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 - 按大小和类型划分细胞器亚群 细胞内细胞器大小和功能的异质性与多种动态密切相关 过程，包括融合、裂变、生物分子合成和细胞器内的储存、生物分子 运输、氧化应激和降解（例如通过线粒体自噬）。疾病、衰老和药物治疗，令人不安 这些过程的稳态平衡对每种细胞器类型的影响不同，并且对于给定类型可能会导致 具有不同大小、功能或形态的细胞器亚群。因此，分离细胞器势在必行。 特定类型及其亚群（如果存在），因为这是表征其特征的第一步 分子组成，这对于解码功能和分子途径的改变至关重要 被不确定的亚细胞定位所掩盖。然而，最常见的技术无法隔离 基于大小的细胞器。此外，高纯度细胞器分离需要多次、繁琐和时间 消耗过程容易造成样品损失，并且仍然遭受细胞器共隔离的影响，表现出类似的物理特性 特性。必须考虑亚细胞定位并依赖于样本的重要生物分子研究 因此，由于缺乏针对基于大小的细胞器亚群的合适技术，质量受到严重限制 和功能不同的细胞器。这项研究将通过开发一种新颖的分馏来解决这一瓶颈 能够按尺寸和类型分离足够大量且高纯度的细胞器的技术。 用于分离细胞器的新颖、尖端的微流体技术基于迁移机制 在非平衡条件下发生的现象需要定制的微环境和定制的驱动力。如果 正确设计的这些“棘轮”装置对细胞器的大小、类型、速度和速度表现出独特的选择性 高通量能力，通过电动和介电泳的微妙相互作用实现 力以及微流体装置的几何形状。数值模拟工具将基于 实验观察到的特定目标 (SA) 1 中的迁移参数。这项计算机研究是必要的，因为 棘轮装置通常遵循“非直观”的迁移方案，需要详细的参数研究才能适应 它们用于生物学应用。通过数值模拟获得的优化参数集将是 经过实验验证野生型（正常）和通过基因产生的小线粒体和扩大线粒体 敲除作为 SA2 中基于尺寸的分离的模型。野生型线粒体以及酸性细胞器将 作为基于类型的分离（SA2）的模型系统。然后，新技术将被扩大规模以构建 用于高通量分级分离和收集不同大小或类型的细胞器级分的装置， 允许研究分级线粒体亚群的表型和高纯度 使用标准表征方法 (SA3) 分离细胞器。随着小说的成功开发 分馏技术，该项目将为未来研究高纯度提供独特且关键的工具 细胞器组分来揭示生物疾病途径，其中细胞器大小和类型起着关键作用。