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|Author(s):||Georgeta Crivat; Fuyuki Tokumasu; Juliana M. Sa; Thomas E. Wellems; Jeeseong Hwang;|
|Title:||Tetracysteine-Based Fluorescent Tags to Study Protein Localization and Trafficking in Plasmodium falciparum-Infected Erythrocytes|
|Published:||August 10, 2011|
|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. Using a tetracysteine (TC) motif and TC-binding biarsenical fluorophores (BAFs) including fluorescein arsenical hairpin (FlAsH) and resorufin arsenical hairpin (ReAsH), we detected knob-associated histidinerich 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 endogeneous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. While TC/BAF labelling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labelling 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.|
|Keywords:||Biarsenical fluorophores, FlAsH, infectious disease, knob-associated histidine rich protein KAHRP, malaria, microscopy, molecular imaging protein trafficking, ReAsH|
|Research Areas:||Biological Physics, Quantitative Molecular Imaging|