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First published online 5 October 2004
doi: 10.1242/jcs.01404
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Research Article |
1 Institut für Biochemie, Medizinische Fakultät, Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany
2 Cytoskeletal Laboratory, Abteilung für Anatomie und Embryologie, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, G ermany
* Author for correspondence (e-mail: t.huff{at}biochem.uni-erlangen.de)
Accepted 8 July 2004
Thymosin ß4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin ß4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin ß4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin ß4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin ß4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin ß4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.
Key words: ß-thymosins, Fluorescent labelling, Microinjection, Nuclear actin
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