Fractionating Organelle Subpopulations by Size and Type

按大小和类型划分细胞器亚群

• 批准号: 9897641
• 负责人: Alexandra Ros
• 金额: $ 28.36万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897641
• 关键词: Affect; Aging; Biochemical; Biological; Biological Models; Biology; Cell Line; Centrifugation; Collection; Consumption; Cryoelectron Microscopy; Development; Devices; Diffusion; Disease; Disease Pathway; Endosomes; Equilibrium; Etiology; Exhibits; Fractionation; Functional disorder; Future; Genes; Geometry; Goals; Healthcare Systems; Hela Cells; Heterogeneity; Investigation; Knock-out; Lysosomes; Methods; Microchip Analytical Procedures; Microfluidic Microchips; Mitochondria; Mitochondrial DNA; Modeling; Molecular; Morphology; Organelles; Oxidative Stress; Particle Size; Pathologic; Pathway interactions; Performance; Pharmacotherapy; Phenotype; Play; Preparation; Procedures; Process; Protein Analysis; Resolution; Role; Sampling; Scheme; Societies; Speed; System; Techniques; Technology; Time; Western Blotting; Work; base; biophysical properties; cost; design; driving force; in silico; light scattering; microfluidic technology; migration; new technology; novel; particle; physical property; scale up; tool

Abstract - Fractionating Organelle Subpopulations by Size and Type Intracellular organelle heterogeneity in size and function is intimately associated with multiple dynamic processes, including fusion, fission, biomolecular synthesis and storage within the organelle, biomolecular transport, oxidative stress, and degradation (e.g. via mitophagy). Disease, aging, and drug treatment, perturbing the homeostatic balance of such processes, affect each organelle type differently and for a given type may result in organelle subpopulations with distinct size, function, or morphology. It is thus imperative to isolate organelles of specific type and, when present, their subpopulations, because this is the first step in characterizing their molecular composition, which is essential to decode alterations in function and molecular pathways that are obscured by uncertain subcellular localization. However, the most common techniques are not capable to isolate organelles based on size. Moreover, high purity organelle isolations require multiple, cumbersome and time consuming processes prone to sample loss and still suffer from organelle co-isolation exhibiting similar physical properties. Important biomolecular studies that must account for subcellular localization and that rely on sample quality, are thus severely limited by the lack of suitable technologies for size-based organelle subpopulations and functionally distinct organelles. This study will close this bottleneck by developing a novel fractionation technology capable of separating organelles by size and type in sufficiently large amounts and with high purity. The novel, cutting-edge microfluidic technology to fractionate organelles is based on migration mechanisms that occur under non-equilibrium conditions, require tailored microenvironments and tailored driving forces. If correctly designed these ‘ratchet’ devices exhibit unique selectivity for organelles by size and type, speed, and high throughput capabilities, here realized through a subtle interplay of electrokinetic and dielectrophoretic forces as well as the microfluidic device geometry. Numerical modeling tools will be developed based on experimentally observed migration parameters in specific aim (SA) 1. This in silico study is necessary since ratchet devices often follow ‘non-intuitive’ migration schemes and require detailed parameter studies to adapt them for biological applications. The optimized parameter set obtained from numerical modeling will then be experimentally validated for wild type (normal) and small as well as enlarged mitochondria generated via gene knock-out as a model for size-based separation in SA2. Wild type mitochondria as well as acidic organelles will serve as the model system for type-based separation (SA2). The novel technology will then be scaled-up to build a device for high throughput fractionation and collection of organelle fractions of different sizes or types, allowing the investigation of the phenotype of fractionated mitochondria subpopulations and highly pure organelle isolations with standard characterization methods (SA3). With the successful development of the novel fractionation technology, this project will provide a unique and pivotal tool for future inquiry into highly pure organelle fractions to unravel biological disease pathways in which organelle size and type play a critical role.

摘要 - 按大小和类型按大小和类型的细胞器子分类子 细胞内细胞器的大小和功能异质性与多键动态有关 过程，包括融合，裂变，生物分子合成和储存器中的过程，双分子 运输，氧化应激和降解（例如，通过线粒体），衰老和药物治疗 此类过程的体内平衡，对每个细胞器类型的影响都不同，并且可能导致给定类型 在具有不同大小，功能或形态的细胞器子中。 特定类型的类型，当存在时，是因为它们表征他们的第一步 分子组成，这对于解码功能和分子途径的改变至关重要。 但是，最常见的技术无法隔离。 基于大小的细胞器。 容易发生样品损失的过程，仍然患有细胞器共隔离，表现出相似的物理 物体。 因此，质量受到限制，由于缺乏适合基于尺寸的细胞器亚群的技术 在功能上不同的细胞器中。 能够按大小和类型分离细胞器的技术，并具有很高的纯度。 新型的，尖端的微流体技术到分级细胞器的基于迁移机制 在非平衡内容物下发生的，需要定制的微环境和量身定制的驱动力。 正确设计的这些设备可以按大小和类型，速度和类型显示独特的选择性。 高吞吐量功能，尽管对电动和介电性的微妙解释，这里实现了 作为微流体设备的几何形状的力。 在特定目标（SA）1中观察到的实验性观察到的迁移参数。在计算机研究中，这是必要的 棘轮设备通常遵循'迁移方案，需要详细的参数研究以适应 它们用于生物学应用。 实验性验证了通过基因产生的野生型（正常）和小的线粒体的实验性验证 作为SA2中尺寸分离的模型，酸性细胞器将会 用作基于模型系统的模型分离（SA2）。 一种用于高级分馏和收集不同尺寸或类型的细胞器分数的设备， 允许研究分离的Mitchondria亚匹配和高度纯的表型 具有标准表征方法的细胞器隔离（SA3）。 分馏技术，该项目将提供一个独特而关键的工具，将来对高度纯净的纯纯纯纯纯纯纯纯纯纯纯纯纯纯纯纯净 细胞器分数可阐明生物疾病途径，其中细胞器大小和类型起着至关重要的作用。