Frasoński, Mikołaj: Binding partners of the neuroexophilin-neurexin complex identified in Nxph1-GFP tg/- and Nxph3-GFP tg/- mice. 2022
Inhalt
- Binding partners of the neurexophilin-neurexin complex identified in Nxph1-GFPtg/- and Nxph3-GFPtg/- mice
- Content
- 1. Introduction
- 1.1 Synapses
- 1.2 Cell adhesion molecules
- 1.2.1 Synaptic cell adhesion molecules: Neurexins
- 1.2.2 The function of neurexins
- 1.2.3 The structure of neurexins and their binding partners
- 1.3 Neurexophlins
- 1.3.1 Expression of neurexophilins
- 1.3.2 The structure and properties of neurexophilins
- 1.3.3 Function of neurexophilins
- 1.4 The aim of the study
- 2. Materials and Methods
- 2.1 Materials
- 2.1.1 Animals
- 2.1.2 Antibodies
- 2.1.3 Apparatus
- 2.1.4 Cell cultures
- 2.1.5 Chemicals
- 2.1.6 Media and Supplements
- 2.1.7 Solutions and media for cell culture
- 2.1.8 Molecular biology kits
- 2.1.9 Oligonucleotides
- 2.1.10 Plasmids
- 2.1.11 Software
- 2.2 Methods
- 2.2.1 Molecular biology methods
- 2.2.1.1 Polymerase chain reaction for cloning
- 2.2.1.2 In vitro site-directed mutagenesis using QuikChange Kit
- 2.2.1.3 Heat pulse transformation and culturing on plates
- 2.2.1.4 Express Mini (Holmes and Quigley, 1981)
- 2.2.1.5 Restriction enzyme digestion of DNA
- 2.2.1.6 Agarose gel electrophoresis
- 2.2.1.7 Purification of DNA by QIAEX® II Gel Extraction Kit
- 2.2.1.8 Dephosphorylation of 5’ DNA ends
- 2.2.1.9 Ligation
- 2.2.1.10 Electrotransformation of bacteria with plasmid DNA
- 2.2.1.11 Plasmid DNA mini-preparation (NucleoSpin® Plasmid)
- 2.2.1.12 Plasmid DNA maxi-preparation (NucleoBond® PC500)
- 2.2.1.13 Concentration analysis of plasmid DNA
- 2.2.1.14 DNA-sequencing and sequence analysis
- 2.2.1.15 Preparation of mouse genomic DNA for PCR
- 2.2.1.16 PCR for genotyping of Nxph1-GFPtg/- and Nxph3-GFPtg/-
- 2.2.2 Biochemical procedures
- 2.2.2.1 Cryonic storage and re-cultivation of HEK293 cells
- 2.2.2.2 Expression of proteins in HEK293 cells
- 2.2.2.3 Preparation of HEK293 homogenates
- 2.2.2.4 Membrane protein extraction from rodent brain
- 2.2.2.5 Pull-down experiments with the GFP-Trap
- 2.2.2.6 Pull-down experiments with Fc-tagged proteins
- 2.2.2.7 Polyacrylamide gel electrophoresis (SDS-PAGE)
- 2.2.2.8 Western blotting
- 2.2.2.9 Coomasie staining
- 3. Results
- 3.1 The differences between neurexophilins
- 3.2 All neurexophilin isoforms bind to the same epitope of Neurexin1α
- 3.3 Searching for novel binding partners of the neurexophilin/neurexin complex
- 3.4 GluN1 binds to neurexins in adult brains
- 3.5 NMDAR, GABABR, LRRTM2, Nlgn1, mGluR3 and mGluR5 interact with Nxph3-GFP/αNrxn complex during different stages of development
- 3.6 Recombinant GluN1-GFP binds to neurexins
- 4. Discussion
- 4.1 All neurexophilins bind to the same epitope of Nrxn1α
- 4.2 NMDAR is a novel binding partner of neurexins
- 4.3 NMDAR interacts with α-neurexin/neurexophilin complex in mature brains
- 4.4 Other age-dependent interactions
- 4.5 Limitations and recommendations
- 4.6 Conclusion and outlook
- 5. References
- 6. Summary
- 7.
- 8. Abbrevations
- 9. List of figures
- 10. List of tables
- 11.
- |Acknowledgment