| Project/Area Number |
22K19272
|
|---|
| Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
|
| Allocation Type | Multi-year Fund |
|---|
| Review Section |
Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
Carlton Peter 京都大学, 生命科学研究科, 准教授 (20571813)
|
|---|
| Project Period (FY) |
2022-06-30 – 2025-03-31
|
| Project Status |
Granted (Fiscal Year 2023)
|
| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2023: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2022: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
|
|---|
| Keywords | Meiosis / Chromosomes / C. elegans / Length sensing / 減数分裂 / 線虫 / 染色体ダイナミクス / シナプトネマ複合体 / in vivoイメージング / ライブイメージング |
|---|
| Outline of Research at the Start |
線虫の減数分裂における染色体分離は、交叉（相同染色体間の組み換えが起こる場所）から末端までの長さを測って、短い方を分離面として行われる。本研究は、この「染色体の長さを測るメカニズム」を解明し、「細胞ができること」の1つに「長さを比べる」という新しい概念を加えることを目指す。
|
| Outline of Annual Research Achievements |
We performed live imaging experiments to test our assumption that crossover sites act as borders to diffusion of SC proteins. Using a strain carrying the SC protein SYP-3 fused to the photoconvertible protein mMaple3, we aimed to track the diffusion of SYP-3 by photoconverting a small chromosome region and following the spread of the converted fraction of molecules. This was achieved with a laser scanning confocal microscope, by irradiating a tiny region of single chromosomes next to a crossover site. We found that crossover sites could be visualized with the fusion protein GFP-COSA-1, even though it emits in the same green channel as un-converted mMaple3, since it is brighter and makes a larger focus on the chromosome. While we had performed similar experiments in the previous fiscal year, in FY2023 the experimental technique had improved greatly and we were able to obtain useable data. Analysis of this data required manual tracing of chromosome axes, normalization and averaging of intensity levels on each side of the crossover, and sliding-window analyses on composite images in which all timepoints were averaged. We are making the code for this analysis available as part of a planned publication. The analysis of these live imaging traces showed that crossover sites do indeed act as at least partial barriers to SYP-3 diffusion. This data was the last required for a manuscript which is to be submitted in early FY2024.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
While the data acquisition on the confocal microscope was difficult (many worms do not survive the imaging process, and movement of nuclei during imaging makes many time courses unsuitable for analysis), we had expected this from the beginning since we knew that we were working at the limits of possibility for live imaging. Therefore, although it took time, we were able to collect information that no other group has been able to: quantitative analysis of the movement of proteins within the chromosome axis on both sides of a known crossover site. This was the last part of the data needed for our manuscript, which is currently being read critically by colleagues and will be submitted soon.
|
| Strategy for Future Research Activity |
We have found the underlying principle that specifies short and long arm identity in C. elegans meiotic chromosomes, as a response to a factor that is contributed additively by crossover designation sites. We still do not know the identity of the factor, but have several candidates in mind. We will test the candidates through knockdown and targeted mutation of phosphorylatable sites.
Further, we plan to enhance our existing live imaging capabilities using the recently-developed photoconvertible JF dyes that are brighter than mMaple3, and can withstand longer imaging treatment without photobleaching. By imaging SYP-3 protein tagged with JF dyes we plan to examine how often single SYP-3 molecules can move through the crossover region.
|
