Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP‐fusion proteins and multimode FRET microscopy

Abstract

SummaryMultimode 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 ( Le CPK1) 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 acceptorbleached 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.

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

“…This fragment was digested with ClaI and NcoI and cloned into ClaI/NcoI-digested pMON-YFP-Zm7hvr, which encodes the yellow fluorescent protein (YFP) fused to the 40 C-terminal amino acids (the hypervariable region) of Rho of plant 7 from Zea mays (Zm7hvr) (Vermeer et al, 2004).…”

“…For this, YFP fused to the 40 C-terminal amino acids (the hypervariable region) of Rho of plant 7 from Z. mays (Zm7hvr) (Vermeer et al, 2004), which contains a myristylation site, was used as a marker protein. As expected (Vermeer et al, 2004), protoplasts transfected with pMON-YFP-Zm7hvr, which contains the YFP-Zm7hvr coding region under the control of a double 35S promoter, showed fluorescence mainly in the plasma membrane (Fig. 3a).…”

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

“…As ECFP is not sensitive to photobleaching [35,36], no obvious fluorescence loss was observed for all four ECFP fusion proteins even after 100 iterations of photobleaching with the maximum laser power (data not shown). However, the problem considered most in FRET analysis of living cell is the acceptor mobility, as photobleaching of acceptor fluorophore will cause inhomogeneity in the cellular fluorescent population [37]. Our results showed that the low mobility of acceptor, Myc-E47-EYFP fusion protein, will not interfere with FRET detection in living mammalian cells by the acceptor photobleaching method.…”

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements

“…Combining GFP variants to form good FRET pairs takes some amount of prudence because the excitation and detection ranges are usually quite close so that spectral crosstalk may hinder the accurate determination of FRET efficiencies. These difficulties caused by spectral overlap can be overcome by the detection of the entire spectrum and mathematically unmixing the components (40,41). Another limitation is that most available laser lines are not optimal for excitation, and the GFP variants mostly cover the lower part of the visible spectrum, where autofluorescence greatly limits the sensitivity.…”