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Heimo Wolinski, PhD
University Graz
Senior Scientist
Humboldtstrasse 50/II
8010 Graz, Austria
Phone: +43 316 380 5489
Fax: +43 316 380 9854 -------------------------------
Web: IMB-Graz MCF
Web: H. Wolinski Research

Localization studies using GFP technology and reference dyes

Green-fluorescent-protein (GFP) fusions are indispensable tools for studying the distribution and dynamics of proteins and organelles in vivo. We use GFP and variants thereof to determine the spatial subcellular localization of proteins involved in the lipid metabolism of the yeast. In this respect, we study the expression of GFP tagged proteins in single cells, in larger cell populations (quantitative analysis) or globally using commercially available or in-house produced strain libraries (1,2). Generated localization data are partly public (YPL, Yeast Protein Localization Database (3,4)) and can be accessed via Saccharomyces cerevisiae genome database (SGD, Stanford University). In addition to GFP fusions we apply commerically available reference dyes for studying interactions of proteins and organelles.

Double labeling.
GFP fusion protein, MitoTracker Red CM-H2XRos.
Triple labeling.
GFP fusion protein, DAPI, FM4-64.

FM4-64 (endocytosis marker). Plasma membranes
and vesicles (30 min after adding the dye).
FM4-64. Vacuolar membranes
(60 min after adding the dye).

DAPI. Nuclei and mitochondrial DNA.
ConcavalinA. Cell walls.

DASPMI. Mitochondria.
Nile Red. Lipid droplets (overlay:
interference contrast image).

Vacuolar lumen, ER.
Vacuolar membranes.

Budding yeast cells.
Differential interference contrast.
Mammalian cell line.
Lipid bodies, nucleus.

Yeast heart :).

GFP-tagged protein located
at the periphery of lipid bodies
(BODIPY C-12 labelled) of a COS-7 cell.

Fat galaxy. Nuclei (pink/magneta),
ER (red), lipid bodies (green).

.....and more!

1. Natter K, Leitner P, Faschinger A, Wolinski H, McCraith S, Fields S, 2. Kohlwein SD, The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy. Mol Cell Proteomics. 2005; 4(5):662-72.
2. Kohlwein SD. The beauty of the yeast: live cell microscopy at the limits of optical resolution. Microsc Res Tech. 2000; 51, 511-29.
3. Habeler G, Natter K, Thallinger GG, Crawford ME, Kohlwein SD, Trajanoski Z., YPL.db: the Yeast Protein Localization database. Nucleic Acids Res. 2002 ;30(1):80-3.
4. Kals M, Natter K, Thallinger GG, Trajanoski Z, Kohlwein SD, YPL.db2: the Yeast Protein Localization database, version 2.0. Yeast. 2005; 22(3):213-8.
Images/movies (C) H.W., S.D.K., YGMBG, University Graz, Austria.

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