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Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos.
PLOS Genetics ( IF 4.0 ) Pub Date : 2020-04-06 , DOI: 10.1371/journal.pgen.1008735
Boyang Liu 1, 2 , Ingo Gregor 3 , H-Arno Müller 4 , Jörg Großhans 1, 2
Affiliation  

The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.

中文翻译:


荧光波动分析揭示了活体胚胎中 PpV 依赖性 Cdc25 蛋白动态。



蛋白磷酸酶 Cdc25 通过激活 Cdk-细胞周期蛋白复合物而成为细胞周期的关键调节因子。 Cdc25 受其表达水平和翻译后机制的调节。在早期果蝇胚胎发生中,Cdc25/Twine 驱动快速且同步的核周期。细胞周期的暂停和重塑为具有间隙期的更通用的细胞周期模式是由早期间期 14 中的 Twine 失活和破坏决定的,响应合子基因组激活。尽管假激酶 Tribbles 有助于及时降解 Twine,但 Twine 水平受到额外但未知的翻译后机制的控制。在这里,我们应用基于荧光波动分析(FFA)的非侵入性方法以分钟级分辨率记录单个活胚胎中 Twine 的绝对浓度分布。利用该测定,我们发现蛋白磷酸酶 V (PpV)(人 PP6 催化亚基的同源物)可确保在间期 14 开始时适当降低 Twine 蛋白水平。PpV 直接或间接控制 Twine 在多个丝氨酸和通过磷酸位点作图揭示苏氨酸残基。突变分析证实,这些位点参与了 Twine 蛋白动力学的控制,并且部分磷酸位点突变胚胎的细胞周期重塑被延迟。我们的数据揭示了一种控制 Twine 蛋白水平的新机制及其对胚胎细胞周期重塑的意义。
更新日期:2020-04-06
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