2018 Fiscal Year Annual Research Report
Mechanisms that promote the correct completion of meiosis by breaking symmetry of chromosomes
| Project/Area Number |
18H02373
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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) |
2018-04-01 – 2021-03-31
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| Keywords | 減数分裂 / 染色体 / 線虫 / 超解像度顕微鏡 |
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| Outline of Annual Research Achievements |
We have uncovered new aspects of the dynamics of the synaptonemal complex involved in specifying the domains where chromosome cohesion is lost in a stepwise fashion during meiosis I and II. By tagging non-phosphorylatable SYP-1 protein with an HA tag, we have shown that the partitioning of phosphorylated SYP-1 to the short arm chromosome domain does not involve a repulsion between phospho-SYP-1 and non-phospho-SYP-1, but likely involves net movement from the long to the short arms at different rates, with phospho-SYP-1 moving faster or earlier. Additionally, we have examined formation of short and long arm domains on triple fusion chromosomes, and found that short-arm domains can be created between two crossovers, showing that a chromosome end is not required for short arm domain formation.
Further, we have used FRAP (fluorescence recovery after photobleaching) to determine the relative speed of synaptonemal complex turnover on chromosomes in both the wild type and in mutants with non-phosphorylatable SYP-1. We have successfully acquired data using this experimentally-challenging system in live animals; however, the current trend of the data does not indicate a substantial dependence on SYP-1 phosphorylation for overall synaptonemal complex dynamics. Therefore, we hypothesize that if the enrichment of phosphorylated SYP-1 on the short arm is due to its movement from the long arm, then it is because it is moving earlier, not faster.
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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
Our work is progressing mostly along the lines we set out in our proposal. A slight change is that instead of tagging SYP-1 with mScarlet, we have used the HA tag, since larger protein tags such as GFP have been shown to interfere with SYP-1 function. With the smaller HA tag, we can track the different types of SYP-1 (wild-type and non-phosphorylatable) in fixed material without interfering at all with SYP-1 function.
We have successfully used FRAP to track the recovery times of SYP-3:GFP protein in both the wild type and animals with non-phosphorylatable SYP-1. So far, there has not been a significant difference found between these two conditions; however, the throughput of the technique has limited the number of nuclei we can successfully track. Using a different microscope or improved protein (such as a photoconvertible or photoactivatable protein) fused to SYP-3 should improve the throughput of this method.
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| Strategy for Future Research Activity |
Our research in the coming fiscal year will focus on two aspects of SYP-1 dynamics: (1) the dynamic phosphorylation-dependent movement of SYP-1, and (2) the molecular mechanisms of short arm accumulation by phosphorylated SYP-1.
For the first part, we will continue to use microscopy to determine the dynamic movement of the synaptonemal complex in conditions where SYP-1 either can or cannot be phosphorylated. Until now, we have used FRAP microscopy for this purpose; however, in this year we will create a photoconvertible version of SYP-3, a synaptonemal complex protein, which will allow single-particle tracking of the SC, leading to more sensitive determination of SC dynamics.
For the second part, our results to date implicate a feedback mechanism operating on either or both chromosome arms that inhibits phospho-SYP-1 on the long arm and promotes it on the short arm. Since we have also shown that phospho-SYP-1 recruits the polo-like kinase PLK-2, we have developed several hypotheses for possible feedback mechanisms by analogy to known positive feedback loops involving PLK-2. In particular, we will determine whether CDC-25 phosphatase and/or WEE-1 kinase interact with the pathway of SYP-1 phosphorylation enrichment, since these proteins are known to use PLK-2 in their feedback loop.
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Research Products
(6 results)
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[Journal Article] The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing2019
Author(s)
Billmyre KK, Doebley A-L, Spichal M, Heestand B, Belicard T, Sato-Carlton A, Flibotte S, Simon M, Gnazzo M, Skop A, Moerman D, Carlton PM, Sarkies P, Ahmed S
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Journal Title
PLOS Genetics
Volume: 15
Pages: e1008004
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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