Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
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2016
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| 245 | 1 | 0 | |a Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 |c vorgelegt von Atul Kumar |
| 264 | 1 | |a Köln |c 2016 | |
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| adam_text | Titel: Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2
Autor: Kumar, Atul
Jahr: 2016
Table of contents
Chapter 1
1. Introduction 1
1.1 The Cytoskeleton 1
1.1.1 The Cytoskeleton 1
1.1.2 The Actin Cytoskeleton 2
1.1.3 Actin in Neuron 4
1.1.4 Synaptic Cytoskeleton 5
1.1.4.1 Organization of presynaptic actin 6
1.1.4.2 Organization of postsynaptic actin 6
1.2 Actin binding proteins 8
1.3 Cyclase associated protein (CAP) 9
1.4 Aim of research 12
Chapter 2
2. Results
2.1 Generation of CAP2 knockout mice 13
2.2 CAP2 is expressed in cortex, hippocampus and cerebellum 14
2.3 Mutant mice do not show gross anatomical difference in brain 17
2.4 CAP2 localizes to dendrites, presynaptic and postsynaptic terminal 17
2.5 Altered spine density in CAP2 mutant neurons 19
2.6 Altered dendritic complexity in CAP2 mutant neurons 22
2.7 Actin filament turnover is impaired in CAP2 deficient neurons 25
2.8 CAP1 does not compensate the loss of CAP2 in brain 29
2.9 CAP2 mutant neurons show reduce density of surface dendritic GluRl 29
2.10 CAP2 regulates synaptic actin dynamics 32
2.11 CAP2 interacts with n-cofilin 34
2.12 CAP2 affects phosphorylated n-cofilin levels and leads to its aggregation 37
2.13 CAP2 interacting partners in neurons 41
Chapter 3
3. Discussion 43
3.1 CAP2: Role in neuronal development 43
3.2 CAP2: Role in presynaptic terminal 44
3.3 CAP2: Role in postsynaptic terminal 45
3.4 CAP2 interacts with n-cofilin 46
3.5 CAP2 and neurological disorder: Future perspective 47
Chapter 4
4. Materials and Methods 50
4.1. Materials 50
4.1.1 Enzymes for molecular biology 50
4.1.2 Antibodies 50
4.1.3 Antibiotics 51
4.1.4 Reagents 51
4.1.5 Kits 52
4.1.6 Bacterial host strain 53
4.1.7 Media for E.coli culture 53
4.1.8 SOC medium 53
4.1.9 Eukaryotic cells 53
4.1.10 Media for cell culture 54
4.1.11 Oligonucleotides 54
4.1.12 Buffers and other solutions 55
4.2. Methods 56
4.2.1 Analysis of mutant mice 56
4.2.1.1 Transgenic animals 56
4.2.1.2 Genotyping of mice 56
4.2.1.3 Immunohistochemistry and histology 57
4.2.1.4 X-gal staining 57
4.2.1.5 Nissl staining 57
4.2.1.6 Golgi-Cox staining 58
4.2.2 Cell culture techniques 58
4.2.2.1 Passage of cells, trypsinization and determination of cell number 58
4.2.2.2 Thawing cells 59
4.2.2.3 Freezing cells 59
4.2.2.4 Differentiation of cells 59
4.2.2.5 Primary cortical neuron culture 59
4.2.2.6 Transaction and immunofluorescence 60
4.2.3 Morphometric analysis of Purkinje neurons 60
4.2.3.1 Vibratome section of brain 60
4.2.3.2 Labeling of Purkinje neurons 61
4.2.3.3 Imaging of Purkinje neurons 61
4.2.4 Biochemical assay 61
4.2.4.1 Synaptosome preparation 61
4.2.4.2 Determination of the F/G-actin ratio 62
4.2.4.3 KC1 depolarization assay 62
4.2.4.4 Surface biotinylation assay 62
4.2.4.5 Induction of chemical LTP 63
4.2.5 Protein analysis methods 63
4.2.5.1 Extraction of protein from cells 63
4.2.5.2 Extraction of protein from mouse tissue 63
4.2.5.3 Measurement of protein concentration 64
4.2.5.4 P-cofilin and LIMK analysis 64
4.2.5.5 SDS-polyacrylamide gel electrophoresis and western blot analysis 64
4.2.6 Protein-Protein interaction 65
4.2.6.1 Cofilin depletion assay 65
4.2.6.2 Expression of different domains of CAP2 65
4.2.6.3 Pull-down assay 66
4.2.6.4 Immunoprecipitation using GFP trap beads 66
4.2.7 Molecular biology techniques 67
4.2.7.1 Plasmid-DNA isolation from E. coli by alkaline lysis (miniprep) 67
4.2.7.2 Plasmid-DNA isolation with a kit from Macherey-Nagel 67
4.2.7.3 DNA agarose gel electrophoresis 67
4.2.7.4 Elution of DNA fragments from agarose gels 68
4.2.7.5 Measurement of DNA concentrations 68
4.2.7.6 Restriction digestion of DNA 68
4.2.7.7 Polymerase chain reaction (PGR) 68
4.2.7.8 Cloning of Rah GDI beta 69
4.2.7.9 Transformation of E.coli cells with plasmid DNA 69
4.2.8 Miscellaneous methods 69
4.2.8.1 Computer programs 69
4.2.8.2 Statistical analysis 69
References 70
Summary/ Zusammenfassnng 79/80
Erklärung 81
Curriculum vitae 82
Lebenslauf 83
Publication 84
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| discipline | Biologie |
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| indexdate | 2024-12-24T05:55:32Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-029668594 |
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| physical | 1 Mikrofiche (IX, 85 Seiten) Illustrationen 21 cm |
| publishDate | 2016 |
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| spellingShingle | Kumar, Atul 1986- Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 |
| subject_GND | (DE-588)4113937-9 |
| title | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 |
| title_auth | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 |
| title_exact_search | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 |
| title_full | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 vorgelegt von Atul Kumar |
| title_fullStr | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 vorgelegt von Atul Kumar |
| title_full_unstemmed | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 vorgelegt von Atul Kumar |
| title_short | Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2 |
| title_sort | neuronal actin dynamics spine density and neuronal dendritic complexity are regulated by cap2 |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/112003938X/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029668594&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kumaratul neuronalactindynamicsspinedensityandneuronaldendriticcomplexityareregulatedbycap2 |