Elucidating the mechanisms of chromosome length sensing by the synaptonemal complex

2023 Fiscal Year Research-status Report

• Project/Area Number: 22K19272
• 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
• Keywords: Meiosis / Chromosomes / C. elegans / Length sensing

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

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.

Causes of Carryover

Our initial plans for the grant included two items that were not used, since mMaple3 protein single-molecule imaging was not possible in live animals: the GPU-enabled processor for image analysis and a camera attachment for our microscope that should have enabled single-molecule analysis of mMaple3:GFP. We are going to employ the bright photoconvertible Halo-tag dyes developed at the Janelia institute for this purpose in the next fiscal year, which will require funds to purchase the dyes as well as for imaging.