Noncanonical regulatory mechanisms in cell biology

细胞生物学中的非常规调节机制

• 批准号: 10616490
• 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/10616490
• 关键词: Ablation; Adult; Animals; Area; Behavioral Assay; Biological; Biological Assay; Biological Phenomena; Biology; Biotinylation; Cell Communication; Cell division; Cell membrane; Cells; Cellular biology; Central Nervous System; 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; Modeling; Molecular; Nerve; 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; intercellular connection; 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.

项目摘要：这个项目在几十年成功阐明基因和分子的基础上进一步扩展。 雌性配子发育中细胞间通讯和细胞骨架重塑的基础 还有雄性。我们建议在两个有趣的领域进行研究：1)细胞脱落的抑制 生殖细胞分裂的胞质分裂；2)高密度脂蛋白的分子调控及其功能意义 在蛋白质翻译过程中高效的组织特异性终止密码子阅读。动物生殖系细胞的增殖 卵巢和睾丸不能完成脱落，导致细胞团保持连接。 通过细胞间的桥梁，称为环状运河。不完全胞质分裂也发生在几个淋巴增生性疾病中。 强调了了解如何控制有丝分裂的这一非规范终点的重要性。 利用果蝇睾丸生殖系有丝分裂的实时成像，我们发现了一种以前未知的 环状通道形成的中间步骤，包括改建成通道的中体型结构 在子代细胞之间。来了解这个成熟步骤是如何发生的，以及驱动 脱落被抑制从定位到中体，我们将使用定位的生物素化来识别中体 并探索已知在细胞质分裂过程中起作用的蛋白质的遗传要求。我们还将 使用局部生物素化来鉴定质膜-细胞骨架界面的环管蛋白。我们的 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架(ORF)，中间有一个单一的终止密码子。在……里面 卵巢，翻译终止于停止产生环管蛋白，没有明显的功能 第二个ORF。相比之下，我们观察到神经中的终止密码子阅读效率非常高 在幼虫和成虫的中枢神经系统(CNS)，产生丰富的ORF1+ORF2蛋白。 此外，果蝇中的kelch和其他几个基因显示了有效的终止密码子阅读 在中枢神经系统中，提示存在许多具有未知功能的羧基末端延伸的蛋白质。 我们将利用核糖体图谱系统地分析终止密码子阅读的范围和规模 神经细胞组织和总蛋白裂解产物的质谱学鉴定通读多肽。要定义 机制，我们将使用遗传筛选来研究两个刺激顺式作用序列 侧翼是通读基因和反式作用因子中的终止密码子。最后，要理解的功能 通过阅读神经元终止密码子，我们将使用基因编辑来去除kelch和其他几个基因的ORF2 并使用细胞生物学和行为分析仔细分析表型。这项研究计划将引领 关于调节重要的基本细胞生物学和遗传机制的发现 非规范生物现象--产生动物合胞体的胞质分裂的另一种结局 生殖细胞和核糖体读出终止密码子，扩展神经元蛋白质组。