Spindle Assembly and Scaling via Microfluidic Encapsulation of Xenopus Nuclei

描述（由申请人提供）：为了准确分离其染色体，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，这些动态的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进行组织，最终产生具有特征性纺锤体形状的稳态结构。因此，纺锤体组装基本上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，但对潜在的力学机制知之甚少。解决我们理解中的这一差距将需要直接的物理扰动和对主轴装配过程中产生的力的定量测量。我们建议开发一种创新的方法来操纵和分析有丝分裂纺锤体组件。具体来说，我们将开发和应用一种基于微流体的平台，用于将细胞核封装在悬浮在连续矿物油相中的水性液滴中。这种方法将使我们能够研究有限的细胞质体积如何影响双极纺锤体组装的物理约束。关于细胞质体积和纺锤体缩放之间的关系的这个问题是基本的，但直到现在，没有答案。然而，我们的技术，将使我们能够精确地调节限制，从而量化的力量在主轴组装和研究的内在机制主轴磁极合并在双极化。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，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体，这是肿瘤转化的标志和染色体出生缺陷的原因，因此填补这一空白在人类疾病的背景下具有重要意义。为此，本提案的具体目的是：1）使用明确的提取物-矿物油乳液分析细胞质体积与纺锤体大小之间的关系，以及2）开发和应用基于高通量微流体的方法来调节纺锤体：液滴比和限制，用于纺锤体极聚结的体外分析。 公共卫生相关性：为了准确地分离其染色体，分裂细胞必须首先组装有丝分裂纺锤体，这基本上是一种生物力学现象。尽管这一过程进行得非常精确，但确实会发生错误，导致非整倍体，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人类疾病具有深远的重要意义。在这个项目中，我们提出了一个微流体实验平台的发展，这将使我们能够封装核，反过来，建立物理环境和生物力学之间的关系主轴组件。这些信息可用于开发更准确和预测性的纺锤体组装模型，这反过来可能导致治疗癌症和其他与纺锤体功能不正常有关的疾病的新策略。

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.