Investigating phosphoregulation of meiotic recombination using superresolution microscopy

2016 Fiscal Year Annual Research Report

• Project/Area Number: 15H04328
• 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): 2015-04-01 – 2018-03-31
• Keywords: 減数分裂 / 線虫 / リン酸化による制御

Outline of Annual Research Achievements

Our phosphoproteomics screen discovered that the synaptonemal complex protein SYP-1 was phosphorylated at 12 amino acid residues, including a threonine at position 452 which creates a binding site for Polo-like kinase (C. elegans PLK-2) when phosphorylated. By creating a transgenic animal in which this residue is mutated to Alanine and thus cannot be phosphorylated, we determined that PLK-2 is very likely to bind this site on SYP-1, and further that this binding is required for several critical aspects of progression through meiotic prophase. We showed that PLK-2 relocalization to SYP-1 is required to end the zygonema stage of meiotic prophase in a timely manner.
Furthermore, we showed that after crossing-over has occurred, phosphorylated SYP-1 moves to one restricted domain of the chromosome, bringing PLK-2 with it in a mutually-dependent manner. This restriction is required to set up the functional difference between two specialized chromosome domains: the "short arm", which contains phospho-SYP-1 and PLK-2, and the "long arm", which contains other proteins (LAB-1 and protein phosphatase 1). We showed that the distinction between these two domains is required for correct chromosome segregation in the first meiotic division, and showed that the mechanism involves specifying the localization of protein phosphatase 1. This work is unique in linking modification of an early meiotic structure (the synaptonemal complex) to correct segregation in later meiosis.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

While we have not definitively established that SYP-1 is an actual PPH-4.1 target, our overall goal was to identify proteins whose phosphoregulation is important for meiosis. In this regard, our work on SYP-1 (Sato-Carlton et al., JCB, 2017) was a successful outcome of our research plan. Our unpublished work does show that SYP-1 persists in a hyperphosphorylated state in pph-4.1 mutants, so we still consider it highly likely to be a target of PPH-4.1, in agreement with our phosphoproteome data. However, to show a direct connection between SYP-1 and PPH-4.1 (which would have merited a rank of (1) above) was not possible within the allotted time period.

Strategy for Future Research Activity

In the current period we will continue to characterize how SYP-1 phosphorylation orchestrates multiple events of meiotic prophase and how these relate to the activity of PPH-4.1. We will determine whether SYP-1 and PPH-4.1 act in the same or different pathways by genetic analysis of hemizygous pph-4.1 mutants, which are normally viable, to test whether this exacerbates the phenotype of SYP-1 phospho-dead mutant T452A. We also plan to elucidate the mechanisms of restriction of phospho-SYP-1 and PLK-2 to the "short arm" of the chromosome, and how this restriction sets up the functional distinction between the short and long arms.
Since our findings show that a large number (~60%) of embryos can survive despite failure to localize phospho-SYP-1 to the short arm, we suspect that redundant mechanisms operate to ensure correct segregation. We will investigate the operation of these mechanisms by combining heterozygous mutations in genes known to affect segregation with homozygous syp-1 mutations, to see if there is a synergistic effect. If <<60% viability results from such a genetic combination, it is likely to represent a gene involved in a complementary mechanism.