Determining the Role of the Conserved TRPM Ion Channel in Egg Activation, Using the Drosophila Model

使用果蝇模型确定保守 TRPM 离子通道在卵激活中的作用

• 批准号: 10271276
• 负责人: Mariana Federica Wolfner
• 金额: $ 7.74万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-26 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10271276
• 关键词: Animals; Antibodies; Biochemical; Biological Models; Biological Process; CRISPR/Cas technology; Calcineurin; Calcium; Cell Cycle; Cell Cycle Progression; Cell membrane; Cells; Characteristics; Complement; Coupled; Data; Development; Diagnosis; Drosophila genus; Egg Proteins; Embryo; Embryonic Development; Environment; Event; Family; Fertility; Fluorescence; Future; Genetic; Human; Infertility; Insecta; Investigation; Ion Channel; Mammals; Mediating; Medical; Meiosis; Membrane; Methods; Modeling; Molecular; Mus; Oocytes; Oogenesis; Organism; Orthologous Gene; Peptide Initiation Factors; Phosphoric Monoester Hydrolases; Process; Protein Biosynthesis; Proteins; Proteome; Proteomics; RNA chemical synthesis; Rana; Reagent; Role; Series; Source; System; Time; Tissues; Totipotency; Translations; Vertebrates; Xenopus; base; calcineurin phosphatase; egg; experimental study; female fertility; fly; follow-up; mechanical force; sperm cell; tool; uptake; zygote

SUMMARY Fertility requires that a mature oocyte become competent to transform into an embryo. This “egg activation” initiates with increases of Ca2+ in the oocyte. In mammals, the fertilizing sperm triggers a local rise in cytoplasmic Ca2+ that induces a wave of Ca2+ release from internal stores that moves across the egg. Oscillations in Ca2+ then occur via Ca2+ uptake from the external environment through TRPM7 channels. Drosophila egg activation also begins with a local rise in Ca2+ that induces a wave via Ca2+ release from internal stores. The initial Ca2+ rise is due to uptake of Ca2+ from the external environment. Recently, we found that TRPM, the Drosophila ortholog of TRPM7, mediates this Ca2+ uptake. Thus Ca2+ influx through a TRPM- channel is fundamental to fertility from flies to mammals, and dissecting its mechanism and effects is of relevance to human infertility diagnosis and its alleviation. However, how TRPM channels operate in oocytes, and how the Ca2+ rise relates to the macromolecular changes that make the oocyte competent to develop, is unknown in any organism. Drosophila’s genetic advantages, and large eggs, make it the ideal model system with which to determine such mechanisms and identify molecules critical for this transition in all animals. We will do so via two aims: First, we will investigate why TRPM channels cause a local rather than global calcium rise: are the channels localized at the oocyte poles or are they uniformly distributed around the oocyte plasma membrane and only activated at the poles? In addition to revealing how TRPM channels activate during egg activation, our results are relevant to how these channels act locally in several additional medically-relevant biological processes. Depending on which hypothesis we find to be correct, our future studies will determine how TRPM is localized to the poles during oogenesis or, alternatively how a non-localized channel is activated to act only locally. Second, we will follow up our surprising discovery that, similar to the situation in mouse, despite egg activation abnormalities TRPM-deficient Drosophila oocytes can initiate early embryogenesis, although development later becomes abnormal. We will investigate the relationship of TRPM-mediated Ca2+ influx to the phospho- modulation of essential maternal proteins that occurs during egg activation. We and others have shown that the Ca2+-activated phosphatase calcineurin is essential for egg activation in both flies and frogs. Exploiting genetic and biochemical advantages unique to Drosophila, we then showed that calcineurin activity regulates changes in the phospho-state of cell cycle regulators and translation initiation factors during egg activation. We propose here to determine whether these phosphoproteome changes depend on TRPM- mediated Ca2+ influx. If they do, our future studies will aim to identify how development can initiate in the absence of this phosphomodulation. If the calcineurin-based changes to the phosphoproteome do not depend on TRPM- mediated Ca2+ influx, we will investigate the source of the Ca2+ that activates calcineurin at this time.

总结 生育力需要成熟的卵母细胞能够转化为胚胎。这种“卵子激活” 随着卵母细胞中Ca 2+的增加而启动。在哺乳动物中，精子引发了局部的 细胞质中的Ca 2+，诱导钙从内部存储的释放波，移动到整个鸡蛋。 然后通过TRPM 7通道从外部环境中摄取Ca 2+，从而发生Ca 2+的振荡。 果蝇卵激活也开始于Ca 2+的局部升高，通过Ca 2+释放诱导波， 内部存储。最初的Ca 2+上升是由于从外部环境中摄取Ca 2+。最近，我们发现 TRPM，果蝇TRPM 7的直系同源物，介导这种Ca 2+摄取。因此，Ca 2+流入通过TRPM- 通道是从蝇类到哺乳动物生殖的基础，剖析其机制和作用， 与人类不孕症诊断及其缓解的相关性。然而，TRPM通道如何在卵母细胞中运作， 以及Ca 2+升高与使卵母细胞能够发育的大分子变化之间的关系， 在任何生物体中都是未知的。果蝇的遗传优势，以及巨大的卵，使其成为理想的模式系统 用它来确定这种机制，并确定在所有动物中这种转变的关键分子。我们 将通过两个目标来实现： 首先，我们将研究为什么TRPM通道引起局部而不是整体钙升高： 通道位于卵母细胞的两极，还是均匀分布在卵母细胞质膜周围 只在两极被激活除了揭示TRPM通道如何在卵子激活过程中激活外， 我们的研究结果与这些通道如何在几个额外的医学相关的生物学中局部作用有关。 流程.根据我们发现哪个假设是正确的，我们未来的研究将确定TRPM如何 在卵子发生过程中定位于两极，或者替代地，非定位通道如何被激活以仅 在附近 其次，我们将继续我们的惊人发现，类似于小鼠的情况，尽管卵 激活异常TRPM缺陷的果蝇卵母细胞可以启动早期胚胎发生，尽管 后来的发展变得不正常。我们将研究TRPM介导的Ca 2+内流与细胞凋亡的关系。 在卵激活过程中发生的必需母体蛋白的磷酸化调节。我们和其他人已经 表明钙激活磷酸酶钙调神经磷酸酶是苍蝇和青蛙卵激活所必需的。 利用果蝇独有的遗传和生化优势，我们发现钙调神经磷酸酶活性 调节卵细胞发育过程中细胞周期调节因子和翻译起始因子磷酸化状态的变化 activation.我们建议在这里确定这些磷酸化蛋白质组的变化是否取决于TRPM- 介导的Ca 2+内流。如果他们这样做，我们未来的研究将旨在确定如何在 缺乏这种磷酸调节。如果磷酸化蛋白质组的钙调神经磷酸酶变化不依赖于 在TRPM介导的Ca 2+内流中，我们将研究此时激活钙调磷酸酶的Ca 2+的来源。