Spindle Assembly and Scaling via Microfluidic Encapsulation of Xenopus Nuclei

描述（由申请人提供）：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。在纺锤装配过程中，这些动态的细胞骨架聚合物在空间和时间上被基于微管的马达和其他分子力组织起来，最终形成具有纺锤形状特征的稳态结构。因此，主轴装配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，但对潜在的力学知之甚少。解决这一差距在我们的理解将需要直接的物理扰动和在主轴装配过程中产生的力的定量测量。我们建议发展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如何影响双极主轴装配的物理约束。这个关于细胞质体积和纺锤体缩放之间的关系的问题是基本的，但直到现在还没有答案。然而，我们的技术将使我们能够精确地调节约束，从而量化主轴装配过程中的力，并研究双极化过程中主轴极合并的内在机制。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，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)使用定义明确的提取物矿物油乳剂分析细胞质体积与纺锤体尺寸之间的关系；2)开发并应用基于高通量微流体的方法来调节纺锤体：液滴比和约束，用于纺锤体极结的体外分析。

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.