Fractionating Organelle Subpopulations by Size and Type

摘要-按大小和类型划分细胞器亚群 细胞内细胞器在大小和功能上的异质性与多种动态变化密切相关。 过程，包括融合，裂变，生物分子合成和储存在细胞器内，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 这些过程稳态平衡不同地影响每种细胞器类型，且对于给定类型，可能导致 在具有不同大小、功能或形态的细胞器亚群中。因此分离细胞器是非常必要的 的特定类型，并在存在时，其亚群，因为这是第一步，在其特征 分子组成，这是必不可少的解码改变的功能和分子途径， 被不确定的亚细胞定位所掩盖。然而，最常见的技术不能分离 细胞器的大小。此外，高纯度的细胞器分离需要多次、繁琐和时间 消耗过程易于样品损失，并且仍然遭受细胞器共分离，表现出类似的物理特性。 特性.重要的生物分子研究，必须考虑亚细胞定位和依赖于样品 因此，由于缺乏基于大小的细胞器亚群的合适技术， 和功能不同的细胞器。这项研究将通过开发一种新的分馏方法来关闭这一瓶颈。 能够按大小和类型分离足够大量和高纯度的细胞器的技术。 新的，尖端的微流控技术，以压裂细胞器是基于迁移机制 在非平衡条件下发生的，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 高通量能力，这里通过电动和介电泳的微妙相互作用实现 力以及微流体装置几何形状。数值建模工具将基于 实验观察到的特定目标（SA）1中的迁移参数。这项计算机模拟研究是必要的，因为 棘轮装置通常遵循“非直观的”迁移方案， 用于生物应用。然后，从数值建模获得的优化参数集将是 实验验证了野生型（正常）和小以及扩大线粒体产生的基因 敲除作为SA 2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（SA 2）的模型系统。这项新技术将被扩大规模， 一种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， 允许研究分级分离的线粒体亚群的表型， 用标准表征方法分离细胞器（SA 3）。随着小说的成功发展 分馏技术，该项目将提供一个独特的和关键的工具，为未来的调查高纯度 细胞器组分，以解开生物疾病的途径，其中细胞器的大小和类型发挥关键作用。

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.