Elucidating the mechanisms of Orb2 mediated neural stem cell asymmetry and division

阐明 Orb2 介导的神经干细胞不对称和分裂的机制

• 批准号: 10752115
• 负责人: Joseph Buehler
• 金额: $ 4.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752115
• 关键词: Apical; Auras; Binding; Biological Models; Brain; CPE-binding protein; Cell Cycle; Cell division; Cells; Cellular biology; Centrioles; Centrosome; Centrosome Pathway; Chromosome Segregation; Cytokinesis; Data; Defect; Disease; Disease Progression; Drosophila genus; Exhibits; Failure; Genomic Instability; Goals; Human; Image; Interphase; Intervention; Lead; Malignant Neoplasms; Malignant neoplasm of brain; Mediating; Membrane; Messenger RNA; Metaphase; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Natural regeneration; Organelles; Orthologous Gene; Post-Transcriptional Regulation; Prognosis; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Regulation; Reporting; Repression; Role; Testing; Transcript; Translational Regulation; Translational Repression; Translations; aurora kinase A; cancer cell; crosslinking and immunoprecipitation sequencing; daughter cell; insight; nerve stem cell; neuroregulation; recruit; segregation; self-renewal; stem cell model; stem cells; trafficking; tumor progression; tumorigenesis

Project Summary: The centrosome is a membraneless organelle comprising a pair of centrioles surrounded by pericentriolar material, which nucleates microtubules to direct cellular trafficking and mitosis. Cancer cells frequently possess extra or aberrant centrosomes, which are associated with poor prognosis. Multiple mechanisms for centrosome overexpansion in cancer cells are proposed, including failures in cytokinesis and unregulated centrosome duplication. Centrosome amplification produces erroneous mitotic spindles, which lead to chromosomal segregation defects, contributing to tumorigenesis and cancer progression. Drosophila neural stem cells (NSCs) represent a genetically tractable model to study mechanisms by which centrosomes assure proper mitotic potency. High grade brain cancers frequently exhibit centrosome amplifications, illustrating how the Drosophila NSC model system informs foundational cancer cell biology. NSCs undergo repeated rounds of asymmetric cell division along an invariant apical-basal polarity axis to regenerate a self-renewing stem cell and a daughter cell fated for differentiation. Our lab recently discovered that loss of the RNA-binding protein Orb2 results in centrosome amplification, dysregulation of centrosome asymmetry, and spindle alignment errors in NSCs, but the mechanisms behind these defects remain elusive. Orb2 is a conserved cytoplasmic polyadenylation element binding protein (CPEB) ortholog involved in the translational regulation of mRNAs. I hypothesize that Orb2 represses the translation of specific centrosome and spindle-associated RNAs to control NSC asymmetric cell division. Importantly, my preliminary data show that the basal centrosome is hyperactivated in orb2 null NSCs. To determine the mechanism by which Orb2 influences asymmetric centrosome maturation (Aim1), I will 1) test whether Orb2 represses the translation of the centrosome activation targets aurA, polo, cnb, or wdr62, and 2) determine if Orb2 requires RNA-binding activity to promote NSC centrosome asymmetry. To determine the mechanism by which Orb2 influences spindle alignment and centrosome segregation (Aim2), I will 1) test whether Orb2 represses the translation of spindle stability targets msps, tacc, or eb1; and 2) live image control and orb2 null NSCs to characterize the formation of dysmorphic spindles and supernumerary centrosomes. This proposed study will reveal how centrosome and mitotic asymmetry is guided by translational control of centrosome and spindle proteins, providing insight into how post transcriptional regulation of the centrosome cycle can lead to cancer.

项目概要： 中心体是一种无膜细胞器，由一对被中心粒周围包围的中心粒组成 使微管成核以指导细胞运输和有丝分裂的材料。癌细胞经常具有 额外或异常的中心体，与不良预后相关。中心体的多种机制 提出癌细胞过度扩张，包括胞质分裂失败和中心体不受调节 复制。中心体扩增产生错误的有丝分裂纺锤体，从而导致染色体 分离缺陷，导致肿瘤发生和癌症进展。果蝇神经干细胞 （NSC）代表了一种遗传上易于处理的模型，用于研究中心体确保适当的机制 有丝分裂效力。高级别脑癌经常表现出中心体扩增，这说明了 果蝇 NSC 模型系统为基础癌细胞生物学提供信息。 NSC 经历反复轮次 沿着不变的顶端-基底极性轴不对称细胞分裂以再生自我更新的干细胞 和一个注定要分化的子细胞。我们的实验室最近发现 RNA 结合蛋白的丢失 Orb2 导致中心体扩增、中心体不对称性失调和纺锤体排列 NSC 中存在错误，但这些缺陷背后的机制仍然难以捉摸。 Orb2 是一个保守的细胞质 多聚腺苷酸化元件结合蛋白 (CPEB) 直系同源物参与 mRNA 的翻译调节。我 假设 Orb2 抑制特定中心体和纺锤体相关 RNA 的翻译 控制 NSC 细胞不对称分裂。重要的是，我的初步数据表明基底中心体是 orb2 null NSC 中过度激活。确定 Orb2 影响不对称的机制 中心体成熟（目标1），我将1）测试Orb2是否抑制中心体的翻译 激活目标 aurA、polo、cnb 或 wdr62，以及 2) 确定 Orb2 是否需要 RNA 结合活性来促进 NSC 中心体不对称。确定 Orb2 影响主轴对准的机制 中心体分离（Aim2），我将1）测试Orb2是否抑制纺锤体稳定性目标的翻译 msps、tacc 或 eb1； 2) 实时图像对照和 orb2 null NSC 来表征畸形的形成 纺锤体和多余的中心体。这项拟议的研究将揭示中心体和有丝分裂如何 不对称性由中心体和纺锤体蛋白的翻译控制引导，从而深入了解如何 中心体周期的转录后调控可导致癌症。