Edda Ballweber

Nuclear localisation of the G-actin sequestering peptide thymosin β4

Thymosin β4 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 β4 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 β4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin β4 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 β4 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 β4 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.

Thymosin β4 serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin1

Thymosin β4 possesses actin‐sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin β4 serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln‐23 and Gln‐36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln‐39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G‐actin and could be cross‐linked to G‐actin by 1‐ethyl‐3‐[3‐(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin β4 may serve as a useful tool for further investigations in cell biology. Thymosin β4 could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin β4 concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin β4 may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase‐related processes.

Plant profilin induces actin polymerization from actin:β‐thymosin complexes and competes directly with β‐thymosins and with negative co‐operativity with DNase I for binding to actin

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin β4 and β9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tβ4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tβ4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle α‐actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross‐linking indicated that birch profilin competes with Tβ4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross‐linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co‐operativity between the profilin and DNase I binding sites on actin.

Interaction of ADP‐ribosylated actin with actin binding proteins

Actin ADP‐ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP‐ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP‐ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP‐ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin β4, cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.

Cross-linked long-pitch actin dimer forms stoichiometric complexes with gelsolin segment 1 and/or deoxyribonuclease I that nonproductively interact with myosin subfragment 1.

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.

Interaction of isolated cross‐linked short actin oligomers with the skeletal muscle myosin motor domain

The cyclical interaction between F‐actin and myosin in muscle cells generates contractile force. The myosin motor domain hydrolyses ATP, resulting in conformational changes that are amplified by the myosin lever arm that links the motor domain to the rod domain. Recent cryo‐electron microscopic data have provided a clear picture of the myosin‐ATP–F‐actin complex, but structural insights into other stages of the myosin‐actin interaction have been less forthcoming. To address this issue, we cross‐linked F‐actin subunits between Cys374 and Lys191, and separated them by gel filtration. Purified actin‐dimers, ‐trimers and ‐tetramers retained the ability to polymerize and to stimulate myosin‐subfragment 1 (myosin‐S1) ATPase activity. To generate stable actin oligomer:myosin‐S1 complexes, we blocked actin polymerization with gelsolin and Clostridium botulinum iota toxin‐mediated ADP‐ribosylation. After polymerization inhibition, actin‐trimers and ‐tetramers retained the ability to stimulate the myosin‐S1‐ATPase, whereas the actin‐dimer showed very little ATPase stimulation. We then analysed the stoichiometry and binding affinity of myosin‐S1 to actin oligomers. Actin‐trimers and ‐tetramers bound myosin‐S1 in the absence of nucleotide; the trimer contains one myosin‐S1 binding site. We calculated a dissociation constant (Kd) of 1.1 × 10−10 m and 1.9 × 10−10 m for binding of native F‐actin and the actin‐trimer to myosin‐S1, respectively. EM of the actin‐trimer:myosin‐S1 complex demonstrated the presence of single particles of uniform size. Image reconstruction allowed a reasonable fit of the actin‐trimer and myosin‐S1 into the obtained density clearly showing binding of one myosin‐S1 molecule to the two long‐pitch actins of the trimer, supporting the kinetic data.