School of Biomedical Sciences - Research Publications

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Author: Hatters, Daniel × Author: MOHAMED RAMDZAN, YASMIN ×

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    ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
    Irtegun, S ; Ramdzan, YM ; Mulhern, TD ; Hatters, DM (JOURNAL OF VISUALIZED EXPERIMENTS, 2011-08)
    Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells. The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).
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    Tracking protein aggregation and mislocalization in cells with flow cytometry
    Ramdzan, YM ; Polling, S ; Chia, CPZ ; Ng, IHW ; Ormsby, AR ; Croft, NP ; Purcell, AW ; Bogoyevitch, MA ; Ng, DCH ; Gleeson, PA ; Hatters, DM (NATURE PUBLISHING GROUP, 2012-05)
    We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.