The Role of Cell-Cell Interactions During Drosophila Development

果蝇发育过程中细胞间相互作用的作用

• 批准号: RGPIN-2014-05229
• 负责人: Boulianne, Gabrielle
• 金额: $ 5.17万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=645988
• 关键词: Role; Cell; Interactions; During; Drosophila

Normal development requires the commitment of individual cells to the appropriate cell fate and their subsequent differentiation. The long-term goal of our research program is to understand how individual cell fates are acquired during development. Towards this goal, we have been studying the role of the Notch signaling pathway, which functions to determine the fate of most cell types in all complex animals. Specifically, we have focused on understanding the function of one core member of this pathway, neuralized (neur), which encodes a protein with a C-terminal RING domain and two Neur homology repeats (NHR1 & NHR2). We initially showed that Neur is required for a subset of Notch signaling events during development. We also showed that both NHR and RING finger domains are absolutely required for Neur function in vivo. Specifically, we showed that the RING finger domain in Neur confers E3-ubiquitin ligase activity while the NHR1 domain is required for its interaction with the Notch ligands, Delta & Serrate. We also found that the NHR2 domain mediates formation of intra- and inter-molecular dimers, which regulate Neur activity and hence, Notch activation. Finally, through in vitro binding assays, we identified an interaction between Neur and phosphoinositides (PIPs), membrane lipids that mediate membrane trafficking and signaling. Specifically, we found that the PIP-binding motif in Neur is required for Delta internalization. Altogether, our studies have elucidated the molecular function of Neur and its specific role within the Notch signaling pathway. However, despite intensive analysis, we still know very little as to how Neur itself is regulated. Recently, we have found that Neur is phosphorylated at several key residues flanking the PIP-binding and NHR domains. We also found that Neur can associate with other NHR domain containing proteins suggesting that Neur function may be regulated by protein phosphorylation and oligomerization. Interestingly, we have also uncovered a novel role for Neur in epithelial cell polarity and germ line stem cell maintenance. Importantly, neither Notch nor its ligands Delta and Serrate, are required for these processes, suggesting a novel, Notch-independent function for Neur during development. To uncover the specific roles of Neur in these two developmental processes, we used a combination of biochemical and genetic approaches. To date, we have shown that Neur can bind to another NHR domain containing protein, called CG3894. We also found that loss of function mutations in CG3894 give rise to similar defects in cell polarity as those observed in neur mutants. This raises the exciting possibility that Neur and CG3894 act together to regulate epithelial cell polarity. Finally, we have begun to uncover the potential role of Neur in the male germ line. Specifically, we found that Neur is a direct target of the JAK/STAT pathway, which has previously been shown to play a key role in the maintenance of germ line stem cells. We also found that Neur can bind and ubiquitinate STAT92E, providing additional evidence that the function of Neur in the germ line is mediated by JAK/STAT signaling. In this proposal, we will: 1) Determine how phosphorylation affects Neur function; 2) Determine the role of Neur in epithelial polarity; and, 3) Determine the role of Neur in germline stem cells. Altogether, these experiments will uncover the mechanisms that regulate Neur function in Notch signaling and also reveal novel functions for Neur in cell polarity and germ line stem cell maintenance, two processes that play critical roles in the development of multicellular organisms.

正常的发育需要单个细胞承担适当的细胞命运和随后的分化。我们研究计划的长期目标是了解在发育过程中个体细胞命运是如何获得的。为了实现这一目标，我们一直在研究Notch信号通路的作用，它决定了所有复杂动物中大多数细胞类型的命运。具体来说，我们专注于了解该途径的一个核心成员neuralized （neur）的功能，该成员编码具有c端环结构域和两个neur同源重复序列（NHR1和NHR2）的蛋白质。我们最初表明，在发育过程中，Neur是Notch信号事件子集所必需的。我们还发现NHR和无名指结构域在体内都是Neur功能所必需的。具体来说，我们发现Neur的RING finger结构域赋予e3 -泛素连接酶活性，而NHR1结构域则是与Notch配体Delta和Serrate相互作用所必需的。我们还发现NHR2结构域介导分子内和分子间二聚体的形成，从而调节Neur活性和Notch激活。最后，通过体外结合试验，我们确定了Neur与磷脂肌苷（PIPs）之间的相互作用，PIPs是介导膜运输和信号传导的膜脂。具体来说，我们发现Neur中pip结合基序是Delta内在化所必需的。总之，我们的研究已经阐明了Neur的分子功能及其在Notch信号通路中的特定作用。然而，尽管进行了深入的分析，我们对Neur本身是如何调控的仍然知之甚少。最近，我们发现Neur在pip结合和NHR结构域两侧的几个关键残基上被磷酸化。我们还发现Neur可以与其他含有NHR结构域的蛋白结合，这表明Neur的功能可能受到蛋白磷酸化和寡聚化的调节。有趣的是，我们还发现了Neur在上皮细胞极性和生殖系干细胞维持中的新作用。重要的是，Notch及其配体Delta和Serrate都不是这些过程所必需的，这表明Neur在发育过程中具有新颖的、与Notch无关的功能。为了揭示Neur在这两种发育过程中的具体作用，我们采用了生化和遗传相结合的方法。到目前为止，我们已经证明Neur可以结合另一种含有NHR结构域的蛋白，称为CG3894。我们还发现，CG3894中功能突变的缺失会导致与其他突变体相似的细胞极性缺陷。这就提出了Neur和CG3894共同作用调节上皮细胞极性的可能性。最后，我们已经开始揭示Neur在雄性生殖系中的潜在作用。具体来说，我们发现Neur是JAK/STAT通路的直接靶点，而JAK/STAT通路先前已被证明在生殖系干细胞的维持中发挥关键作用。我们还发现Neur可以结合并泛素化STAT92E，这进一步证明了Neur在种系中的功能是由JAK/STAT信号介导的。在本提案中，我们将：1)确定磷酸化如何影响Neur功能；2)确定Neur在上皮极性中的作用；3)确定Neur在种系干细胞中的作用。总之，这些实验将揭示在Notch信号中调控Neur功能的机制，并揭示Neur在细胞极性和生殖系干细胞维持中的新功能，这两个过程在多细胞生物的发育中起着关键作用。