High resolution approaches to defining organelle heterogeneity in Trypanosoma brucei

摘要 糖体是动质体内特殊的过氧化物体，具有多种生化途径。 突显这些细胞器的重要性，糖体完整性的破坏是致命的。尽管它们是必不可少的 自然，我们对维持糖体动态平衡的过程的了解是有限的。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独的 糖体或被分成不同的糖体群体。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可用的实验工具 研究细胞器生物学。糖体是异质性的，我们假设这种异质性反映了 功能专门化(代谢酶在不同糖体群体中的定位)和 糖体生物发生过程中形成的囊泡中间体。生化分级与广阔的领域 荧光成像显示代谢酶和蛋白质参与糖体/过氧化酶体的生物发生 所谓的过氧化物素(PEX)表现出不同的局部化模式。这些研究表明，糖体在它们的 功能能力及其成熟状态。然而，这些方法的局限性阻碍了 将一种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来 量化显示不同定位模式的糖体蛋白定位到不同 糖体。布氏锥虫的亚细胞器很难生化分解， 目前的方法存在严重的交叉污染。在目标2中，我们将开发使用 荧光激活细胞器分选(FAOS)是一种新的、有效的纯化和表征方法 包括糖体在内的亚细胞器。这项工作将极大地促进我们对寄生虫的了解 在几个方面的细胞生物学。它将能够高效、快速地分离出比目前纯度更高的细胞器 方法，根据细胞器的内部组成分离细胞器，以及分析单个细胞器， 这对今后糖体异质性的研究将是有用的。本文定义的方法可用于 从其他真核细胞中提纯细胞器，并可扩展到包括功能染料或荧光 生物传感器跟踪新陈代谢功能。糖体是功能专门化的最终发现将 提供对新陈代谢的洞察，并为定义靶向序列和细胞器奠定基础 受体参与了这种划分的建立和维持。证明了糖小体 异质性代表了生物发生途径中的中间体，这将为解决 不同的糖体生物发生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 了解糖体生物学，并开发可应用于 其他真核生物中的细胞器研究。

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.