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Fluorescence‐based RNAi screen for mutants in fatty acid uptake in African trypanosomes
Author(s) -
Paul Kimberly Sue,
McCall Barbara,
Winston Brette,
Vigueira Patrick,
McCallister Jennifer
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.268
Subject(s) - bodipy , fatty acid , biochemistry , fluorophore , chemistry , fluorescence , fluorescence microscope , moiety , biology , stereochemistry , physics , quantum mechanics
Trypanosoma brucei has two options to supply itself with fatty acids: it can synthesize fatty acids via an ER‐localized elongase pathway, or it can conserve the energy used for synthesis and instead, take up fatty acids from the host. The bloodstream form is known to take up fatty acids from the host in lipoprotein particles and other fatty acid conjugates. Less is known about the mechanisms governing uptake of free fatty acids. We have developed a fluorescence‐based uptake assay utilizing a C12 fatty acid conjugated at the acyl end to the green fluorophore, BODIPY. T. brucei cells were incubated with BODIPY‐C12 and uptake was monitored by flow cytometry. BODIPY‐C12 uptake was linear with respect to time and concentration, and appeared saturable at higher BODIPY‐C12 concentrations. The uptake of BODIPY‐C12 was inhibited at low temperatures and by exposure of the cell surface to trypsin prior to uptake. Uptake of unconjugated BODIPY was only ∼10% of BODIPY‐C12, suggesting that uptake of BODIPY‐C12 is largely mediated by the fatty acid moiety. Also, co‐incubation of BODIPY‐C12 with a 100‐fold excess of non‐BODIPY‐labeled C16 fatty acid inhibited the uptake of BODIPY‐C12 by ∼70%. Finally, fluorescence microscopy shows BODIPY‐C12 labeling of internal cellular structures including the ER and cytoplasmic puncta. Having developed a fluorescence‐based assay for fatty acid uptake, we are now poised to screen the RNAi library. Identification of the genes governing fatty acid uptake and elucidation of these uptake mechanisms will enhance our understanding of the T. brucei host‐parasite relationship.