Verfasser (DE-588)1050671953 aut Nuclear actin polymerization by formins vorgelegt von Haicui Wang 2014 VII, 88 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Marburg, Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027673269&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wang, Haicui 1983- Nuclear actin polymerization by formins |
| subject_GND | (DE-588)4113937-9 |
| title | Nuclear actin polymerization by formins |
| title_auth | Nuclear actin polymerization by formins |
| title_exact_search | Nuclear actin polymerization by formins |
| title_full | Nuclear actin polymerization by formins vorgelegt von Haicui Wang |
| title_fullStr | Nuclear actin polymerization by formins vorgelegt von Haicui Wang |
| title_full_unstemmed | Nuclear actin polymerization by formins vorgelegt von Haicui Wang |
| title_short | Nuclear actin polymerization by formins |
| title_sort | nuclear actin polymerization by formins |
| topic_facet | Hochschulschrift |
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