Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes |
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Authors: | Anjali Gupta Danqin Lu Harikrushnan Balasubramanian Zhang Chi Thorsten Wohland |
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Affiliation: | 1. Department of Biological Sciences and Centre for Bioimaging Sciences (CBIS), National University of Singapore (NUS), Singapore, Singapore;2. School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, China;3. Department of Chemistry, National University of Singapore (NUS), Singapore, Singapore |
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Abstract: | The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer–induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10–15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes. |
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Keywords: | fluidizers phase separation phases membranes heptanol epidermal growth factor receptor alcohols assay clustering MFIC CTxB" },{" #name" :" keyword" ," $" :{" id" :" kwrd0065" }," $$" :[{" #name" :" text" ," _" :" cholera toxin subunit B DiI-C18" },{" #name" :" keyword" ," $" :{" id" :" kwrd0075" }," $$" :[{" #name" :" text" ," _" :" 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate DMEM" },{" #name" :" keyword" ," $" :{" id" :" kwrd0085" }," $$" :[{" #name" :" text" ," _" :" Dulbecco's Modified Eagle Medium DOPC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0095" }," $$" :[{" #name" :" text" ," _" :" 1,2-dioleoyl-sn-glycero-3-phosphocholine DPPC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0105" }," $$" :[{" #name" :" text" ," _" :" 1,2-dipalmitoyl-sn-glycero-3-phosphocholine EGFR" },{" #name" :" keyword" ," $" :{" id" :" kwrd0115" }," $$" :[{" #name" :" text" ," _" :" epidermal growth factor receptor EMCCD" },{" #name" :" keyword" ," $" :{" id" :" kwrd0125" }," $$" :[{" #name" :" text" ," _" :" electron multiplying charge-coupled device FCS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0135" }," $$" :[{" #name" :" text" ," _" :" fluorescence correlation spectroscopy GPI" },{" #name" :" keyword" ," $" :{" id" :" kwrd0145" }," $$" :[{" #name" :" text" ," _" :" glycosylphosphatidylinositol mEGFP" },{" #name" :" keyword" ," $" :{" id" :" kwrd0155" }," $$" :[{" #name" :" text" ," _" :" monomeric enhanced green fluorescence protein MFIC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0165" }," $$" :[{" #name" :" text" ," _" :" membrane fluidizer-induced clustering PM" },{" #name" :" keyword" ," $" :{" id" :" kwrd0175" }," $$" :[{" #name" :" text" ," _" :" plasma membrane trLAT" },{" #name" :" keyword" ," $" :{" id" :" kwrd0185" }," $$" :[{" #name" :" text" ," _" :" transmembrane domain of linker for activation of T-cells |
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