Noncanonical regulatory mechanisms in cell biology

细胞生物学中的非常规调节机制

• 批准号: 10206358
• 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/10206358
• 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.

项目摘要：该项目扩大了数十年的成功阐明遗传和分子 女性配子发育中的细胞间交流和细胞骨架重塑的基础 和男性。我们在两个有趣的领域提出了研究：1）在 分裂种系细胞的细胞因子和2）高度的分子调节和功能意义 蛋白质翻译过程中有效的组织特异性停止密码子读取。动物中的种系细胞增殖 卵巢和测试的特征是无法完成脱落，导致细胞簇保持连接 通过细胞间桥，称为环管。不完整的细胞因子也发生在几种淋巴增生 疾病，强调了解如何控制这种非规范终点对有丝分裂的重要性。 使用果蝇睾丸中种系有丝分裂的实时成像，我们发现了以前未知的 中间步骤中的环管形成，涉及中型结构，该结构重塑为通道 在子细胞之间。了解这种成熟步骤是如何发生的以及如何驱动的分子机械 脱落被抑制了定位到中部的脱落，我们将使用局部生物素化来识别中部 成分并探测已知在细胞动力学期间起作用的蛋白质的遗传需求。我们也会 使用局部生物赖以鉴定质膜 - 细胞骨架界面处的环管蛋白。我们的 从我们对kelch基因及其功能的广泛分析中，对停止密码子读取的步骤的兴趣 卵子发生。 Kelch mRNA编码一个大型开放式阅读框（ORF），该框架由一个终止密码子打断。在 卵巢，翻译终止于停止以产生环管蛋白，并且没有明显的功能 第二个ORF。相比之下，我们观察到神经中的效率非常高的停止密码子读取 幼虫和成人的中枢神经系统（CNS），产生丰富的ORF1+ORF2蛋白。 此外，果蝇中的凯尔奇和其他几个基因都显示有效的停止密码子。 在中枢神经系统中，暗示存在许多蛋白质具有未知功能的羧基末端扩展。 我们将系统地分析使用核糖体分析的终止密码子读取的范围和规模 总蛋白质裂解物的神经元组织和质谱法，以鉴定读书宠物。定义 读取机理，我们将使用遗传筛选来研究两个刺激顺式作用序列 读取基因和反式作用因子中的停止密码子的侧翼。最后，了解 神经元终止密码子读取，我们将使用Kelch和其他几个基因的基因编辑对ORF2进行融化ORF2 并使用细胞生物学和行为测定仔细分析表型。该研究计划将领导 有关调节重要的重要细胞生物学和遗传机制的发现 非规范生物学现象 - 产生动物合成症的细胞因子的替代性 终止密码子的生殖细胞和核糖体读取，该终止密码子扩展了神经元蛋白质组。