High resolution approaches to defining organelle heterogeneity in Trypanosoma brucei

摘要 糖体是动质体的特殊过氧化物酶体，具有多种生化途径。 强调这些细胞器的重要性，糖体完整性的破坏是致命的。尽管它们必不可少 本质上，我们对维持糖体稳态过程的理解是有限的。对于 例如，我们不知道不同代谢途径在单一代谢途径中的共同定位程度 糖体或相反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（代谢酶定位到不同的糖体群体）和 糖体生物合成过程中形成的囊泡中间体。生化分级分离和宽场 荧光成像显示代谢酶和蛋白质参与糖体/过氧化物酶体生物发生 称为过氧化物酶（Pexs）的物质表现出独特的定位模式。这些研究表明糖体的不同之处在于 功能能力及其成熟度。然而，这些方法的局限性阻碍了 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 定量表现出不同定位模式的糖体蛋白定位到不同的程度 糖体。布氏锥虫的亚细胞器很难通过生化方法解析，并且 目前的方法会发生严重的交叉污染。在目标 2 中，我们将开发使用方法 荧光激活细胞器分选 (FAOS)，一种新颖而强大的方法，用于纯化和表征 亚细胞细胞器，包括糖体。这项工作将极大地增进我们对寄生虫的理解 细胞生物学有多种方式。它将能够高效、快速地分离比目前纯度更高的细胞器 方法，根据细胞器的内部组成进行分离，以及单个细胞器的分析， 这将有助于未来糖体异质性的研究。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 跟踪代谢功能的生物传感器。糖体具有功能特化的明确发现将 深入了解新陈代谢并为定义靶向序列和细胞器奠定基础 参与建立和维持这种区室化的受体。糖体的证明 异质性代表生物发生途径中的中间体，将为解决这一问题奠定基础 不同的糖体生物发生途径及其在寄生虫生物学中发挥的作用。这项工作将推动我们 了解糖体生物学并开发可应用于 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.