High resolution approaches to defining organelle heterogeneity in Trypanosoma brucei

摘要 糖体是动质体中具有多种生化途径的特殊过氧化物酶体。 强调这些细胞器的重要性，糖体完整性的破坏是致命的。尽管他们的基本 在自然界中，我们对维持糖体稳态的过程的理解是有限的。为 例如，我们不知道不同的代谢途径在多大程度上集中在一个单一的代谢途径中。 糖体或相反分离成不同的糖体群体。此外，我们不知道这些 细胞器形成。在很大程度上，这种知识差距是由于缺乏实验工具， 研究细胞器生物学糖体是异质的，我们假设这种异质性反映了 功能特化（代谢酶定位于不同的糖体群体）和 在糖体生物发生过程中形成的囊泡中间体。生化分馏和宽场 荧光成像显示参与糖体/过氧化物酶体生物合成的代谢酶和蛋白质 称为过氧化物酶（Pexs）的蛋白质呈现不同的定位模式。这些研究表明，糖体在它们的 功能性能力及其成熟状态。然而，这些方法的局限性阻止了 我们将蛋白质分配给特定的糖体。在目标1中，我们将使用超分辨率成像技术， 定量显示不同定位模式的糖体蛋白定位于不同位置的程度， 糖体来自布氏锥虫的亚细胞器难以用生物化学方法解析， 当前的方法会发生严重的交叉污染。在目标2中，我们将开发使用 荧光激活细胞器分选（FAOS），一种新的和强大的方法，以纯化和表征， 包括糖体在内的亚细胞器。这项工作将极大地推进我们对寄生虫的理解 细胞生物学在几个方面。它将使高效，快速分离细胞器的纯度高于目前 方法，基于其内部组成的细胞器的分离，以及单个细胞器的分析， 这将是有用的，在未来的糖体异质性的研究。本文定义的方法可用于 从其他真核细胞中纯化细胞器，并且可以扩展到包括功能性染料或荧光染料。 生物传感器来跟踪代谢功能。糖体功能特化的决定性发现将 提供对代谢的深入了解，并为定义靶向序列和细胞器建立立足点 受体参与建立和维持这种区室化。证明了糖体 异质性代表了生物起源途径中的中间体，这将为解决 不同的糖体生物合成途径及其在寄生虫生物学中的作用。这项工作将推动我们的 糖体生物学的理解，以及开发实验方法，可以应用于 其他真核生物的细胞器研究。

ABSTRACT Glycosomes are specialized peroxisomes of kinetoplastids that harbor multiple biochemical pathways. Highlighting the importance of these organelles, disruption of glycosome integrity is lethal. Despite their essential nature, our understanding of the processes involved in maintaining glycosome homeostasis is limited. For example, we do not know the extent to which different metabolic pathways are localized together within a single glycosome or are instead separated into distinct glycosome populations. Additionally, we do not know how these organelles are formed. In large part, this gap in knowledge is due to a lack of experimental tools available for studying organelle biology. Glycosomes are heterogeneous and we hypothesize that this heterogeneity reflects both functional specialization (the localization of metabolic enzymes to different glycosome populations) and vesicular intermediates formed during glycosome biogenesis. Biochemical fractionations and widefield fluorescence imaging show that metabolic enzymes and proteins involved in glycosome/peroxisome biogenesis called peroxins (Pexs) exhibit distinct localization patterns. These studies suggest that glycosomes differ in their functional capabilities as well as their maturation status. However, the limitations of these approaches prevent us from assigning a protein to a specific glycosome. In Aim 1, we will use superresolution imaging techniques to quantitate the extent to which the glycosome proteins that exhibit distinct localization patterns localize to different glycosomes. Subcellular organelles from Trypanosoma brucei are difficult to resolve biochemically and significant cross-contamination occurs with current approaches. In Aim 2, we will develop methods to use fluorescence activated organelle sorting (FAOS), a novel and powerful method, to purify and characterize subcellular organelles including glycosomes. This work will dramatically advance our understanding of parasite cell biology in several ways. It will enable the efficient, rapid isolation of organelles of higher purity than current approaches, the separation of organelles based on their internal composition, and analysis of single organelles, which will be useful in future studies of glycosome heterogeneity. The methods defined herein can be used to purify organelles from other eukaryotic cells and can be expanded to include functional dyes or fluorescent biosensors to follow metabolic function. The definitive finding that glycosomes are functionally specialized will provide insight into metabolism and establish a foothold into defining the targeting sequences and organelle receptors involved in establishing and maintaining this compartmentalization. The demonstration that glycosome heterogeneity represents intermediates in the biogenesis pathway would lay the groundwork for resolving different glycosome biogenesis pathways and the role they play in parasite biology. This work will forward our understanding of glycosome biology as well as develop experimental approaches that can be applied to organelle studies in other eukaryotes.