2019 Fiscal Year Annual Research Report
| Project/Area Number |
19J20655
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
Guzman Christine 沖縄科学技術大学院大学, 科学技術研究科, 特別研究員(DC1)
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| Project Period (FY) |
2019-04-25 – 2022-03-31
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| Keywords | Neurexin / synapse / evolution / basal animals / protein interaction |
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| Outline of Annual Research Achievements |
During Yr 1, I have improved the phylogenetic tree analysis of Neurexin (Nrxn) protein. I have also performed phylum-wide survey and phylogenetic analyses of synaptic proteins and known postsynaptic ligands of Neurexin. Different predictions tools were used to identify conserved domains and functional sites. Domain identification and sequence alignment were complemented with structure homology search and 3D modelling. I have also checked the cell type-specific expression of Nrxn and other synaptic proteins using publicly available single cell RNA-seq data. For the identification of components of Nrxn protein complexes in non bilaterian animals, custom-made antibodies against each species’ Nrxns were generated. I performed substantial experiments to check the specificity of the antibodies on each target species.
Findings during Yr1 include:
1) Several Nrxn copies are found in non-bilaterian animals (Cnidaria and Placozoa). 2) Most known postsynaptic ligands of Nrxn is absent in non-bilaterian animals. Only few ligands are present, but important domains and binding sites are not well-conserved. 3) Most synaptic proteins, including Nrxns, are expressed in cell types other than neuronal or peptidergic cells suggesting their possible ancestral functions outside the nervous system. 4) In Nematostella (Cnidaria), NvNrxn1 is expressed ubiquitously in all cell types whereas shorter copies of Nrxns have expressions primarily on neuron cells. 5) siRNA-mediated knockdown of Nematostella Neurexin confirmed that the custom-made antibody can be used for co-immunoprecipitation experiments.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Most of the experimental works performed have focused on Nematostella which is readily available in the host research lab. Meanwhile, works on the ctenophore Bolinopsis is affected by the availability of the samples. Bolinopsis are only collected when they become abundant during late summer to mid-autumn.
Upon performing Western blotting for NvNrxn1, I found out that the antibody has other non-specific bindings. Hence, I plan not to use the antibody (anti-NvNrxn1) in performing Immunohistochemistry assay.
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| Strategy for Future Research Activity |
In the next year, I will continue to optimize the co-immunoprecipitation experiments. I will also try to employ crosslinking methods prior to co-immunoprecipitation. I plan to obtain Mass Spectrometry analysis results which is necessary to identify binding partners of ancestral Neurexins. I will continue analyzing the single cell expressions of synaptic proteins to obtain insights on how early synapse functions. I also plan to knockdown other Neurexin genes in Nematostella and check their phenotypic effects on the animal.
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Research Products
(2 results)
