2022 Fiscal Year Research-status Report
Elucidating the mechanisms of chromosome length sensing by the synaptonemal complex
| Project/Area Number |
22K19272
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| Research Institution | Kyoto University |
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Principal Investigator |
Carlton Peter 京都大学, 生命科学研究科, 准教授 (20571813)
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| Project Period (FY) |
2022-06-30 – 2024-03-31
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| Keywords | 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 |
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| Outline of Annual Research Achievements |
In this fiscal year we have improved our imaging techniques by making several adjustments to the in vivo conditions. We have succeeded in following the dynamic movement of mMaple3-labeled SYP-3 protein within and between meiotic chromosomes of C. elegans, while simultaneously visualizing the position of crossover designation sites by using eGFP-labeled COSA-1 protein. While both COSA-1 and non-activated mMaple3-SYP-3 are fluorescent in the green channel, we have found conditions where the COSA-1 foci that mark crossovers are unambiguously visible against the background of the linear signal characterizing SYP-3 staining. Thus, we were able to overcome the limitation of having only two fluorescent channels to measure three desired signals (non-activated mMaple3-SYP-3, activated mMaple3-SYP-3, and COSA-1). Further, we have developed an updated set of image analysis tools (https://github.com/carltonlab/carltonlab_imagej_tools) and simulation software (https://github.com/carltonlab/scsim) that we use to generate and test predictions of our diffusion and accumulation model.
The graduate student leading this project has presented our work-in-progress at the Meiosis Gordon Conference in FY2022, in accordance with the research plan, and received valuable feedback from the research community.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The live imaging experiments are still very challenging and do not always produce usable data, so last year the condition was slightly delayed; however, with our recent improvements to the imaging conditions, we are able to obtain good data almost every time, and the number of observations of SYP-3 diffusion has approached what we require to make statistically significant observations. However, some of the strains we intended to make for these imaging experiments proved difficult to obtain, likely due to genetic interactions. We therefore will need to spend extra time constructing strains combining mMaple3-SYP-3, GFP-COSA-1, and the syp-1(T452A) mutation, as well as the proposed AID degron alleles of cdc-25. These experiments are still ongoing and with our improved in vivo imaging techniques, should catch up to our intended point by the end of FY2023.
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| Strategy for Future Research Activity |
We have found so far that our model of chromosome partitioning is in fairly good agreement with our observations, but our model does not yet specify the identity of some of the molecules involves; specifically, the "signal" that starts at the crossover has not yet been determined. There are two parallel pathways described in the literature that give rise to partitioned chromosomes: one, which we described in 2020, involves recruitment of PLK-2 to SYP-1; the other involves the RING finger proteins ZHP-1 and -2. We will determine whether these pathways are acting in parallel, and the nature of cross-talk between them, by examining the localization of ZHP-2 in a non-phosphorylatable SYP-1 mutant in which PLK-2 does not localize to the synaptonemal complex.
Furthermore, we intend to test whether the phosphatase cdc-25 is involved in the feedback loop we propose, by constructing AID-degron strains that can be specifically depleted for cdc-25 activity. CDC-25 exists as four separate isoforms in C. elegans, with likely redundancy between various subunits; thus, checking for the possibilities exhaustively would require constructing 16 different strains. We will streamline this process by first constructing each single mutant, and only proceeding to double and higher mutant combinations in the case where single mutants do not show any effect on chromosome partitioning.
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| Causes of Carryover |
We will not be able to analyze all the mutant strains we had hoped to analyze during the current fiscal year, since combining them caused lethality. The scope of the current project therefore had to change to focus on a more limited question, whether SYP-3 ever diffused past the crossover point. For a more complete story we need to determine whether this happens also when SYP-1 is unable to be phosphorylated, and when cdc-25 phosphatase activity is absent, but the strains needed to address this question will need to be constructed and analyzed into the next FY. We will therefore use the amount to be used next fiscal year for construction of the strains (CRISPR/Cas9 reagents), and usage fees for shared microscopy for live imaging analysis.
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| Remarks |
The above website is a Github repository containing the simulation code for making predictions about where phospho-SYP-1 localizes, depending on the chromosome length and position of crossovers.
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