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Heimo Wolinski, PhD
University Graz
Senior Scientist
Humboldtstrasse 50/II
8010 Graz, Austria
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Addressing protein dynamics using bleaching technologies

Fluorescence 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).

1. Wolinski H, Natter K, Kohlwein SD (2009). The Fidgety Yeast: Focus on High-Resolution Live Yeast Cell Microscopy. Methods in Molecular Biology, Yeast Functional Genomics and Proteomics, vol. 548.
2. Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH. Photobleaching and photoactivation: following protein dynamics in living cells. Nature cell biology 2003;Suppl:S7-14.
3. Sbalzarini IF, Mezzacasa A, Helenius A, Koumoutsakos P. Effects of organelle shape on fluorescence recovery after photobleaching. Biophysical journal 2005;89(3):1482-92.
4. Wu YX, Masison DC, Eisenberg E, Greene LE. Application of photobleaching for measuring diffusion of prion proteins in cytosol of yeast cells. Methods (San Diego, Calif 2006;39(1):43-9.
5. Prescott M, Battad JM, Wilmann PG, Rossjohn J, Devenish RJ. Recent advances in all-protein chromophore technology. Biotechnology annual review 2006;12:31-66.
6. Betzig E, Patterson GH, Sougrat R, et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 2006;313(5793):1642-5.
7. Petty HR. Fluorescence microscopy: established and emerging methods, experimental strategies, and applications in immunology. Microscopy research and technique 2007;70(8):687-709.
8. Stavreva DA, McNally JG. Fluorescence recovery after photobleaching (FRAP) methods for visualizing protein dynamics in living mammalian cell nuclei. Methods Enzymol 2004;375:443-55.
9. Patterson GH, Lippincott-Schwartz J. Selective photolabeling of proteins using photoactivatable GFP. Methods (San Diego, Calif 2004;32(4):445-50.
10. Lippincott-Schwartz J, Patterson GH. Development and use of fluorescent protein markers in living cells. Science 2003;300(5616):87-91.
11. Wehrle-Haller B. Analysis of integrin dynamics by fluorescence recovery after photobleaching. Methods in molecular biology (Clifton, NJ 2007;370:173-202.

Images (C) H.W. YGMBG, University Graz, Austria.

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