Noncanonical regulatory mechanisms in cell biology

细胞生物学中的非常规调节机制

• 批准号: 10398207
• 负责人: Lynn COOLEY
• 金额: $ 59.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10398207
• 关键词: Adult; Animals; Area; Behavioral Assay; Biological; Biological Assay; Biological Phenomena; Biology; Biotinylation; Cell division; Cell membrane; Cells; Cellular biology; Characteristics; Cis-Acting Sequence; Cytokinesis; Cytoskeleton; Development; Drosophila genus; Excision; Female; Genes; Genetic; Genetic Screening; Germ Cells; Giant Cells; Image; Larva; Learning; Lymphoproliferative Disorders; Mass Spectrum Analysis; Messenger RNA; Mitosis; Molecular; Nerve; Neuraxis; Neurons; ORF2 protein; Oogenesis; Open Reading Frames; Outcome; Ovarian; Ovary; Peptides; Phenotype; Process; Proliferating; Proteins; Proteome; Regulation; Research; Ribosomes; Structure; Terminator Codon; Testis; Tissues; Trans-Activators; Translations; daughter cell; egg; intercellular communication; interest; male; programs; ribosome profiling; sperm cell; stem; success

Project summary: This project expands on decades of success elucidating the genetic and molecular underpinnings of intercellular communication and cytoskeletal remodeling in gamete development in females and males. We propose research in two interesting areas: 1) the inhibition of cellular abscission during cytokinesis of dividing germline cells, and 2) the molecular regulation and functional significance of highly efficient tissue-specific stop codon readthrough during protein translation. Proliferating germline cells in animal ovaries and testes characteristically fail to complete abscission, leading to cell clusters that remain connected by intercellular bridges, called ring canals. Incomplete cytokinesis also occurs in several lymphoproliferative disorders, highlighting the importance of understanding how this noncanonical endpoint to mitosis is controlled. Using live imaging of germline mitosis in the Drosophila testis, we discovered a previously unknown intermediate step in ring canal formation involving a midbody-like structure that remodels into a channel between daughter cells. To learn how this maturation step occurs and how the molecular machinery that drives abscission is inhibited from localizing to midbodies, we will use localized biotinylation to identify midbody components and probe the genetic requirement for proteins known to function during cytokinesis. We will also use localized biotinlylation to identify ring canal proteins at the plasma membrane-cytoskeleton interface. Our interest in stop codon readthrough stems from our extensive analysis of the kelch gene and its function during oogenesis. The kelch mRNA encodes a large open reading frame (ORF) punctuated by a single stop codon. In ovaries, translation terminates at the stop to produce a ring canal protein and there is no apparent function for the second ORF. In contrast, we have observed remarkably high efficiency stop codon readthrough in nerves of the central nervous system (CNS) of larvae and adults, producing abundant ORF1+ORF2 protein. Furthermore, kelch and several other genes in Drosophila display efficient stop codon readthrough specifically in the CNS, suggesting the presence of many proteins with carboxy-terminal extensions of unknown function. We will systematically analyze the scope and scale of stop codon readthrough using ribosome profiling of neuronal tissue and mass spectrometry of total protein lysates to identify readthrough peptides. To define the mechanism of readthrough, we will use genetic screens to investigate both stimulatory cis-acting sequences flanking stop codons in readthrough genes and trans-acting factors. Finally, to understand the function of neuronal stop codon readthrough, we will use gene editing to ablate ORF2 from kelch and several other genes and carefully analyze phenotypes using cell biological and behavioral assays. This research program will lead to discoveries concerning the fundamental cell biological and genetic mechanisms that regulate important noncanonical biological phenomena – an alternate ending to cytokinesis that produces syncytia of animal germline cells and ribosomal readthrough of stop codons that expands the neuronal proteome.

项目概述：该项目扩展了数十年来的成功，阐明了遗传和分子 雌性配子发育中细胞间通讯和细胞骨架重塑的基础 和男性。我们建议在两个有趣的领域进行研究：1）抑制细胞凋亡， 分裂生殖系细胞的胞质分裂，和2）高度的分子调控和功能意义 在蛋白质翻译期间有效的组织特异性终止密码子通读。动物生殖系细胞的扩增 卵巢和睾丸的特征是不能完全脱落，导致细胞簇保持连接 通过细胞间的桥梁，称为环管。不完全胞质分裂也发生在几个淋巴增生性 疾病，强调了解如何控制这种非经典的有丝分裂终点的重要性。 利用果蝇睾丸生殖细胞有丝分裂的实时成像，我们发现了一个以前未知的 涉及重塑成通道的中间体样结构的环管形成的中间步骤 子细胞之间。为了了解这一成熟步骤是如何发生的，以及驱动这一过程的分子机制是如何发生的， 由于中间体的生物素化作用受到抑制，因此我们将利用生物素化的方法来鉴定中间体 这些蛋白质是细胞质分裂过程中已知的功能性蛋白质的遗传需求。我们还将 使用局部生物素化来鉴定质膜-细胞骨架界面处的环管蛋白。我们 对终止密码子通读的兴趣源于我们对kelch基因及其功能的广泛分析， 卵子发生。kelch mRNA编码一个大的开放阅读框（ORF），由一个单一的终止密码子打断。在 在卵巢中，翻译终止于终止处以产生环管蛋白，并且对于 第二个ORF。相比之下，我们已经在神经中观察到显著高效率的终止密码子通读， ORF 1 + ORF 2蛋白在幼虫和成虫的中枢神经系统（CNS）中表达丰富。 此外，kelch和其他几个基因在果蝇中显示出高效的终止密码子通读特异性 在中枢神经系统中，这表明存在许多未知功能的羧基末端延伸的蛋白质。 我们将使用核糖体分析系统地分析终止密码子通读的范围和规模。 神经元组织和总蛋白裂解物的质谱分析以鉴定通读肽。来定义 通读机制，我们将使用遗传筛选来研究这两个刺激顺式作用序列 侧接通读基因和反式作用因子中的终止密码子。最后，为了了解 神经元终止密码子通读，我们将使用基因编辑来消除kelch和其他几个基因的ORF 2 并使用细胞生物学和行为测定仔细分析表型。这项研究计划将导致 发现有关的基本细胞生物学和遗传机制，调节重要的 非典型生物现象-细胞质分裂的交替结束，产生动物的合胞体 生殖细胞和核糖体通读终止密码子，扩大神经元蛋白质组。