Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2002
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 122 Bl. Ill., graph. Darst. : 30 cm |
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MARC
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| 100 | 1 | |a Garvalov, Boyan |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A |c [vorgelegt von Boyan Garvalov] |
| 264 | 1 | |c 2002 | |
| 300 | |a 122 Bl. |b Ill., graph. Darst. : 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Heidelberg, Univ., Diss., 2002 | ||
| 650 | 0 | 7 | |a Actin-bindende Proteine |0 (DE-588)4359634-4 |2 gnd |9 rswk-swf |
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| adam_text |
TABLE OF CONTENTS
LIST OF FIGURES 1
LIST OF ABBREVIATIONS 2
SUMMARY 3
ACKNOWLEDGEMENTS 4
1. INTRODUCTION 5
1.1. ACTIN-RELATED PROTEINS (ARP'S) 5
1.1.1. THE ARP SUPERFAMILY 5
1.1.2. ARP FAMILIES (CLASSES) 6
1.1.3. WELL-STUDIED ARPS 8
1.1.3.1. ARP1 8
1.1.3.2. ARP2 AND ARP3 9
1.1.3.3. NUCLEAR ARPS 10
1.1.4. HUMAN ARP7A AND ARP7B 12
1.2. FOCAL ADHESIONS. KNOWN AND NOVEL FOCAL ADHESION COMPONENTS 14
1.2.1. THE DIVERSITY AND IMPORTANCE OF CELL ADHESIONS 14
1.2.2. THE MOLECULAR COMPLEXITY OF FOCAL ADHESIONS 15
1.2.3. THE ASSEMBLY AND FUNCTIONS OF FOCAL ADHESIONS 18
1.2.4. SELECTED FOCAL ADHESION COMPONENTS 21
1.2.4.1. MENA AND VASP 21
1.2.4.2. ZYXIN 22
1.2.4.3. PAXILLIN 24
1.2.4.4. A-ACTININ 25
1.2.5. THE TRIPLE LIM DOMAIN PROTEIN TES 27
2. RESULTS 31
2.1. CELLULAR DISTRIBUTION AND PROPERTIES OF ARP7A AND ARP7B 31
2.1.1. STRUCTURAL FEATURES OF ARP7A AND ARP7B 31
2.1.2. CLONING, EXPRESSION AND PURIFICATION OF ARP7A AND ARP7B
CONSTRUCTS. ANTIBODY GENERATION 32
2.1.3. CHARACTERIZATION OF THE ANTI-ARP7A ANTIBODIES 32
2.1.4. ARP7A IS DIFFERENTIALLY EXPRESSED IN VARIOUS ORGANS AND
CELL LINES 34
2.1.5. THE DIFFERENT ANTI-ARP7A ANTIBODIES SHOW DISPARATE
STAINING PATTERNS BY IMMUNOFLUORESCENCE 35
2.1.6. TRANSFECTED ARP7A AND ARP7B CONSTRUCTS LOCALIZE
DIFFUSELY 37
2.1.7. ARP7A FRACTIONATES AS A COMPLEX BY GEL FILTRATION 39
2.1.8. THE N-TERMINAL DOMAIN OF ARP7A INTERACTS WITH TES 40
2.2. SUBCELLULAR LOCALIZATION, BIOCHEMICAL AND FUNCTIONAL
CHARACTERIZATION OF TES 41
2.2.1. TRANSFECTED TES2 CONSTRUCTS LOCALIZE AT SITES OF FOCAL
ADHESION 41
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/966219058
2.2.2. TES CONSTRUCTS, PURIFICATION OF TES AND TES DOMAINS.
ANTIBODY GENERATION 41
2.2.3. ENDOGENOUS TES LOCALIZES AT FOCAL ADHESIONS 44
2.2.4. THE C-TERMINAL PORTION OF TES TARGETS TO FOCAL ADHESIONS
WHILE THE N-TERMINAL ONE ASSOCIATES WITH THE ACTIN
CYTOSKELETON 45
2.2.5. THE ISOLATED LIM1 IS RECRUITED TO FOCAL ADHESIONS 46
2.2.6. TES IS AN ACTIN-BINDING PROTEIN WHICH ALSO ASSOCIATES WITH
MENA AND VASP 46
2.2.7. TES, MENA AND VASP CO-LOCALIZE AT FOCAL CONTACTS 48
2.2.8. MENA AND VASP BIND THE LIM3 DOMAIN IN THE C-TERMINAL
PORTION OF TES 50
2.2.9. LIM3 IS ESSENTIAL FOR THE PROPER LOCALIZATION OF TES 51
2.2.10. THE N- AND C-TERMINAL PORTIONS OF TES HAVE DIFFERENT
BINDING PROPERTIES FROM THE INTACT PROTEIN 53
2.2.11. THE N- AND C-TERMINAL HALVES OF TES INTERACT WITH EACH
OTHER 55
2.2.12. FOCAL ADHESION COMPONENTS ARE LOCALIZED NORMALLY IN
TES-DEFICIENT CELLS 58
2.2.13. TES OVEREXPRESSION DISRUPTS THE LOCALIZATION OF MENA
AND VASP 60
3. DISCUSSION 62
3.1. CHARACTERIZATION OF THE HUMAN ACTIN-RELATED PROTEINS 7A AND 7B 62
3.1.1. ARP7A IS NOT A UBIQUITOUSLY EXPRESSED PROTEIN 62
3.1.2. THE IMMUNOFLUORESCENCE STAINING FOR ARP7A - A PANOPLY
OF ARTEFACTS? 62
3.1.3. LOCALIZATION OF ARP7A AND ARP7B 65
3.1.4. ARP7A INTERACTS WITH TES 66
3.2. TES IS A NOVEL FOCAL ADHESION PROTEIN, THAT INTERACTS WITH VARIOUS
COMPONENTS OF FOCAL CONTACTS AND IS INVOLVED IN MENAA/ASP
TARGETING. 67
3.2.1. HUMAN TES IS A FOCAL ADHESION PROTEIN 67
3.2.2. TES INTERACTS WITH FOCAL ADHESION COMPONENTS 68
3.2.3. THE SEPARATE DOMAINS OF TES HAVE DIFFERENT INTERACTION
AND LOCALIZATION PROPERTIES WHEN EXPRESSED ALONE AND
WHEN PRESENT IN THE FULL-LENGTH PROTEIN 69
3.2.4. INTRAMOLECULAR INTERACTIONS WITHIN TES. MODELS FOR THE
REGULATION OF TES STRUCTURE 70
3.2.5. TARGETTING OF TES TO FOCAL ADHESIONS 72
3.2.6. FUNCTIONS OF TES 73
3.3. PERSPECTIVES 76
4. MATERIALS AND METHODS 78
4.1. REAGENTS 78
4.2. GENERAL BUFFERS AND BACTERIAL MEDIA 78
4.3. CLONING AND RELATED TECHNIQUES 78
4.3.1. CLONING OF ARP7A, ARP7B AND ARP7A DELETION CONSTRUCTS 78
4.3.2. CLONING OF TES2 AND TES2 CONSTRUCTS BY PCR 79
4.3.3. PREPARATION OF COMPETENT
E. COLI
81
4.3.4. TRANSFORMATION OF COMPETENT E. COLI 81
4.3.5. RESTRICTION DIGESTS 81
4.3.6. LIGATION OF DNA 82
4.3.7. AGAROSE GEL ELECTROPHORESIS 82
4.3.8. "MINI" AND "MIDI" PLASMID PREPARATION 82
4.3.9. POINT MUTANT GENERATION 82
4.4. TISSUES CULTURE AND RELATED TECHNIQUES 83
4.4.1. CELL LINES 83
4.4.2. CALCIUM PHOSPHATE TRANSFECTION 84
4.4.3. CATIONIC LIPID-MEDIATED TRANSFECTION 85
4.4.4. IMMUNOFLUORESCENSCE 85
4.4.5. MICROSCOPY 86
4.5. PROTEIN PURIFICATION AND ANALYSIS 86
4.5.1. PROTEIN EXPRESSION AND PURIFICATION FROM THE SOLUBLE
FRACTION. AFFINITY COLUMN PREPARATION FOR PULLDOWNS 86
4.5.2. PROTEIN PURIFICATION FROM INCLUSION BODIES. AFFINITY COLUMN
PREPARATION FOR PULLDOWNS 88
4.5.3. PREPARATION OF AFFINITY COLUMNS FOR ANTIBODY PURIFICATION 88
4.5.4. MEASUREMENT OF PROTEIN CONCENTRATION 89
4.5.5. SODIUM DODECYLSULPHATE POLYACRYLAMIDE GEL
ELECTROPHORESIS (SDS PAGE) 89
4.5.6. SILVER STAINING OF POLYACRYLAMIDE GELS 89
4.5.7. WESTERN BLOTTING (IMMUNOBLOTTING) 90
4.5.8. MASS SPECTROMETRY ANALYSIS 90
4.6. ANTIBODIES 90
4.6.1. GENERATION OF ANTIBODIES AGAINST ARP7A AND TES 90
4.6.2. AFFINITY PURIFICATION OF ANTIBODIES 91
4.6.2.1. PURIFICATION OF THE ANTI-ARP7A-N PEPTIDE ANTIBODY 91
4.6.2.2. PURIFICATION OF THE ANTI-ARP7A/FULL/ AND ANTI-
ARP7A/1 -65/ ANTI BOD IES 91
4.6.2.3. PURIFICATION OF THE ANTI-TES/FULL/ ANTIBODY 92
4.6.3. COMMERCIAL ANTIBODIES AND GIFTS 92
4.7. CELL AND TISSUE EXTRACTS, PULLDOWN EXPERIMENTS 93
4.7.1. MOUSE ORGAN EXTRACTS 93
4.7.2. EXTRACT FOR GEL FILTRATION 93
4.7.3. PULLDOWNS WITH NI-HIS-TES AFFINITY RESINS 94
4.7.4
IN VITRO INTERACTIONS BETWEEN THE N-TEMINAL PORTION OF TES
AND THE C-TERMINAL PORTION/LIM DOMAINS 94
4.8. BIOINFORMATIC TOOLS 94
5. PUBLICATIONS 96
6. REFERENCES 97 |
| any_adam_object | 1 |
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| institution | BVB |
| language | English |
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| physical | 122 Bl. Ill., graph. Darst. : 30 cm |
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| spelling | Garvalov, Boyan Verfasser aut Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A [vorgelegt von Boyan Garvalov] 2002 122 Bl. Ill., graph. Darst. : 30 cm txt rdacontent n rdamedia nc rdacarrier Heidelberg, Univ., Diss., 2002 Actin-bindende Proteine (DE-588)4359634-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Actin-bindende Proteine (DE-588)4359634-4 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010272632&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Garvalov, Boyan Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A Actin-bindende Proteine (DE-588)4359634-4 gnd |
| subject_GND | (DE-588)4359634-4 (DE-588)4113937-9 |
| title | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A |
| title_auth | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A |
| title_exact_search | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A |
| title_full | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A [vorgelegt von Boyan Garvalov] |
| title_fullStr | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A [vorgelegt von Boyan Garvalov] |
| title_full_unstemmed | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A [vorgelegt von Boyan Garvalov] |
| title_short | Characterization of the novel focal adhesion protein Tes and of its interacting partner, actin related protein 7A |
| title_sort | characterization of the novel focal adhesion protein tes and of its interacting partner actin related protein 7a |
| topic | Actin-bindende Proteine (DE-588)4359634-4 gnd |
| topic_facet | Actin-bindende Proteine Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010272632&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT garvalovboyan characterizationofthenovelfocaladhesionproteintesandofitsinteractingpartneractinrelatedprotein7a |