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Addressing protein dynamics using bleaching technologiesFluorescence recovery after photobleaching (FRAP) and related techniques (FLIP, FLAP, photoconversion, photoactivation) are applied to study the dynamics of fluorescently labeled proteins in living cells. Although such advanced imaging techniques are "well established" to address protein kinetics e.g. in mammalian cells, the application of bleaching methods to yeast cells is still a challenge. Beside general limitations of bleaching technologies (1,2), the small size of yeast cells, the movement of subcellular structures during image acquisition and particularly rapid changes of the cell physiology under preparative conditions can hamper the acquisition of reliable data from bleaching experiments. Thus, the application of this technology to yeast cells presumes a particularly careful experimental design.
Fig.1. Testing microscope features to illuminate user-defined regions of arbitrary shape. The letters "HW" were defined as a region of interest (ROI) within a dense population of yeast cells expressing a cytosolic GFP-tagged protein. The defined ROI was illuminated with high laser intensity resulting in a significant decrease of fluorescence.
Fig.2. Qualitative FRAP. Application of FRAP to study the dynamics of a GFP-tagged protein localized at a bar-like subcellular structure. Recovery of fluorescence 5 min. after bleaching (red arrow).
Fig. 3. Laser induced relocalization of a GFP-tagged yeast protein. Most likely the protein is localized to a cytoskeleton structure. Strong laser illumination may cause an alternation of the morphology of this subcellular structure. Unbleached structures do not show this effect (data not shown).
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Images (C) H.W. YGMBG, University Graz, Austria.
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