Novel molecular mechanisms of cell migration in C. elegans

线虫细胞迁移的新分子机制

• 批准号: RGPIN-2016-06644
• 负责人: Culotti, Joseph
• 金额: $ 2.4万
• 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=616434
• 关键词: Novel; molecular; mechanisms; cell; migration

Genetic analysis in C. elegans provides a powerful approach for illuminating the molecular mechanisms underlying developmental cell migration. In this proposal we focus on two novel genes required for the directed migration of distal tip cells (DTCs) during the formation of the C. elegans gonad. One gene, unc-129, encodes a TGF-beta that regulates the activity of UNC-6/Netrin signaling required for axon guidance and DTC migration. UNC-129 forms a high dorsal - low ventral gradient in the developing nematode that promotes ventral to dorsal DTC migration. Preliminary studies suggest that UNC-129 enhances UNC-6/netrin repulsive signaling at low concentrations of UNC-6 (graded opposite to UNC-129) by diverting signaling from less sensitive UNC-5 alone to more sensitive UNC-5+UNC-40 signaling. Our studies are directed at understanding the signaling mechanisms activated by UNC-129, which is novel since it does not involve canonical TGF-beta receptors. Our approach relies on known properties of UNC-130, a transcription factor that when disabled de-represses ventral unc-129 gene expression - disrupting its dorsoventral gradient and causing failures of ventral to dorsal DTC migration. unc-129 loss of function does not induce DTC defects, but does suppress unc-130 mutant DTC defects. Thus, by isolating mutations that suppress the unc-130 DTC migration defect (like unc-129 mutations do), we identified mutations in 3 genes required for UNC-129 signaling. We propose to identify these 3 genes using one step whole genome sequencing/SNP mapping then characterize them for expression and function. Because these genes affect UNC-129 signaling, their further genetic and molecular analysis will be carried out to identify additional elements of the UNC-129 signaling cascade. The second gene is ngat-1, encoding an N-acetylgalactosaminyltransferase enzyme (NGAT-1) that, based on sequence, is predicted to add N-acetylgalactosamine to glycan substrates, creating a LacdiNAc modification on glycoproteins or glycolipids. The ngat-1 temperature-sensitive mutant was identified by its defect in ventral to dorsal migration of the DTCs, suggesting that addition of LacdiNAc is a signal that promotes DTC migration in the developing gonad. Temperature shift experiments show ngat-1 completes its function in embryogenesis for a migration that occurs during late post-embryonic development - a unique property of known ECM molecules. These findings provide evidence for an as yet unknown embryonic mechanism that manifests at a much later stage of development. We propose to use mass spectrometry and immunoblotting to characterize the nature of the NGAT-1-induced glycan modifications and to identify the target protein(s) or glycolipid(s) for NGAT-1 activity. Our long term goal is to fully understand the mechanisms that direct specific cell migrations during animal development and possibly tumor metastasis.

遗传分析表明，C. elegans提供了一个强有力的方法来阐明发育细胞迁移的分子机制。在这个建议中，我们集中在两个新的基因所需的定向迁移的远端尖端细胞（DTC）的形成过程中的C。秀丽隐翅虫性腺 一个基因，unc-129，编码TGF-β，调节轴突导向和DTC迁移所需的β-6/Netrin信号传导的活性。在发育中的线虫中，DTC-129形成高背侧-低腹侧梯度，促进腹侧到背侧的DTC迁移。初步研究表明，在低浓度的α-干扰素-6（与α-干扰素-129相反分级）下，α-干扰素-129通过将信号从较不敏感的α-干扰素-5单独转移到更敏感的α-干扰素-5 + α-干扰素-40信号来增强α-干扰素-6/netrin排斥信号。我们的研究旨在了解UNC-129激活的信号传导机制，这是新颖的，因为它不涉及典型的TGF-β受体。我们的方法依赖于已知的特性，转录因子，当禁用去抑制腹侧unc-129基因表达-破坏其背腹梯度，并导致腹侧背侧DTC迁移失败。unc-129的功能丧失不诱导DTC缺陷，但抑制unc-130突变型DTC缺陷。因此，通过分离抑制unc-130 DTC迁移缺陷的突变（如unc-129突变所做的），我们确定了3个基因中的突变，这些基因是nc-129信号传导所需的。我们建议使用一步全基因组测序/SNP作图来鉴定这3个基因，然后表征它们的表达和功能。由于这些基因影响UNC-129信号传导，因此将对其进行进一步的遗传和分子分析，以确定UNC-129信号传导级联的其他元件。 第二个基因是ngat-1，编码一种N-乙酰半乳糖胺转移酶（NGAT-1），根据序列，预计该酶可将N-乙酰半乳糖胺添加到聚糖底物上，从而在糖蛋白或糖脂上产生LacdiNAc修饰。ngat-1温度敏感性突变体被确定其缺陷腹背迁移的DTC，这表明，除了LacdiNAc是一个信号，促进DTC迁移在发育中的性腺。温度变化实验表明ngat-1完成了其在胚胎发生中的功能，用于在胚胎后发育后期发生的迁移-这是已知ECM分子的独特性质。这些发现为一种在发育后期表现出来的未知胚胎机制提供了证据。我们建议使用质谱和免疫印迹来表征NGAT-1诱导的聚糖修饰的性质，并鉴定NGAT-1活性的靶蛋白或糖脂。我们的长期目标是充分了解在动物发育过程中指导特定细胞迁移和可能的肿瘤转移的机制。