Spindle Assembly and Scaling via Microfluidic Encapsulation of Xenopus Nuclei

通过非洲爪蟾核的微流体封装进行主轴组装和缩放

• 批准号: 8290793
• 负责人: John S Oakey
• 金额: $ 31.29万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8290793
• 关键词: Address; Aneuploidy; Area; Beryllium; Biological Assay; Biomechanics; Cell Nucleus; Cells; Characteristics; Chromosome Segregation; Chromosomes; Congenital Abnormality; Development; Devices; Disease; Elements; Emulsions; Encapsulated; Genome; Goals; In Vitro; Interphase; Lead; Length; Life; Link; Measurement; Measures; Mechanics; Methodology; Metric; Microfluidic Microchips; Microfluidics; Micromanipulation; Microtubules; Mineral Oil; Mitotic spindle; Molecular; Monitor; Motor; Neoplastic Cell Transformation; Nuclear; Phase; Physical environment; Polymers; Process; Research; Shapes; Structure; System; Techniques; Technology; Testing; Time; Work; Xenopus; aqueous; base; biological research; cancer therapy; cell type; cellular imaging; design; egg; human disease; innovation; predictive modeling; treatment strategy

DESCRIPTION (provided by applicant): To accurately segregate its chromosomes, a dividing cell must first assemble a mitotic spindle, of which the main structural elements are microtubules. During spindle assembly, these dynamic cytoskeletal polymers are organized in space and time by microtubule-based motors and other molecular forces, ultimately giving rise to a steady-state structure with a characteristic spindle-like shape. Thus, spindle assembly is fundamentally a biomechanical phenomenon, and while numerous studies have focused on the underlying molecular mechanisms, little is known regarding the underlying mechanics. Addressing this gap in our understanding will require direct physical perturbations and quantitative measurements of the forces generated during spindle assembly. We propose to develop an innovative approach to the manipulation and analysis of mitotic spindle assemblies. Specifically, we will develop and apply a microfluidic-based platform for encapsulating nuclei within aqueous droplets that are suspended in a continuous mineral oil phase. This approach will allow us to investigate how physical constraints imposed by limited cytoplasmic volume impact bipolar spindle assembly. This question regarding the relationship between cytoplasmic volume and spindle scaling is fundamental but, until now, unanswered. Our techniques, however, will allow us to precisely regulate confinement and thus quantify forces during spindle assembly and study inherent mechanisms of spindle pole coalescence during bipolarization. This work serves our ultimate ambitions, which are to better understand the remarkable fidelity of chromosome segregation by elucidating the biomechanics that govern bipolar spindle assembly and function. Because errors in this process can lead to aneuploidy, a hallmark of neoplastic transformation and the cause of chromosomal birth defects, filling this gap has important implications in the context of human disease. To these ends, the Specific Aims of this proposal are: 1) To analyze the relationship between cytoplasmic volume and spindle size using well-defined extract-mineral oil emulsions, and 2) To develop and apply a high-throughput microfluidic-based approach to regulate spindle:droplet ratio and confinement for in vitro analysis of spindle pole coalescence. PUBLIC HEALTH RELEVANCE: In order to accurately segregate its chromosomes, a dividing cell must first assemble a mitotic spindle, which is fundamentally a biomechanical phenomena. Despite the remarkable fidelity with which this process is conducted, errors do occur that can lead to aneuploidy, a hallmark of neoplastic transformation and the cause of chromosomal birth defects. As such, developing a understanding of the biomechanical process by which nuclei replicate has profoundly important implications in the context of human disease. In this project we propose the development of a microfluidic experimental platform that will allow us to encapsulate nuclei and, in turn, establish relationships between the physical environment and the biomechanics of spindle assembly. This information may be used to develop more accurate and predictive models of spindle assembly, which in turn may lead to new strategies for the treatment of cancers and other diseases linked to improper spindle function.

描述（申请人提供）：为了准确分离染色体，分裂细胞必须首先组装有丝分裂纺锤体，其主要结构元件是微管。在纺锤体组装过程中，这些动态细胞骨架聚合物通过基于微管的马达和其他分子力在空间和时间上组织，最终产生具有特征性纺锤体形状的稳态结构。因此，纺锤体组装从根本上来说是一种生物力学现象，虽然许多研究都集中在潜在的分子机制上，但人们对潜在的力学知之甚少。要解决我们理解中的这一差距，需要对主轴组装过程中产生的力进行直接的物理扰动和定量测量。我们建议开发一种创新方法来操纵和分析有丝分裂纺锤体组件。具体来说，我们将开发和应用基于微流体的平台，用于将核封装在悬浮在连续矿物油相中的水滴中。这种方法将使我们能够研究有限的细胞质体积所施加的物理约束如何影响双极纺锤体组装。关于细胞质体积和纺锤体尺度之间关系的这个问题是根本性的，但迄今为止尚未得到解答。然而，我们的技术将使我们能够精确调节约束，从而量化主轴组装过程中的力，并研究双极化过程中主轴极合并的固有机制。这项工作服务于我们的最终目标，即通过阐明控制双极纺锤体组装和功能的生物力学来更好地理解染色体分离的非凡保真度。由于这一过程中的错误可能导致非整倍性，这是肿瘤转化的标志和染色体出生缺陷的原因，因此填补这一空白对人类疾病具有重要意义。为此，本提案的具体目标是：1) 使用明确的提取矿物油乳剂分析细胞质体积和纺锤体大小之间的关系，2) 开发和应用基于高通量微流体的方法调节纺锤体：液滴比例和限制，用于纺锤体极聚结的体外分析。 公共健康相关性：为了准确分离染色体，分裂细胞必须首先组装有丝分裂纺锤体，这从根本上来说是一种生物力学现象。尽管这一过程的执行非常精确，但确实会发生错误，导致非整倍性，这是肿瘤转化的标志，也是染色体出生缺陷的原因。因此，了解细胞核复制的生物力学过程对于人类疾病具有深远的重要意义。在这个项目中，我们建议开发一个微流体实验平台，该平台将使我们能够封装细胞核，进而建立物理环境和主轴组件的生物力学之间的关系。这些信息可用于开发更准确和更具预测性的纺锤体组装模型，这反过来可能会导致治疗癌症和其他与纺锤体功能不当相关的疾病的新策略。