Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein-tagged Golgi components.

Abstract

Protein-protein interaction at the organelle level can be analyzed by using tagged proteins and assessing Förster resonance energy transfer (FRET) between fluorescent donor and acceptor proteins. Such studies are able to uncover partners in the regulation of proteins and enzymes. However, any organelle movement is an issue for live FRET microscopy, as the observed organelle must not change position during measurement. One of the mobile organelles in plants is the Golgi apparatus following cytoplasmic streaming. It is involved in the decoration of proteins and processing of complex glycan structures for the cell wall. Understanding of these processes is still limited, but evidence is emerging that protein-protein interaction plays a key role in the function of this organelle. In the past, mobile organelles were usually immobilized with paraformaldehyde (PFA) for FRET-based interaction studies. Here, we show that the actin inhibitor Cytochalasin D (CytD) is superior to PFA for immobilization of Golgi stacks in plant cells. Two glycosyltransferases known to interact were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, coexpressed in Nicotiana benthamiana leaves and analyzed using confocal microscopy and spectral imaging. Fixation with PFA leads to reduced emission intensity when compared to CytD treatment. Furthermore, the calculated FRET efficiency was significantly higher with CytD than with PFA. The documented improvements are beneficial for all methods measuring FRET, where immobilization of the investigated molecules is necessary. It can be expected that FRET measurement in organelles of animal cells will also benefit from the use of inhibitors acting on the cytoskeleton.