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 glyc...