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First published online 3 March 2009
doi: 10.1242/jcs.041715
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Research Article |
1 Cellular Neurobiology, University of Braunschweig, 38092 Braunschweig, Germany
2 Cell Biology, Zoological Institute, University of Braunschweig, 38092 Braunschweig, Germany
* Author for correspondence (e-mail: m.rothkegel{at}tu-bs.de)
Accepted 27 November 2008
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Chicken fibroblasts, Evolution, Profilin isoforms, Actin-based motility
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Introduction |
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; Schluter et al., 1997; Witke, 2004). They bind to monomeric actin in a 1:1 complex and thus can shift the balance between actin filaments and their subunits. In animal cells, this process is under the regulation of numerous large, multidomain proteins of the Ena/VASP and formin families that can bind actin, profilin and profilin-actin complexes and are members of various signalling pathways (Krause et al., 2003; Krebs et al., 2001; Watanabe et al., 1997). In addition to binding to actin and the polyproline stretches of formins and Ena/VASP and other proteins, profilins can bind to acidic phospholipids, some of which are also involved in signal transduction. Hence, profilins are thought to be engaged in linking signal transduction to actin filament formation, and in accordance with this view, they were found to localize with actin, VASP, WAVE and formins in membrane-apposed, dynamic regions of cells (Jockusch et al., 2007). However, the wealth of different actin-based motility processes in cells, such as locomotion, membrane trafficking, cytokinesis, morphogenesis of cellular protrusions and adhesion complexes, probably requires regional modulation and specific fine-tuning of profilin-ligand interactions. Thus, different phospholipids, different formins or Ena/VASP proteins, different actin isoforms and different profilins might specialize in various motility processes. Consistent with such a concept is the notion that profilins occur in a number of isoforms that show tissue-specific expression. The structural relationship and evolutionary origin of profilin isoforms and paralogues from vertebrates has been investigated in detail (Polet et al., 2007), but their biochemical and cell biological properties has been analyzed only in a restricted number of cases. In mammals, four discrete profilin genes, one of which is spliced in two ways, give rise to five different isoforms. Profilin-1 (PFN1) is ubiquitously expressed and is apparently engaged in general motility functions, such as cell migration, cytokinesis and adhesion in all cell types (Jockusch et al., 2007). This concept is supported by the finding that loss of PFN1 in mice is lethal very early in development (Witke et al., 2001). Profilin-2a (PFN2a), the major splice form of profilin-2, is primarily expressed in the brain, whereas a minor splice product, PFN2b, was identified in the kidney (Di Nardo et al., 2000; Lambrechts et al., 2000). Furthermore, PFN3 and PFN4 were identified in mammalian testis (Hu et al., 2001; Obermann et al., 2005), where PFN4 seems specifically involved in the maturation of spermatids (Obermann et al., 2005).
Isoform-specific functional roles of mammalian profilins have been mainly investigated in the CNS of mice and rats. PFN1 was suggested to modulate neuronal actin dynamics during neuritogenesis (Lambrechts et al., 2006), whereas two possible functions are under debate for PFN2a. In transfected hippocampal neurons of mice and rats, PFN2a translocates in an activity-dependent manner to dendritic spines and stabilizes their head morphology (Ackermann and Matus, 2003), suggesting an engagement of PFN2a in the structural plasticity of synapses. By contrast, PFN2a was also found to associate with ligands of endocytotic and exocytotic pathways in mouse brain (Witke et al., 1998). The inhibition of the endocytotic machinery by PFN2a-dynamin-1 interaction and the markedly increased vesicle exocytosis in Pfn2-deficient mice strongly support a regulatory function of PFN2a in endo- and exocytosis of neurons (Gareus et al., 2006; Pilo Boyl et al., 2007). Although these data imply specialized functions of PFN2a in synapses, they do not of course exclude the possibility that PFN2a might functionally substitute for PFN1 in the more general tasks of actin-based motility, if expressed in other tissues.
Here, by using isoform-specific antibodies, we show that a homologue of the mammalian neuronal protein PFN2a is widely expressed in birds. In contrast to mammals, PFN2a in the chicken is not tissue-restricted but is coexpressed with PFN1 in all tissues. The ratios of the isoforms differ in embryonic and adult tissues, indicating that their expression is differentially regulated. Knockdown of either PFN1 or PFN2a or both isoforms in embryonic chicken fibroblasts by miRNA showed that PFN2a contributes to a major extent to cell adhesion and locomotion and that selective loss of PFN1 does not markedly impede these processes.
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Results |
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). In mammals, sequence homology of PFN1 is higher than 90%, but drops to approximately 75% when compared with that in birds, and declines to about 50-60% compared with sequences in frogs and fish (Fig. 1A). PFN2a reveals a different picture: this isoform is much better conserved throughout different classes of vertebrates (Fig. 1A). In particular, the sequences of chicken and mouse differ in exactly one amino acid, a conserved substitution of isoleucine to valine in position 38 (Fig. 1B,C). These findings prompted us to reconsider the hypothesis that PFN2a is generally restricted in its tissue expression and serves primarily specialized functions, such as membrane trafficking in the brain.
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). Within this 16-residue peptide, there are only three differences from the mouse to chicken sequence. With this fusion protein as an antigen, a monoclonal mouse antibody was obtained that recognizes the 12 N-terminal amino acids of the immunogenic peptide. Rabbit serum against PFN2a was obtained by using a peptide comprising the 13 C-terminal residues of mouse (and chicken) PFN2a. A weak crossreactivity with PFN1 was removed by cross-adsorption on mouse PFN1. Fig. 2 shows the reactivity of these antibodies in immunoblots with recombinant profilins, and with protein extracts derived from cultivated chicken and mouse fibroblasts and mouse brain.
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PFN1 and PFN2a are expressed in chicken embryonic fibroblasts but differ in intracellular location
The antibodies specific for either PFN1 or PFN2a in chicken were used to analyze these isoforms in embryonic chicken fibroblasts. Fig. 3 shows a representative cell taken from embryonic stage 12, cultivated for 24 hours, after double immunostaining and counterstaining F-actin with phalloidin. Both isoforms were highly concentrated in the nucleus. PFN1 was also seen rather diffusely distributed in the cytosol, whereas PFN2a was associated with actin-positive structures in the extensive leading lamella and especially in the actin-filament arc proximal to it. These observations suggest that PFN2a is involved in actin-based motility in these cells.
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The finding of profilin in the nucleus is consistent with earlier observations on PFN1 in the nuclei of mammalian epithelial cells (Mayboroda et al., 1997; Stüven et al., 2003), fibroblasts (Skare et al., 2003) and oocytes (Rawe et al., 2006), and there is evidence that it associates with actin in this compartment (Jockusch et al., 2007). However, the significance of these findings for the nuclear function of profilins is still unclear. To obtain further information that might be relevant to this question, we analyzed the compartment-specific residence of both isoforms in chicken fibroblasts. Fig. 4 shows that the level of PFN1 changed in cultivated fibroblasts over time. Cells grown for 96 hours contained about twice as much PFN1 than at 24 hours, whereas the level of PFN2a remained constant (Fig. 4A). Furthermore, the ratio of nuclear and cytosolic PFN1 concentration depended on the culture conditions. Under optimal growth conditions, the rise in PFN1 level was correlated with a higher concentration in the cytosol, whereas the opposite was seen under starvation, where the nuclear concentration exceeded the cytosolic level (Fig. 4B,C). PFN2a did not show such fluctuations (data not shown). These observations do not add to our knowledge on the functional significance of such changes, but they indicate different roles for both isoforms in the same cell. Together with images as shown in Fig. 3, they suggest that a rather constant level of cytosolic PFN2a is required for actin-based functions such as cellular motility.
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PFN1 and PFN2a are involved in cellular motility to different degrees
To analyze the putative role of PFN2a in actin-based cellular dynamics, we knocked down each isoform separately. We chose a miRNA-based vector system driven by the non-species-specific CMV promoter. Based on in silico predictions, various vectors were constructed comprising miRNAs directed against different regions of the profilin mRNAs and a GFP reporter sequence for cell monitoring. Furthermore, we used a bicistronic plasmid, containing miRNAs specific for both isoforms, and a control vector encoding a miRNA sequence for β-galactosidase (LacZ). Chicken fibroblasts grown in culture were transfected with these vectors, and a decrease of PFN protein expression in GFP-positive cells was observed by immunostaining with the isoform-specific antibodies. The time course of knockdown was determined by fixing and staining cells every 24 hours and examining profilin fluorescence. These analyses revealed that the PFN1 signal was lowest 96 hours after transfection, whereas PFN2a decreased much more slowly to a minimum 8 days after transfection. Consequently, in all further analyses on the effects of profilin knockdown, these time points were chosen for monitoring. Fig. 5 shows that cells where PFN1 and/or PFN2a had been knocked down still revealed a faint fluorescence. Quantification of this residual staining by densitometry revealed that it amounted to 11-17% of the values seen with untransfected cells in the case of PFN1, and to 24-31% of those for PFN2a. These values include about 5% background fluorescence as seen in cells incubated with the secondary antibody only. Hence, the knockdown by RNA silencing resulted in at least 70% less profilin. Fig. 5 shows that cells subjected to PFN1 knockdown, still expressed normal levels of PFN2a, and vice versa: a microscopically visible knockdown of PFN2a did not affect expression of PFN1. Cells transfected with the bicistronic vector, analyzed 8 days after transfection, showed a drastic reduction of either profilin, but were still viable (Fig. 5). Analysis of their ability to proliferate showed only a slight effect: their doubling time was 1.3 times that measured in control cultures, and binucleate cells, an indicator of defective cytokinesis, were not observed. This is analogous to observations on PFN1-depleted HUVEC cells (Ding et al., 2006).
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We then analyzed the consequence of profilin loss on cell adhesion and spreading, two processes intimately related and based on actin filament dynamics. Fig. 6A,B shows that chicken fibroblasts lacking PFN1, as seen after transfection with two different vectors, were only slightly impeded in their ability to adhere to and spread on collagen-coated coverslips. The effects were statistically not significantly different from cells transfected with the control vector. By contrast, loss of PFN2a, also obtained with two different vectors, had a much more pronounced effect, and cells transfected with the bicistronic vector and devoid of both isoforms were severely impaired in adhesion and spreading. Correlated with these findings were results showing that loss of profilins influence the size of focal contacts, as determined in cells after staining with an antibody against vinculin (Fig. 6C,D). Focal contacts of cells transfected with the control vector displayed a rather wide size spectrum, with a maximum number at around 120 square pixels; the highest number of focal contacts in cells lacking PFN1 shifted to 80-100 square pixels. In both cases, 25% very large focal contacts (more than 300 square pixels) were monitored. By contrast, loss of PFN2a and loss of both profilins resulted in a marked shift to much smaller focal contacts (maximum around 60-80 square pixels) and larger focal contacts in the size class of >300 square pixels were rare.
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Next, we analyzed locomotion of cells reduced in either or both isoforms. Fig. 7 shows that these cells, when seeded into the upper part of a Transwell chamber in medium without FCS, can be monitored microscopically by their GFP fluorescence. Migration to the lower part of the chamber is stimulated by medium complemented with FCS. Statistical analysis revealed that cells lacking PFN1 after knockdown with two different miRNAs, were only slightly affected in their Transwell migration, whereas lack of PFN2a had a much more drastic effect. Knockdown of both isoforms after transfection with the bicistronic vector resulted in a reduction of migrating cells to 
50% of the value obtained with cells transfected with the control vector.
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Discussion |
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) (Fig. 1). The striking similarity between mouse and chicken sequences suggested that the tissue-restricted expression and specialized functions ascribed to mammalian PFN2a might reflect an exceptional rather than a general case. However, many more studies on different species will be needed to support such speculations.
In mammalian epithelial, endothelial and fibroblastic cells, PFN1 is the abundant profilin isoform expressed, and profilin 2a is barely or not detectable. Its essential role in actin-dependent processes required for cell adhesion, locomotion and thus for tissue morphogenesis has been demonstrated in several studies (Ding et al., 2006; Janke et al., 2000; Wittenmayer et al., 2004; Zou et al., 2007). In endothelial cells, PFN1 is engaged in migration and proliferation, two processes essential for capillary morphogenesis, as concluded from defects observed after silencing PFN1 expression by RNA interference (Ding et al., 2006). Studies with human breast cancer cells revealed that their level of PFN1 is significantly lower than that of normal breast epithelial cells (Janke et al., 2000), and lowering PFN1 expression even further by RNA silencing upregulates their motility and invasiveness (Zou et al., 2007). Conversely, raising the PFN1 level in these cells by transfection leads to a restoration of epithelial differentiation and drastically reduces their malignancy in nude mice (Wittenmayer et al., 2004). Furthermore, epithelial normalization of these cells is dependent on a functional actin-binding site in the introduced PFN1 molecule (Wittenmayer et al., 2004; Zou et al., 2007).
Our results clearly show that in chicken fibroblasts, PFN2a substantially contributes to functions so far ascribed to mammalian PFN1, such as cell spreading, adhesion and locomotion. These parameters are linked to the size of focal contacts that determine the dynamics of cell attachment to the extracellular matrix, and the focal contacts of PFN2a-deficient cells are significantly smaller than those of control cells, analogous to that found after PFN1 silencing in mammalian cells (Ding et al., 2006). However, elimination of this isoform yielded contradicting results on cell migration in the Transwell assay. Although PFN1 knockdown in human breast cancer cells led to increased motility (Zou et al., 2007), our data with fibroblasts lacking PFN2a revealed a delay or decrease in locomotion. This might be due to differences in the velocity and mode of locomotion between carcinoma cells and fibroblasts, rather than differences in the function of the profilin isoforms. In contrast to various mammalian tumour lines, chicken as well as mouse or human fibroblasts locomote very slowly when not stimulated, with frequent oscillations of direction. In any case, our data for cells deficient in both profilin isoforms obtained in the Transwell assay indicate that PFN2a and PFN1 cooperate in actin-based motility processes in birds. Of course, this does not exclude the possibility that PFN2a performs additional functions in the synaptic vesicle cycle in chicken neuronal cells, by participating in complexes with specific ligands as shown for rodent hippocampal cells (Pilo Boyl et al., 2007), but so far, there are no data to support this.
Remarkably, we found also some differences in chicken fibroblasts between PFN1 and PFN2a expression and localization. There is a striking difference in half-life between the isoforms: PFN1 was effectively knocked down in chicken cells within 96 hours, and this value is similar to the time point determined for mammalian endothelial cells (Ding et al., 2006). By contrast, PFN2a was silenced only 8 days after transfection. Such a long half-life is also observed for mammalian PFN2a in mouse neurons (K.M., unpublished observation), but the biological significance of this discrepancy between the two isoforms remains unclear. Furthermore, we observed a difference in intracellular localization. Although PFN2a was consistently found in both the cytosolic and the nuclear compartment, the ratio between nuclear and cytosolic location of PFN1 was more variable and depended on the culture conditions. Mammalian PFN1 was identified in nuclear and nucleolar bodies such as small nuclear ribonucleoproteins (snRNPs), spliceosomes, gems and Cajal bodies, and there is biochemical evidence indicating that it interacts there with specific ligands such as SMN (survival of motor neuron) (Giesemann et al., 1999; Sharma et al., 2005) and p80 coilin (Skare et al., 2003). Hence, these findings suggest a role for nuclear PFN1 in pre-mRNA splicing (Skare et al., 2003). Furthermore, it participates in the nuclear export of complexes containing nuclear actin and a specific exportin in HeLa cells (Stüven et al., 2003). It is, however, not known whether the nuclear form of mammalian PFN1 is identical to the cytoplasmic moiety. Likewise, although the antibodies used in our study faithfully differentiate between chicken PFN1 and PFN2a, we cannot tell whether the moieties found in either cellular compartment are identical or differ, for example in post-translational modification. Our data do not contribute to a better understanding of the functional significance of nuclear profilin, but they do show that in chicken fibroblasts, nuclear trafficking of PFN1 and PFN2a is differentially regulated.
In summary, we found that PFN2a in birds is not tissue-restricted but is ubiquitously expressed and participates in general functions of actin-based motility. This opens new perspectives for further research on functional aspects of profilin isoforms in vertebrates.
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Materials and Methods |
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Preparation of cell and tissue extracts
Cultivated chicken fibroblasts were harvested by trypsin-EDTA treatment. For one-dimensional SDS-PAGE, cells were suspended in lysis buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 1% Triton X-100, 0.1% SDS, protease inhibitors), lysed by repetitive freeze-thaw cycles and, after determining the protein concentration by Bradford assay, boiled in 6x SDS-loading buffer with 10% β-mercaptoethanol. Organs were removed from chicken embryos (E15) or adult freshly killed chicken and mechanically homogenized in extraction buffer (20 mM HEPES, pH 7.2, 5 mM MgCl2, 1% Triton X-100, 0.1% SDS, protease inhibitors). Protein concentration was determined by Bradford assay.
Expression and purification of recombinant chicken profilins
For expression in E. coli, codon-usage optimized chPFN1-cDNA (Geneart, Regensburg, Germany) was subcloned into pET21-vector via NdeI-XhoI digestion. To obtain recombinant chPFN2a, a substitution of Val38 to isoleucine was introduced in the murine PFN2-cDNA by site-directed mutagenesis (Quikchange Mutagenesis Kit, Stratagene, La Jolla, CA). Expression and purification of both isoforms from E. coli extracts, using poly-L-proline affinity chromatography, was essentially as described for human PFN1 and mouse PFN2a (Lambrechts et al., 1997; Wittenmayer et al., 2004).
Antibodies and reagents
The monoclonal PFN1 antibody was raised in mice immunized with a fusion protein comprising a peptide sequence spontaneously forming nanoparticles (Schroeder et al., 2008), and an N-terminal peptide of mouse PFN1 (residues 5-18). The reactive epitope was determined on membrane-synthesized peptides with an overlap of 15 amino acids by Pepscan analysis, as described (Schoenenberger et al., 2005) and comprised residues 5-16. Rabbit serum directed against PFN2a was generated by a 13-residue C-terminal peptide of PFN2a. A weak crossreactivity with PFN1 was eliminated by affinity chromatography on immobilised recombinant PFN1. Anti-vinculin (hvin1) was purchased from Sigma (Munich, Germany). Secondary antibodies included HRP-conjugated goat-anti-mouse or goat-anti-rabbit IgG (Sigma) and goat anti-mouse IgG-Cy2 or IgG-Cy3, and goat anti-rabbit IgG-Cy3 (Dianova, Hamburg, Germany) which were cross adsorbed prior to use. Filamentous actin and DNA in cells were stained with Phalloidin Alexa Fluor 647 or Phalloidin Alexa Fluor 546 (Invitrogen, Karlsruhe, Germany) and DAPI (Sigma), respectively.
Immunoblotting
PFN1 and PFN2a in chicken tissue extracts were detected by immunoblotting. 25 µg total protein was subjected to SDS-PAGE and blotted onto nitrocellulose. Immunoreactivity was obtained with anti-PFN1 (dilution 1:100) and anti-PFN2a (1:10,000). Quantitative data for profilin content were obtained by comparing the signals obtained with HRP-coupled secondary antibodies to calibration curves prepared with recombinant chPFN1 and chPFN2a (10-200 ng).
Immunocytochemistry
Chicken fibroblasts grown on coverslips were fixed in 4% (w/v) formaldehyde (PFA)-PBS for 20 minutes, permeabilized with 0.2% (v/v) Triton X-100 for 2 minutes and then incubated with 1% (w/v) BSA (Applichem, Darmstadt, Germany) in PBS. For immunofluorescence on cultivated chicken fibroblasts, cells grown on coverslips were incubated with anti-PFN1 (dilution, 1:50), anti-PFN2a (1:100) or anti-vinculin for 1 hour, followed by the respective secondary antibodies (1:100) for 45 minutes. F-actin or DNA staining was obtained by incubation with phalloidin or DAPI for 45 minutes. Specimens were examined in a confocal laser-scanning microscope (LSM 510 Meta, Zeiss, Göttingen, Germany), using a multitrack technique to monitor the individual channels separately. Quantification of fluorescence was performed with ImageJ software (Research Services Branch, NIMH, Bethesda, MD).
Profilin silencing
RNAi experiments were performed with the pcDNATM6.2-GW/EmGFP-miR vector (Invitrogen), which carries a blasticidin-resistance gene to select for antibiotic tolerance and thus allows enrichment of transfected cells. Potentially functional miRNA sequences were designed after in silico prediction (BLOCK-iTTM RNAi Designer, Invitrogen), synthesized (Eurofins MWG Operon, Ebersberg, Germany) and inserted into linearized pcDNATM6.2-GW/EmGFP-miR according to the manufacturer's protocol. Of the predicted six miRNA sequences, two miRNA constructs were obtained for PFN1, which proved effective in transfected cells. These chPFN1-specific RNAi plasmids correspond to the following coding base pairs of EST clone CO635550: miRNA ch1.3, base pairs 341-362, miRNA ch1.5, base pairs 392-413. The miRNA sequences effective in chPFN2a knock down were directed against the 3'UTR and correspond to the following base pairs: miRNA 2.3, base pairs 809-830; miRNA 2.6, base pairs 855-876. The RNAi control plasmid used here encodes an miRNA specific for β-galactosidase (LacZ). Transfection of primary chicken fibroblasts was carried out using TransIT-LT1(Mirus bio, Madison, WI). Transfected cells were kept for 8 days under 20 µg/ml blasticidin (PAA, Cölbe, Germany).
Spreading and motility assays
Adhesion and spreading of chicken fibroblasts were analyzed by plating cells onto collagen-coated coverslips. Cells were allowed to adhere for 30 minutes and washed three times in PBS before fixation and staining with Phalloidin-Alexa Fluor 546. Microscopic inspection allowed classification of cells into two categories: well-spread flat cells (group 1) and small compact cells that had failed to spread after 30 minutes (group 2). For analysis of locomotion, a Transwell migration assay was performed, based on the chemotactic attraction of cells by FCS. The cells were starved overnight and subsequently plated in the upper chamber of Transwell devices (Greiner bio-one, Frickenhausen, Germany), containing either serum-free or FCS-supplemented medium (control for random migration). Cells were allowed to migrate for 6 hours into the bottom chamber containing serum-supplemented medium before being fixed and then stained with DAPI.
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Footnotes |
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References |
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Gareus, R., Di Nardo, A., Rybin, V. and Witke, W. (2006). Mouse profilin 2 regulates endocytosis and competes with SH3 ligand binding to dynamin 1. J. Biol. Chem. 281, 2803-2811.
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Wittenmayer, N., Jandrig, B., Rothkegel, M., Schluter, K., Arnold, W., Haensch, W., Scherneck, S. and Jockusch, B. M. (2004). Tumor suppressor activity of profilin requires a functional actin binding site. Mol. Biol. Cell 15, 1600-1608.
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