Integration of Xenopus extract and microfluidics to study organelle size scaling

非洲爪蟾提取物和微流体的整合研究细胞器尺寸缩放

• 批准号: 9023558
• 负责人: Daniel Leon Levy
• 金额: $ 26.63万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9023558
• 关键词: Accounting; Address; Affect; Biochemical; Biological; Biological Models; Cell Cycle Progression; Cell Nucleus; Cell Size; Cell physiology; Cells; Cytoplasm; Data; Development; Devices; Diagnosis; Dimensions; Disease Progression; Embryo; Emulsions; Encapsulated; Geometry; Goals; Health; Homeostasis; Hydrogels; In Vitro; Interphase; Knowledge; Lead; Light; Link; Malignant - descriptor; Malignant Neoplasms; Mediating; Microfluidics; Mitotic spindle; Modeling; Molecular; Morphology; Motivation; Nuclear; Organelles; Publishing; Regulation; Research; Shapes; Specific qualifier value; Staging; Techniques; Technology; Testing; Therapeutic; Time; Work; Xenopus; base; biological research; cancer cell; cancer diagnosis; cancer therapy; design; egg; high throughput screening; in vivo; innovation; interest; killings; novel strategies; novel therapeutic intervention; predictive modeling; prevent; treatment strategy

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.

描述（由申请人提供）：如何在细胞内调节有丝分裂纺锤体和相间核的大小仍然在很大程度上未知。这种知识的差距使我们无法理解细胞器大小控制的功能意义，尤其是在各种癌症的背景下，在这些癌症的情况下，细胞器与细胞大小之间的缩放关系已经出现了。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的大小和形态如何影响细胞功能。该建议的目的是阐明细胞器尺寸控制的分子基础。具体而言，我们将解决一个问题，即细胞大小施加的物理约束如何影响有丝分裂纺锤体和相间核的大小，形状和功能。我们的中心假设是，通过限制组件机制介导的核和纺锤体大小的缩放尺寸是介导的。为了检验这一假设，我们开发了一个创新的实验平台，该平台利用基于微流体的技术封装了无细胞提取物，从而使我们能够解决有关细胞器缩放的先前棘手的问题。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更准确和预测模型的信息，这又可能导致治疗癌症的新策略以及与核和螺旋功能不当的其他条件。目标1：确定细胞质体积如何调节核缩放。在此目的中，我们将利用微流体和爪蟾提取物在定义大小，形状和组成的细胞质液滴中组装核，以确定细胞质体积的变化是否足以说明体内核缩放。 AIM 2：使用微流体封装来鉴定MITOTI纺锤体的分子效应和相间核缩放。在此目标中，我们将采用微流体乳液/液滴生成设备来表征细胞质体积和纺锤体/核大小之间缩放关系的分子机制。使用无偏的生化筛选与候选分子方法结合使用，我们希望鉴定成分，即缩放因子，其相对量决定了主轴/核大小。目标3：开发微流体液滴操纵技术，以在四个维度（几何和时间）中对细胞质体积和内容的动态控制。该目标将开发微流体技术，通过该技术可以在指定的时间点更改液滴体积或组成，以诱导和观察细胞器大小的动态变化。预计这些目标中提出的工作的完成将（i）在我们对控制有丝分裂纺锤体和核的机制的基本理解中产生基本进步，并且（ii）证明了将微荧光与一个巨大的实用性和潜力已经有功能强大的生物模型系统，源自爪蟾卵和胚胎的无细胞提取物。这很重要，因为它从根本上可以提高我们对如何调节有丝分裂纺锤体和核的大小的了解，从而为新的治疗方法提供靶标。