title: Using fluorescent proteins to monitor glycosome dynamics in the African trypanosome.
abstract: Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions. Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses. To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.
authors.affiliation: Department of Genetics and Biochemistry, Clemson University Eukaryotic Pathogens Innovation Center.
authors.forename: Sarah
authors.initials: S
authors.lastname: Bauer
authors.affiliation: Department of Genetics and Biochemistry, Clemson University Eukaryotic Pathogens Innovation Center.
authors.forename: Meghan
authors.initials: M
authors.lastname: Conlon
authors.affiliation: Department of Genetics and Biochemistry, Clemson University Eukaryotic Pathogens Innovation Center; mmorri3@clemson.edu.
authors.forename: Meredith
authors.initials: M
authors.lastname: Morris
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D005434:Flow Cytometry; D008164:Luminescent Proteins / Q000737:chemistry*; D008830:Microbodies / Q000737:chemistry / Q000378:metabolism*; D000074437:Peroxisomal Targeting Signal 2 Receptor; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D000074437:Peroxisomal Targeting Signal 2 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria