FRET-FLIM applications in plant systems

Abstract

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.

Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy

“…In addition, FRET-FLIM can be easily used to investigate the modulation of protein oligomerization by other HIV-1 proteins and host proteins. Of course, FRET-FLIM approaches are not limited to oligomerization of HIV-1 proteins and were already used to monitor the oligomerization of a number of other viral proteins [2,19,35,76,94] or mammalian proteins [14,21,36,43,85,98,99].…”

Mechanistic dissection of plant embryo initiation

“…Figure 6A-C). In this assay, protein-protein interaction is inferred from energy transfer from the CFP donor to the YFP acceptor molecule, and is measured as a decrease of fluorescence lifetime of the CFP donor (Bucherl et al, 2014). Co-expression of bHLH49-CFP and bHLH49-YFP caused a significant decrease in the lifetime of CFP fluorescence as compared to the co-expression of free YFP, whose effect was less pronounced ( Figure 6D).…”

Stem cell organization in Arabidopsis : from embryos to roots

“…While error rate is highly dependent on insert size and stretches of repetitive bases, sequences of up to 2 kb in size are usually error-free. Bücherl et al, 2014). The drawback of these types of techniques is that they are limited to test the interaction of the selected proteins, and often require prior knowledge regarding the interactions or require all tested proteins to be labeled, limiting the amount of interactions that can be examined and creating a bias in the measurements.…”

“…The drawback of these types of techniques is that they are limited to test the interaction of the selected proteins, and often require prior knowledge regarding the interactions or require all tested proteins to be labeled, limiting the amount of interactions that can be examined and creating a bias in the measurements. Regardless of this, techniques like FRET-FLIM are powerful as a means to characterize individual interactions, as they allow for testing of dynamic interactions at the subcellular level (Bücherl et al, 2014).…”