title: GFP associates with microfilaments in fixed cells.
abstract: The discovery of the green fluorescent protein (GFP) and its use as a marker for proteins in cells revolutionised cell biology. Among its applications are the intracellular localisation of proteins and the investigation of the organisation, regulation and dynamics of the cytoskeleton. GFP itself is considered to be an inert protein, homogeneously distributed within the cytoplasm. Here we investigated the intracellular distribution of GFP in an amphibian and in various mammalian cell lines (XTH2, CHO-K1, HaCaT, MDCK, NIH-3T3) by confocal laser scanning microscopy. After paraformaldehyde fixation GFP became associated with microfilaments in all the cell lines investigated. This interaction was not impaired by detergent treatment (1% Brij 58 for 10 min). In contrast to the F-actin binding of GFP in fixed cells, association of GFP with stress fibres was not detectable in living cells. The actin-binding property of GFP might contribute also to the interaction of fusion proteins with microfilaments. Thus, careful controls are unavoidable in investigating (weak) actin-binding proteins in fixed cells. Because no association of GFP with microfilaments was detectable in living cells, it is recommended to monitor the intracellular distribution of GFP-tagged proteins in vivo.
authors.affiliation: Kinematic Cell Research Group, Biocentre, Goethe University of Frankfurt/Main, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany. d.schmitz@em.uni-frankfurt.de
authors.forename: H D
authors.initials: HD
authors.lastname: Schmitz
authors.forename: J
authors.initials: J
authors.lastname: Bereiter-Hahn
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D005557:Formaldehyde / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis*; D008322:Mammals; D018613:Microscopy, Confocal / Q000295:instrumentation; D011108:Polymers / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D016707:Tissue Fixation / Q000379:methods; D014982:Xenopus laevis
chemical_list: D000199:Actins; D008164:Luminescent Proteins; D011108:Polymers; D049452:Green Fluorescent Proteins; D005557:Formaldehyde; C003043:paraform