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，由一对中心粒和一对中心粒周粒组成， 一种使微管成核以指导细胞运输和有丝分裂的物质。癌细胞通常具有 额外或异常的中心体，这与不良预后有关。中心体的多重机制 提出了癌细胞中的过度扩张，包括胞质分裂失败和中心体不受调节 重复。中心体扩增产生错误的有丝分裂纺锤体，这导致染色体畸变。 分离缺陷，有助于肿瘤发生和癌症进展。果蝇神经干细胞 （神经干细胞）代表了一种遗传上易于处理的模型，用于研究中心体确保正确的机制 有丝分裂潜能高级别的脑癌经常表现出中心体扩增，这说明了 果蝇NSC模型系统告知基础癌细胞生物学。NSC经历重复的循环， 沿着不变的顶部-底部极性轴的不对称细胞分裂沿着以再生自我更新干细胞 和注定要分化的子细胞我们的实验室最近发现RNA结合蛋白的缺失 Orb 2导致中心体扩增、中心体不对称性失调和纺锤体排列 神经干细胞中的错误，但这些缺陷背后的机制仍然难以捉摸。Orb 2是一个保守的细胞质 多聚腺苷酸化元件结合蛋白（CPEB）的直系同源物参与mRNA的翻译调控。我 假设Orb 2抑制特定中心体和纺锤体相关RNA的翻译 来控制神经干细胞的不对称分裂重要的是，我的初步数据显示，基底中心体是 在orb 2无效的NSC中过度活化。确定Orb 2影响非对称性的机制 中心体成熟（Aim 1），我将1）测试Orb 2是否抑制中心体的翻译 激活靶点aurA、波罗、cnb或wdr 62，以及2）确定Orb 2是否需要RNA结合活性来促进 NSC中心体不对称。为了确定Orb 2影响主轴对准的机制， 中心体分离（Aim 2），我将1）测试Orb 2是否抑制纺锤体稳定性靶点的翻译 msps、tacc或eb 1;和2）实时图像对照和orb 2无效NSC，以表征畸形的形成 纺锤体和额外中心体。这项研究将揭示中心体和有丝分裂 不对称性是由中心体和纺锤体蛋白的翻译控制指导的， 中心体周期的转录后调节可导致癌症。