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First published online 5 October 2004
doi: 10.1242/jcs.01404

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Nuclear localisation of the G-actin sequestering peptide thymosin ß₄

Thomas Huff^1,*, Olaf Rosorius¹, Angela M. Otto^1,, Christian S. G. Müller¹, Edda Ballweber², Ewald Hannappel¹ and Hans Georg Mannherz²

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

View larger version (56K): [in a new window]   Fig. 1. Intracellular localisation of thymosin ß4 in MCF-7 cells detected by immunofluorescence. (A) MCF-7 cells grown to near confluence were stained with the affinity-purified anti-thymosin ß4 antibody. The cells were counterstained with Oregon Green DNase I (B) in order to label specifically monomeric actin and with the chromatin-specific dye Hoechst 33258 (C). Magnification, x400.

View larger version (74K): [in a new window]   Fig. 2. Intracellular localisation of thymosin ß4 in MCF-7 and Vero cells. (A-D) Oregon Green cadaverine (OGC) labelled thymosin ß4 (Tß 1-434) with rabbit IgG (controls) was microinjected into either MCF-7 (A,B) or Vero cells (C,D). Bars, 20 µm.

View larger version (68K): [in a new window]   Fig. 3. Distribution of microinjected labelled thymosin ß4 in different cell lines. (A,B) Ten slices of 28 µm thickness of a continuous Z-scan one hour after microinjection of labelled thymosin ß4 into NIH 3T3 cells; note its presence in the cytoplasm and its accumulation within the nucleus. (C-E and F-H) NIH 3T3 cells fixed 30 minutes after microinjection with 4% paraformaldehyde (C,F) and counterstained with TRITC-phalloidin (D,G); note the high concentration of labelled thymosin ß4 within the cytoplasm and its colocalisation along the stress fibres (yellow in merged images E,H). (I-K) MCF-7 cell fixed 30 minutes after microinjection of labelled thymosin ß4 (I) and counterstained with TRITC-phalloidin (J), with the merged image (K); note the almost even distribution between cytoplasm and nucleus. (L-N) Microinjection of labelled thymosin ß4 into a single A431 cell grown within a cell cluster: preferential staining of the nucleus after two hours (L), phase-contrast image (M) and merged image (N). Insert in L shows nuclei of microinjected cells at higher magnification; note that the nucleoli appear almost unstained. Bars, 20 µm.

View larger version (103K): [in a new window]   Fig. 4. Treatment of digitonin-permeabilised cells with fluorescently labelled thymosin ß4. (A-A'') HeLa cells were permeabilised with digitonin and thereafter incubated with fluorescently labelled thymosin ß4 (A') and the chromatin specific dye chromomycin (A) with the merged image shown in A''. Note the pure cytoplasmic distribution of thymosin ß4. (B-B'') HeLa cells were permeabilised with digitonin and further treated using 0.2% Triton X-100. (B) chromomycin staining, (B') labelled thymosin ß4, (B'') merged image. Bar, 50 µm.

View larger version (100K): [in a new window]   Fig. 5. Microinjection of labelled fragments of thymosin ß4 into Vero cells. (A-D) The labelled fragments thymosin ß 1-264OGC and thymosin ß 27-434OGC were generated by enzymatic cleavage using AsnC-proteinase. After separation and characterisation thymosin ß 1-264OGC was microinjected alone (B) or together with rabbit IgG (A). Similarly thymosin ß 27-434OGC was microinjected alone (C) or together with rabbit IgG (D) into Vero cells. Note the predominantly cytoplasmic staining with the thymosin ß 27-434OGC fragment. Bars, 20 µm.

View larger version (91K): [in a new window]   Fig. 6. Microinjection of EDC crosslinked thymosin ß4:actin complex into A431 cells. (A-D) A431 cells were microinjected with crosslinked complex of thymosin ß4 and ADP-ribosylated actin; note the cytoplasmic distribution of this complex at two hours (A,B) and three hours (C) after microinjection. Microinjected cells are marked with arrows. In C, a microinjected cell is shown whose nucleus is outside the confocal plane (arrow head). (D) A confocal Z-stack image series of a microinjected cell (plane distance 6.4 µm). Bars, 20 µm.

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