title: Tetracysteine-based fluorescent tags to study protein localization and trafficking in Plasmodium falciparum-infected erythrocytes.
abstract: Plasmodium falciparum (Pf) malaria parasites remodel host erythrocytes by placing membranous structures in the host cell cytoplasm and inserting proteins into the surrounding erythrocyte membranes. Dynamic imaging techniques with high spatial and temporal resolutions are required to study the trafficking pathways of proteins and the time courses of their delivery to the host erythrocyte membrane.
  
  
  RESULTS
  Using a tetracysteine (TC) motif tag and TC-binding biarsenical fluorophores (BAFs) including fluorescein arsenical hairpin (FlAsH) and resorufin arsenical hairpin (ReAsH), we detected knob-associated histidine-rich protein (KAHRP) constructs in Pf-parasitized erythrocytes and compared their fluorescence signals to those of GFP (green fluorescent protein)-tagged KAHRP. Rigorous treatment with BAL (2, 3 dimercaptopropanol; British anti-Lewisite) was required to reduce high background due to nonspecific BAF interactions with endogenous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. The fluorescence from FlAsH and ReAsH-labeled protein bleached at faster rates than the fluorescence from GFP-labeled protein.
  
  
  CONCLUSIONS
  While TC/BAF labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods. Our observations are in agreement with the currently-accepted model of KAHRP movement through the cytoplasm, including transient association of KAHRP with Maurer's clefts before its incorporation into knobs in the host erythrocyte membrane.
authors.affiliation: Laboratory of Malaria and Vector Research, Malaria Genetics Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Georgeta
authors.initials: G
authors.lastname: Crivat
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Tokumasu
authors.forename: Juliana Martha
authors.initials: JM
authors.lastname: Sa
authors.forename: Jeeseong
authors.initials: J
authors.lastname: Hwang
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wellems
mesh_terms: D004112:Dimercaprol / Q000633:toxicity; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000469:parasitology; D004912:Erythrocytes / Q000378:metabolism* / Q000469:parasitology*; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D009842:Oligopeptides / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D038761:Photobleaching; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D021381:Protein Transport; D015800:Protozoan Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D009842:Oligopeptides; D010078:Oxazines; D010455:Peptides; D015800:Protozoan Proteins; C042968:knob protein, Plasmodium falciparum; D004112:Dimercaprol; C014180:resorufin; D019793:Fluorescein