Structural studies of trans-synaptic complexes
Neuronal synapses are the functional units of the nervous system, allowing the fast transmission of information between neurons. A plethora of proteins populate the cleft between connecting neurons, and many contribute to the formation of protein complexes than span the synaptic cleft. Such trans-sy...
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| Format: | Thesis |
| Language: | English |
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2019
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| _version_ | 1797101084611706880 |
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| author | Heroven, AC |
| author2 | Aricescu, A |
| author_facet | Aricescu, A Heroven, AC |
| author_sort | Heroven, AC |
| collection | OXFORD |
| description | Neuronal synapses are the functional units of the nervous system, allowing the fast transmission of information between neurons. A plethora of proteins populate the cleft between connecting neurons, and many contribute to the formation of protein complexes than span the synaptic cleft. Such trans-synaptic complexes are of tremendous importance for synaptic function, affecting the neuronal wiring specificity, synaptic plasticity or the differentiation of the pre-and post-synaptic compartments. Two important protein families that form trans-synaptic complexes are ionotropic glutamate receptors (iGluRs) and receptor protein tyrosine phosphatases (RPTPs). In this thesis, I present work conducted on two prototypical protein complexes: the Glutamate Delta 2 (GluD2) receptor, in complex with Cerebellin 1 and b Neurexin 1; and the receptor protein tyrosine phosphatase μ (RPTPμ), which forms a homophilic dimer across cell contacts. Despite previous efforts, it is not well understood how these protein complexes are able to integrate their cell-adhesive properties with their downstream signalling functions. The aim of this thesis is to pave the way towards a mechanistic understanding of the function of these complexes. Using X-ray crystallography and biophysical assays, GluD2-specific nanobodies were identified, leading to the discovery of molecular tools that can stabilize distinct GluD2 conformations. Of particular interest, a nanobody capable of activating the receptor and preventing desensitization was identified, which will be instrumental for unveiling the GluD2 metabotropic signalling mechanism. Single-particle cryo-EM was applied to study the full- length GluD2 receptor, providing the first insights into the general architecture of Delta subtype iGluRs and laying the grounds to obtain a high resolution structure. RPTPμ- mediated cell-cell contacts were analysed by cryo electron tomography, obtaining data from cells thinned by focused ion beam (FIB) milling. The FIB milled sections revealed unprecedented detail of the architecture of insect cells and allowed the analysis of RPTPμ in a native environment. Subtomogram averaging shed light on a potential higher-order organization of RPTPμ at cell-cell contacts. Finally, a protocol for the purification and crystallization of the complete cytoplasmic region of RPTPμ was developed. This data paves the way towards unravelling the structure of the juxtamembrane domain, an essential part for understanding how type IIb RPTPs are regulated. |
| first_indexed | 2024-03-07T05:46:55Z |
| format | Thesis |
| id | oxford-uuid:e784d05f-43ac-4523-ba6f-2d21c0d83d81 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:46:55Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:e784d05f-43ac-4523-ba6f-2d21c0d83d812022-03-27T10:39:27ZStructural studies of trans-synaptic complexesThesishttp://purl.org/coar/resource_type/c_db06uuid:e784d05f-43ac-4523-ba6f-2d21c0d83d81Structural BiologyNeurobiologyEnglishHyrax Deposit2019Heroven, ACAricescu, ASiebold, CGrunewald, KNeuronal synapses are the functional units of the nervous system, allowing the fast transmission of information between neurons. A plethora of proteins populate the cleft between connecting neurons, and many contribute to the formation of protein complexes than span the synaptic cleft. Such trans-synaptic complexes are of tremendous importance for synaptic function, affecting the neuronal wiring specificity, synaptic plasticity or the differentiation of the pre-and post-synaptic compartments. Two important protein families that form trans-synaptic complexes are ionotropic glutamate receptors (iGluRs) and receptor protein tyrosine phosphatases (RPTPs). In this thesis, I present work conducted on two prototypical protein complexes: the Glutamate Delta 2 (GluD2) receptor, in complex with Cerebellin 1 and b Neurexin 1; and the receptor protein tyrosine phosphatase μ (RPTPμ), which forms a homophilic dimer across cell contacts. Despite previous efforts, it is not well understood how these protein complexes are able to integrate their cell-adhesive properties with their downstream signalling functions. The aim of this thesis is to pave the way towards a mechanistic understanding of the function of these complexes. Using X-ray crystallography and biophysical assays, GluD2-specific nanobodies were identified, leading to the discovery of molecular tools that can stabilize distinct GluD2 conformations. Of particular interest, a nanobody capable of activating the receptor and preventing desensitization was identified, which will be instrumental for unveiling the GluD2 metabotropic signalling mechanism. Single-particle cryo-EM was applied to study the full- length GluD2 receptor, providing the first insights into the general architecture of Delta subtype iGluRs and laying the grounds to obtain a high resolution structure. RPTPμ- mediated cell-cell contacts were analysed by cryo electron tomography, obtaining data from cells thinned by focused ion beam (FIB) milling. The FIB milled sections revealed unprecedented detail of the architecture of insect cells and allowed the analysis of RPTPμ in a native environment. Subtomogram averaging shed light on a potential higher-order organization of RPTPμ at cell-cell contacts. Finally, a protocol for the purification and crystallization of the complete cytoplasmic region of RPTPμ was developed. This data paves the way towards unravelling the structure of the juxtamembrane domain, an essential part for understanding how type IIb RPTPs are regulated. |
| spellingShingle | Structural Biology Neurobiology Heroven, AC Structural studies of trans-synaptic complexes |
| title | Structural studies of trans-synaptic complexes |
| title_full | Structural studies of trans-synaptic complexes |
| title_fullStr | Structural studies of trans-synaptic complexes |
| title_full_unstemmed | Structural studies of trans-synaptic complexes |
| title_short | Structural studies of trans-synaptic complexes |
| title_sort | structural studies of trans synaptic complexes |
| topic | Structural Biology Neurobiology |
| work_keys_str_mv | AT herovenac structuralstudiesoftranssynapticcomplexes |