2020 Fiscal Year Research-status Report
Unraveling the role of membrane contact sites in Entamoeba histolytica
| Project/Area Number |
20K16233
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| Research Institution | The University of Tokyo |
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Principal Investigator |
サントス ハルベルト・ヒメネス 東京大学, 大学院医学系研究科(医学部), 助教 (90793779)
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| Project Period (FY) |
2020-04-01 – 2022-03-31
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| Keywords | membrane contact site / Entamoeba histolytica / mitosome / endosome |
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| Outline of Annual Research Achievements |
Imaging analyses (indirect fluorescence assay, immunoelectron microscopy, live imaging) and fractionation of proteins involved in mitosome endosome contact sites (MECSs) have been completed. Fractionation experiments have been completed and corroborate the imaging data. Immunoprecipitation to detect other interacting partners of MECS was also finished. Blue-Native PAGE was completed and protein complexes were determined and analyzed. Plasmids for BioID-based proteomics are also ready. Functional characterization including liposome-based assays are being optimized. Preliminary results were presented in the Japan Society of Parasitology held last April 16-17, 2021. A mini review article was submitted and accepted by Parasitology International on April 27, 2021.
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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
Optimization of liposome-based assays is taking time. Silencing of ETMP1 is not possible as it is essential to the parasite. Establishment of dominant-negative mutant for EHD1 is also challenging as the parasite growth is severely hampered.
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| Strategy for Future Research Activity |
1. Characterize some candidate interacting proteins found in MECSs.
2. Complete functional characterization by analyzing effect of overexpression or silencing MCS-related components on endocytic pathway-related processes (endocytosis, phagocytosis, exocytosis etc).
3. Assess the effect of protein overexpression or silencing on mitosome function (sulfate activation).
4. Test whether contact sites involving EHD1 enhance lipid transport by lipid transfer proteins by liposome-based assay.
5. Perform RNAseq analysis of transcriptionally-silenced strain(s).
6. Complete and submit manuscript for publication.
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| Causes of Carryover |
Due to the coronavirus pandemic, the planned use of the budget for conference attendance was not used.
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