Integration of Xenopus extract and microfluidics to study organelle size scaling

描述（由申请人提供）：细胞内有丝分裂纺锤体和间期核的大小是如何调节的，这在很大程度上仍是未知的。这种知识上的差距使我们无法理解细胞器大小控制的功能意义，特别是在细胞器和细胞大小之间的比例关系出错的各种癌症的背景下。我们的长期目标是确定细胞器大小调节的机制，以便更好地了解细胞器大小和形态如何影响细胞功能。这个建议的目的是阐明细胞器大小控制的分子基础。具体来说，我们将解决的问题，如何由细胞大小的物理约束的影响大小，形状和功能的有丝分裂纺锤体和间期核。我们的中心假设是，缩放的核和纺锤体的大小与细胞大小是通过一个限制组件机制介导的。为了验证这一假设，我们开发了一种创新的实验平台，该平台利用基于微流体的技术封装无细胞提取物，使我们能够解决以前关于细胞器缩放的棘手问题。完成这项研究的基本原理是提供可用于开发更准确和预测细胞器组装和功能模型的信息，这反过来可能导致治疗癌症和其他与核和纺锤体功能不正常相关的疾病的新策略。目的1：确定细胞质体积如何调节核缩放。在这个目标中，我们将利用微流体和爪蟾提取物组装细胞核的细胞质液滴的定义的大小，形状和组成，以确定细胞质体积的变化是否足以解释在体内核缩放。目的2：利用微流控微囊技术鉴定影响丝裂纺锤体和间期核缩放的分子效应物。在这个目标中，我们将采用微流控乳液/液滴生成装置来表征细胞质体积和纺锤体/核大小之间的缩放关系的分子机制。使用一个公正的生化屏幕结合候选分子的方法，我们希望确定组件，即缩放因子，其相对量决定纺锤体/核的大小。目标3：开发微流控液滴操作技术，以实现对细胞质体积和四维（几何形状和时间）含量的动态控制。这一目标将开发微流控技术，通过该技术可以在特定时间点改变液滴体积或组成，以诱导和观察细胞器大小的动态变化。完成这些目标中提出的工作预计（i）在我们对控制有丝分裂纺锤体和细胞核大小的机制的基本理解中产生根本性的进步，以及（ii）展示将微流体与已经强大的生物模型系统，来自非洲爪蟾卵和胚胎的无细胞提取物相结合的巨大实用性和潜力。这是重要的，因为它将从根本上推进我们对有丝分裂纺锤体和细胞核大小如何调节的认识，为新的治疗方法提供靶点。

DESCRIPTION (provided by applicant): How the sizes of the mitotic spindle and interphase nucleus are regulated within a cell remains largely unknown. This gap in knowledge prevents us from understanding the functional significance of organelle size control, particularly in the context of various cancers in which the scaling relationship between organelle and cell size has gone awry. Our long-term goal is to identify mechanisms of organelle size regulation in order to better understand how organelle size and morphology impact cell function. The objective of this proposal is to elucidate the molecular basis of organelle size control. Specifically, we will address the question of how physical constraints imposed by cell-size impact the size, shape, and function of both the mitotic spindle and interphase nucleus. Our central hypothesis is that scaling of nuclear and spindle size with cell size is mediated through a limiting component mechanism. To test this hypothesis, we have developed an innovative experimental platform that utilizes microfluidic-based technology to encapsulate cell-free extracts, allowing us to address previously intractable questions regarding organelle scaling. The rationale for completion of this research is to provide information that can be used to develop more accurate and predictive models of organelle assembly and function, which in turn may lead to new strategies for treatment of cancers and other conditions linked to improper nuclear and spindle function. Aim 1: To determine how cytoplasmic volume regulates nuclear scaling. In this aim we will utilize microfluidics and Xenopus extracts to assemble nuclei in cytoplasmic droplets of defined size, shape, and composition to determine whether changes in cytoplasmic volume are sufficient to account for in vivo nuclear scaling. Aim 2: To identify molecular effectors of mitoti spindle and interphase nuclear scaling using microfluidic encapsulation. In this aim, we will employ microfluidic emulsion/droplet-generating devices to characterize the molecular mechanisms of the scaling relationship between cytoplasmic volume and spindle/nuclear size. Using an unbiased biochemical screen in combination with candidate molecule approaches, we expect to identify components, i.e. scaling factors, whose relative amounts determine spindle/nuclear size. Aim 3: To develop microfluidic droplet manipulation techniques to enable dynamic control over cytoplasm volume and content in four dimensions (geometry and time). This aim will develop microfluidic techniques by which droplet volume or composition may be changed at specified time points to induce and observe dynamic changes in organelle size. Completion of the work proposed in these aims is expected to (i) produce a fundamental advance in our basic understanding of the mechanisms that control the size of the mitotic spindle and nucleus and (ii) demonstrate the tremendous utility and potential of combining microfluidics with an already powerful biological model system, cell-free extracts derived from Xenopus eggs and embryos. This is significant because it will fundamentally advance our knowledge of how the size of the mitotic spindle and nucleus are regulated, providing targets for new therapeutic approaches.