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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611513
• 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致力于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个体细胞命运是如何在发育过程中获得的。为了这个目标，我们一直在研究Notch信号通路的作用，它的功能决定了所有复杂动物中大多数细胞类型的命运。具体地说，我们重点了解了这一途径的核心成员神经化(Neur)的功能，它编码一个带有C-末端环区和两个Nur同源重复序列的蛋白质(NHR1和NHR2)。我们最初表明，在发育过程中，Notch信号事件的子集需要NURR。我们还表明，NHR和环指结构域都是体内神经功能所必需的。具体地说，我们发现NURR中的环指结构域赋予E3-泛素连接酶活性，而NHR1结构域是其与Notch配体Delta和Serrate相互作用所必需的。我们还发现，NHR2结构域调节分子内和分子间二聚体的形成，从而调节神经活性，从而激活Notch。最后，通过体外结合实验，我们确定了Neur和磷脂酰肌醇(PIPs)之间的相互作用，PIPs是介导膜转运和信号传递的膜脂。具体地说，我们发现Neur中的PIP结合基序是Delta内化所必需的。总之，我们的研究阐明了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，我们仍然对NURE本身是如何监管的知之甚少。最近，我们发现Nur在PIP结合区和NHR结构域两侧的几个关键残基上被磷酸化。我们还发现，Nur可以与其他含有NHR结构域的蛋白质结合，这表明Nur的功能可能受到蛋白质磷酸化和寡聚化的调节。有趣的是，我们还发现了NURR在上皮细胞极性和生殖系干细胞维持中的一个新角色。重要的是，Notch及其配体Delta和Serrate都不是这些过程所必需的，这表明在Nur发育过程中有一种新的Notch不依赖于Notch的功能。为了揭示NURE在这两个发育过程中的具体作用，我们结合使用了生化和遗传学方法。到目前为止，我们已经证明Nur可以与另一个包含蛋白质的NHR结构域结合，称为CG3894。我们还发现，CG3894功能突变的丢失会导致与neur突变体中观察到的类似的细胞极性缺陷。这提出了一种令人兴奋的可能性，即Neur和CG3894共同作用于调节上皮细胞的极性。最后，我们已经开始发现NURE在男性生殖系中的潜在作用。具体地说，我们发现Neur是JAK/STAT通路的直接靶点，此前已证明JAK/STAT通路在生殖系干细胞的维持中发挥关键作用。我们还发现NURR可以结合和泛素化STAT92E，这为NURR在生殖系中的功能是由JAK/STAT信号介导的提供了进一步的证据。在这个方案中，我们将：1)确定磷酸化如何影响神经功能；2)确定神经在上皮极性中的作用；以及3)确定神经在生殖系干细胞中的作用。总之，这些实验将揭示在Notch信号中调节Neur功能的机制，并揭示Nur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体的发育中发挥关键作用。