Fluorescence Recovery After Photo-Bleaching (FRAP) and Fluorescence Loss in Photo-Bleaching (FLIP) Experiments to Study Protein Dynamics During Budding Yeast Cell Division

Abstract

The easiness of tagging any protein of interest with a fluorescent marker together with the advance of fluorescence microscopy techniques enable researchers to study in great detail the dynamic behavior of proteins both in time and space in living cells. Two commonly used techniques are FRAP (Fluorescent Recovery After Photo-bleaching) and FLIP (Fluorescence Loss In Photo-bleaching). Upon single bleaching (FRAP) or constant bleaching (FLIP) of the fluorescent signal in a specific area of the cell, the intensity of the fluorophore is monitored over time in the bleached area and in surrounding regions; information is then derived about the diffusion speed of the tagged molecule, the amount of mobile versus immobile molecules as well as the kinetics with which they exchange between different parts of the cell. Thereby, FRAP and FLIP are very informative about the kinetics with which the different organelles of the cell separate into mother- and daughter-specific compartments during cell division. Here, we describe protocols for both FRAP and FLIP and explain how they can be used to study protein dynamics during cell division in the budding yeast Saccharomyces cerevisiae. These techniques are easily adaptable to other model organisms.

Yeast ceramide synthases, Lag1 and Lac1, have distinct substrate specificity

“…Therefore, the nuclear barrier is much more transient than the cortical ER and its formation requires rapid build-up of ceramides and specifically PH forms. To follow the diffusion barrier between the mother and daughter either in the cortical ER or in the nuclear envelope (Luedeke et al, 2005; Shcheprova et al, 2008), we used fluorescent loss in photobleaching (FLIP) experiments (Bolognesi et al, 2016; Ellenberg et al, 1997) where the ER is bleached in the mother cell continuously. If the daughter can exchange ER membrane proteins with the mother then its ER will also become rapidly photobleached.…”

Heat stress promotes longevity in budding yeast by relaxing the confinement of age-promoting factors in the mother cell

“…Thus, we probed the diffusion barrier in the outer nuclear membrane using Fluorescence Loss In Photobleaching (FLIP [ Bolognesi et al, 2016 ]) in cells grown at 30°C or 37°C in medium containing 2% or 0.1% glucose. In cells expressing the nucleoporin Nup49 tagged with GFP (Nup49-GFP, nuclear membrane reporter), a small region of the mother part of anaphase nuclei was constantly photo-bleached over time and the fluorescence decay in both mother and daughter nuclear compartments was measured ( Figure 4A,B ).…”

Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein

“…The mean gray value for each ROI was measured and exported to Excel (Microsoft). The background value was subtracted from each measurement, and all values were normalized to the prebleach value for that experiment ( Bolognesi et al, 2016 ). The normalized values were averaged for all experiments.…”