Functional characterization of Autophagy 9 in Dictyostelium discoideum:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2012
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache |
| Beschreibung: | X, 95 S. Ill., graph. Darst. |
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Datensatz im Suchindex
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| adam_text | Titel: Functional characterization of Autophagy 9 in Dictyostelium discoideum
Autor: Tung, Sze Man
Jahr: 2012
Table of contents
1. Introduction...........................................................................1
1.1 Development of Dictyostelium discoideum.....................................................1
1.1.2 The model organism Dictyostelium discoideum..................................2
1.2 The Ubiquitin Proteasome System....................................................................3
1.3 Linking Autophagy and the UPS.......................................................................5
1.4 Mechanism of Autophagy..................................................................................6
1.5 Autophagy related Protein 9 (ATG9)...............................................................10
1.6 Aim of this work................................................................................................12
1.7 Preliminary publications..................................................................................13
2. Results.................................................................................14
2.1 D. discoideum ATG9.........................................................................................14
2.2 Generation and verification of mutant strains...............................................14
2.3 Subcellular localization of ATG9.....................................................................16
2.3.1 Localization analysis.............................................................................16
2.3.2 ATG9 containing vesicles traffic along microtubules........................18
2.4 Analysis of the ATG9 knock-out mutant.........................................................20
2.4.1 Analysis of autophagy..........................................................................20
2.4.1.1 ATG8 levels were increased............................................................20
2.4.1.2 The autophagic flux is impaired in ATG9~ cells.............................21
2.4.2 Analysis of the ubiquitin proteasome system....................................22
2.4.2.1 Expression of the proteasomal subunits 4 and 5 is not affected 22
2.4.2.2 Detection of ubiquitinated proteins in total cell lysates...............24
2.4.2.3 Detection of ubiquitin and ubiquitinated proteins by immuno-
fluorescence.................................................................................................25
2.4.3 Cell growth, phagocytosis and pinocyosis.........................................26
2.4.3.1 The ATG9 mutant displays growth defects...................................26
2.4.3.2 Phagocytosis andpinocytosis are decreased in the ATGST strain
.......................................................................................................................28
2.4.4 Analysis of development......................................................................30
2.4.4.1 Development is impaired in theATG9 mutant..............................30
2.4.4.2 Aggregation of ATGff cells is delayed...........................................33
2.4.4.3 Phototactic ability is impaired in the ATG9 mutant.....................34
2.4.4.4 Spore viability is reduced................................................................34
2.4.5 DIF-1 partially rescues some of the developmental defects of the
ATG9 mutant..................................................................................................35
2.4.5.1 The induction of DIF-1 synthesis genes is blocked in the A TGfT
mutant...........................................................................................................36
2.4.5.2 Addition of DIF-1 partially rescues fruiting body generation in the
ATG9 strain..................................................................................................37
2.4.5.3 The presence of DIF-1 improved spore viability of ATG9 ............38
3 Discussion...........................................................................39
3.1 Subcellular localization of ATG9.....................................................................39
3.2 Autophagy is blocked in the ATG9 mutant....................................................40
3.3 Analysis of the link between autophagy and the UPS...................................41
3.5 ATG9 displayed impaired Cell growth, phagocytosis and pinocyosis.......42
3.6 Development is impaired in the ATG9 mutant..............................................43
3.7 Developmental defects in ATG9 mutant is partially rescued in presence of
DIF-1........................................................................................................................44
4. Summary.............................................................................46
5. Zusammenfassung.............................................................47
6. Materials and Methods.......................................................49
6.1 Materials............................................................................................................49
6.1.1 Bacterial strains.....................................................................................49
6.1.2 Dictyostelium discoideum strains.......................................................49
6.1.3 Kits..........................................................................................................49
6.1.4 Enzymes, antibodies and antibiotics...................................................50
6.1.4.1 Enzymes for molecular biology......................................................50
6.1.4.2 Primary antibodies...........................................................................50
6.1.4.3 Secondary antibodies......................................................................50
6.1.4.4 Antibiotics........................................................................................51
6.1.5 Plasmids.................................................................................................51
6.1.6 Oligonucleotides...................................................................................51
6.1.7 Chemicals...............................................................................................51
6.1.8 Media and buffers..................................................................................53
6.1.8.1 Media and agar plates for E. coli culture.......................................54
6.1.8.2 Media and agar plates for D. discoideum cell culture..................54
6.1.9 Buffers....................................................................................................56
6.2 Methods.............................................................................................................56
6.2.1 Microbiological methods......................................................................56
6.2.1.1 Growth of bacteria...........................................................................56
6.2.1.2 Preservation of bacterial strains....................................................57
6.2.1.3 Preparation of transformation competent E. coli cells.................57
6.2.1.4 Transformation of competent E. coli cells.....................................58
6.2.1.5 Blue-white selection........................................................................58
6.2.2 Cell biological methods........................................................................58
6.2.2.1 Growth ofD. discoideum in liquid nutrient medium (Claviez etal,
1982)..............................................................................................................58
6.2.2.2 Growth ofD. discoideum on SM agar plates.................................59
6.2.2.3 Development ofD. discoideum on phosphate agar plates..........59
6.2.2.4 Preservation ofD. discoideum cells..............................................60
6.2.2.5 Preservation ofD. discoideum spores...........................................60
6.2.2.6 Transformation ofD. discoideum cells..........................................60
6.2.2.7 Imaging of D. discoideum fruiting bodies......................................61
6.2.2.8 Spore Viability Assay......................................................................62
6.2.2.9 Phototaxis Assay.............................................................................62
6.2.2.10 Aggregation analysis in suspension culture...............................63
6.2.2.11 Phagocytosis ofpHrodo™ E. coli bioparticles®...........................63
6.2.2.12 Phagocytosis ofTRITC labeled yeast..........................................63
6.2.2.13 Pinocytosis ofTRITC labeled dextran..........................................64
6.2.3 Molecular biology methods..................................................................65
6.2.3.1 Isolation of Plasmid-DNA from E. coli (Birnboim undDoly, 1979)65
6.2.3.2 Isolation of Plasmid-DNA from E. coli using Qiagen Mini/Midi Kit
.......................................................................................................................65
6.2.3.3 Determination ofDNA and RNA concentrations...........................65
6.2.3.4 Dephosphorylation ofDNA-5 -Ends (rAPid)..................................66
6.2.3.5 Phenol/Chloroform extraction (Sambrook etal., 1989)................66
6.2.3.6 Precipitation ofDNA........................................................................66
6.2.3.7 Ligation ofDNA fragments..............................................................66
6.2.3.8 Digestion with restriction enzymes................................................67
6.2.3.9 DNA Sequencing..............................................................................67
6.2.3.10 Polymerase chain reaction (PCR).................................................67
6.2.3.11 DNA agarose gel electrophoresis.................................................68
6.2.3.12 Isolation ofDNA fragments from agarose gels...........................69
6.2.3.13 Fast isolation of D. discoideum genomic DNA for PCR analysis
.......................................................................................................................69
6.2.3.14 Colony PCR....................................................................................69
6.2.3.15 Southern blotting...........................................................................70
6.2.3.16 Isolation of total RNA from D. discoideum cells.........................70
6.2.3.17 RNA formaldehyde agarose gel electrophoresis........................71
6.2.3.18 Generation of cDNA.......................................................................72
6.2.3.19 Real time PCR................................................................................72
6.2.4 Biochemical methods............................................................................73
6.2.4.1 SDS Polyacrylamide Gelelectrophoresis (SDS-PAGE).................73
6.2.4.2 Coomassie blue staining of SDS-polyacrylamide gels.................73
6.2.4.3 Preparation of total D. discoideum cell lysates.............................74
6.2.4.4 Western Blotting..............................................................................74
6.2.4.5 Immunodetection of blotted proteins.............................................75
6.2.4.6 Determination of protein concentration.........................................75
6.2.5 Immunostaining and imaging...............................................................76
6.2.5.1 Indirect immunofluorescence microscopy ofD. discoideum cells
.......................................................................................................................76
6.2.5.2 Immunolabeling...............................................................................76
6.2.5.3 Confocal microscopy......................................................................77
6.2.5.4 Live cell imaging..............................................................................77
7. References..........................................................................78
8. Appendix.............................................................................88
9. Abbrevations.......................................................................90
10. Erklarung...........................................................................93
11. Lebenslauf.........................................................................94
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| spelling | Tung, Sze Man 1982- Verfasser (DE-588)1032616563 aut Functional characterization of Autophagy 9 in Dictyostelium discoideum vorgelegt von Sze Man Tung 2012 X, 95 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Köln, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025965519&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
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| title | Functional characterization of Autophagy 9 in Dictyostelium discoideum |
| title_auth | Functional characterization of Autophagy 9 in Dictyostelium discoideum |
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| title_full_unstemmed | Functional characterization of Autophagy 9 in Dictyostelium discoideum vorgelegt von Sze Man Tung |
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| title_sort | functional characterization of autophagy 9 in dictyostelium discoideum |
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