Integration of Xenopus extract and microfluidics to study organelle size scaling

描述(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，这在很大程度上还不清楚。这种知识上的差距阻碍了我们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现错误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。这项建议的目的是阐明细胞器大小控制的分子基础。具体地说，我们将解决由细胞大小施加的物理限制如何影响有丝分裂纺锤体和间期核的大小、形状和功能的问题。我们的中心假设是，细胞核和纺锤体大小与细胞大小的比例是通过限制成分机制来调节的。为了验证这一假设，我们开发了一个创新的实验平台，利用基于微流控的技术来封装无细胞提取物，使我们能够解决以前难以解决的细胞器缩放问题。完成这项研究的基本原理是提供可用于开发细胞器组装和功能的更准确和预测模型的信息，这反过来可能导致治疗癌症和其他与核和纺锤体功能异常有关的疾病的新策略。目的1：确定细胞质体积如何调节核鳞片。在这个目标中，我们将利用微流体和非洲爪哇提取物将细胞核组装在确定的大小、形状和组成的细胞质液滴中，以确定细胞质体积的变化是否足以解释体内的核缩放。目的2：利用微流控技术鉴定丝裂原纺锤体和间期核鳞片的分子效应。在这一目标中，我们将使用微流控乳液/液滴生成设备来表征细胞质体积与纺锤体/核大小之间比例关系的分子机制。使用无偏见的生化筛选结合候选分子方法，我们希望确定组件，即比例因子，其相对数量决定纺锤体/核的大小。目的3：开发微流控液滴操纵技术，以实现对细胞质体积和含量的四个维度(几何和时间)的动态控制。这一目标将发展微流控技术，通过它可以在特定的时间点改变液滴的体积或组成，以诱导和观察细胞器大小的动态变化。这些目标中提出的工作的完成有望(I)在我们对控制有丝分裂纺锤体和核大小的机制的基本理解方面取得根本性进展，(Ii)展示将微流体与已经强大的生物模型系统、非洲爪哇卵子和胚胎的无细胞提取物相结合的巨大效用和潜力。这一点意义重大，因为它将从根本上提高我们对有丝分裂纺锤体和核的大小是如何调节的了解，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.