| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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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