title: Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.
abstract: Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.
authors.affiliation: Section Molecular Cytology, Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, The Netherlands.
authors.forename: J E M
authors.initials: JE
authors.lastname: Vermeer
authors.forename: E B
authors.initials: EB
authors.lastname: Van Munster
authors.forename: N O
authors.initials: NO
authors.lastname: Vischer
authors.forename: T W J
authors.initials: TW
authors.lastname: Gadella
mesh_terms: D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins