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Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the detailed molecular mechanisms underlying FA mechanosensitivity are still unclear. Besides talin, another key candidate for regulating FA-associated mechanosensing, is vinculin, a prominent FA component, which possesses either closed ("auto-inhibited") or open ("active") conformation. A direct experimental demonstration, however, of the conformational transition between the two states is still absent. In this study, we combined multiple structural and biological approaches to probe the transition from the auto-inhibited to the active conformation, and determine its effects on FA structure and dynamics. We further show that the transition from a closed to an open conformation requires two sequential steps that can differentially regulate FA growth and stability.

Polymerization of actin monomers into filaments requires the initial formation of nuclei composed of a few actin subunits; however, their instability has hindered their detailed study. Therefore we used chemically crosslinked actin oligomers to analyse their effect on actin polymerization. Actin dimer (upper dimer, UD), trimer and tetramer intermolecularly crosslinked by phenylene-bismaleimide along the genetic helix (between Lys199 and Cys374) were isolated by gel filtration and found to increasingly stimulate actin polymerization as shown by the pyrene assay and total internal reflection fluorescence microscopy. In contrast, the so-called lower actin dimer (LD) characterized by a Cys374-Cys374 crosslink stimulated actin polymerization only at low but inhibited it at high concentrations. UD and trimer stimulated the repolymerization of actin from complexes with thymosin β4 (Tβ4) or profilin, whereas the LD stimulated repolymerization only from the profilin : actin but not the actin : Tβ4 complex. In vivo, actin polymerization is stimulated by nucleation factors. Therefore the interaction and effects of purified LD, UD and trimer on the actin-nucleating activity of gelsolin, mouse diaphanous related (mDia) formin and the actin-related protein 2/3 (Arp2/3) complex were analysed. Native gel electrophoresis demonstrated binding of LD, UD and trimer to gelsolin and its fragment G1-3, to the FH2 domains of the formins mDia1 and mDia3, and to Arp2/3 complex. UD and trimer increased the nucleating activity of gelsolin and G1-3, but not of the mDia-FH2 domain nor of the Arp2/3 complex. In contrast, LD at equimolar concentration to Arp2/3 complex stimulated its nucleating activity, but inhibited that of mDia-FH2 domains, gelsolin and G1-3, demonstrating differential regulation of their nucleating activity by dimers containing differently oriented actin subunits., (© 2015 FEBS.)

We established an expression and purification procedure for recombinant protein production in Neurospora crassa (N. crassa). This Strep-tag® based system was successfully used for purifying recombinant N. crassa nitrate reductase (NR), whose enzymatic activity was compared to recombinant N. crassa NR purified from Escherichia coli. The purity of the two different NR preparations was similar but NR purified from N. crassa showed a significantly higher nitrate turnover rate. Two phosphorylation sites were identified for NR purified from the endogenous expression system. We conclude that homologous expression of N. crassa NR yields a higher active enzyme and propose that NR phosphorylation causes enhanced enzymatic activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tβ4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tβ4 decreased treadmilling, presumably by sequestering monomers. Tβ4 added together with ADF/cofilin also inhibited the treadmilling, relative to cofilin alone, but both the rate and extent of depolymerization were markedly enhanced in the presence of both these proteins. Arp2/3 complex reversed the sequestering activity of Tβ4 when equimolar to actin, but not in the additional presence of cofilin-1 or ADF. Transfection experiments to explore the effects of changing the intracellular concentration of Tβ4 in HeLa cells showed that an increase in Tβ4 resulted in reduced actin filaments bundles and narrower lamellipodia, and a conspicuous decrease of cell migration as seen by two different assays. In contrast, cells transfected with a vector leading to Tβ4 knockdown by small interfering RNA (siRNA) displayed prominent actin filament networks within the lamellipodia and the leading lamella and enhanced migration. The experiments reported here demonstrate the importance of the interplay of these different classes of actin-binding proteins on cell behaviour., (Copyright © 2013 Wiley Periodicals, Inc.)

The existence of a cellular machinery that is based on the reversible polymerization of globular nucleotide-bound protomers into polar microfilaments is a persistent feature from prokaryotes to higher vertebrates. However, while in bacteria, actin-like proteins with such properties have evolved into a large family with divergent sequences and polymeric structures, eukaryotes express only a small number of highly conserved actins. Indeed, the sequence of actin is one of the best conserved among eukaryotes and yet actin carries out many different functions at distinct cellular sites. Because of the notorious conservation and lack of suitable tools to examine structural plasticity, the vast majority of studies on cellular actin functions consider mainly two structural states, G-actin and F-actin. However, there is more to the structural plasticity of actin than first meets the eye. On one hand, more than 200 actin-binding proteins shape the conformation of actin and thereby regulate functional diversity. On the other hand, unconventional actin conformations that differ from monomeric G-actin are stepping into the limelight. In addition, supramolecular actin structures that extend beyond classical F-actin are emerging. Herein, we recapitulate the current knowledge on the structure and conformations of monomeric actin and its polymerization into higher order structures, paying special attention to less known forms and their involvement in actin function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The dynamic rearrangement of the actin cytoskeleton plays a key role in several cellular processes such as cell motility, endocytosis, RNA processing and chromatin organization. However, the supramolecular actin structures involved in the different processes remain largely unknown. One of the less studied forms of actin is the lower dimer (LD). This unconventional arrangement of two actin molecules in an antiparallel orientation can be detected by chemical crosslinking at the onset of polymerization in vitro. Moreover, evidence for a transient incorporation of LD into growing filaments and its ability to inhibit nucleation of F-actin filament assembly implicate that the LD pathway contributes to supramolecular actin patterning. However, a clear link from this actin species to a specific cellular function has not yet been established. We have developed an antibody that selectively binds to LD configurations in supramolecular actin structures assembled in vitro. This antibody allowed us to unveil the LD in different mammalian cells. In particular, we show an association of the antiparallel actin arrangement with the endocytic compartment at the cellular and ultrastructural level. Taken together, our results strongly support a functional role of LD in the patterning of supramolecular actin assemblies in mammalian cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

This article addresses the multiple activities of actin. Starting out with the history of actin's discovery, purification and structure, it emphasizes the close relation between structure and function. In this context, we also point to unconventional actin conformations. Their existence in living cells is not yet well documented, however, they seem to play a special role in the supramolecular patterning that underlies some of the physiological functions of actin. Conceivably, such conformations may contribute to actin's diverse activities in the nucleus that are poorly understood so far., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Actin-like proteins comprise a large group of polymorphic proteins that readily form filaments engaged in cytoskeletal functions. Various members have been identified in prokaryotic and eukaryotic cells, e.g. MreB, ParM and Ta0583, and actin and the actin-related proteins, ARPs, respectively. Therefore, it is assumed that an ancestor of actin/MreB/ParM already existed in the last common progenitor of all cells. In eubacteria and archaea, actin-like proteins are either membrane-associated or freely soluble, and their activities are related to motility, cell shape maintenance, subcellular organization and cell cycle progression. In eukaryotes, all these functions are executed by actin in various isoforms. Additional functions have been described for actin and ARPs in the nucleus of the eukaryotic cell, and some of those were also discovered in prokaryotes. In the current essay, we compare structures and selected functions of prokaryotic and eukaryotic actins and discuss various aspects on how actins may have found their way into bacteria, into the eukaryotic cytoplasm and into the nuclear compartment.

Two profilin isoforms (PFN1 and PFN2a) are expressed in the mammalian brain. Although profilins are essential for regulating actin dynamics in general, the specific role of these isoforms in neurons has remained elusive. We show that knockdown of the neuron-specific PFN2a results in a significant reduction in dendrite complexity and spine numbers of hippocampal neurons. Overexpression of PFN1 in PFN2a-deficient neurons prevents the loss of spines but does not restore dendritic complexity. Furthermore, we show that profilins are involved in differentially regulating actin dynamics downstream of the pan-neurotrophin receptor (p75(NTR)), a receptor engaged in modulating neuronal morphology. Overexpression of PFN2a restores the morphological changes in dendrites caused by p75(NTR) overexpression, whereas PFN1 restores the normal spine density. Our data assign specific functions to the two PFN isoforms, possibly attributable to different affinities for potent effectors also involved in actin dynamics, and suggest that they are important for the signal-dependent fine-tuning of neuronal architecture.

Lamellipodial extension depends essentially on the polymerisation cycle of actin. In this cellular compartment the rate and extent of actin polymerisation is tightly regulated by a large number of actin-binding proteins. The main regulators comprise proteins of the actin-depolymerising factor (ADF)/cofilin family, which stimulate actin cycling, but there are also minor constituents like gelsolin and certain variants of tropomyosin that have so far not been considered to be lamellipodial constituents. A number of cell lines express ADF and cofilin simultaneously as shown here for the fibroblastic normal rat kidney (NRK) cell line. Both proteins co-localise in the lamellipodial region. We furthermore demonstrate the presence of gelsolin in lamellipodia by immunostaining with anti-gelsolin antibodies and transfection with EGFP-tagged gelsolin constructs. The presence of tropomyosins in lamellipodia has recently been reported (Hillberg et al., 2006. Tropomyosins are present in lamellipodia of motile cells. Eur. J. Cell Biol. 85, 399-409). In order to evaluate the effect of the simultaneous presence of ADF and cofilin together with tropomyosin and/or gelsolin on the polymerisation cycle of actin, we analysed their effect or combinations of these actin-binding proteins on the steady-state F-actin-ATPase activity in biochemical assays. Our results demonstrate stimulatory effects of ADF/cofilin on actin cycling and a further modulation of ADF/cofilin-stimulated F-actin-ATPase activity by gelsolin and tropomyosin in a complex manner., (2010 Elsevier GmbH. All rights reserved.)

Profilins are small actin-binding proteins expressed in all eukaryotes. They are involved in the regulation of actin filament dynamics and various signalling pathways. The identification of a variety of profilin isoforms led to the assumption that there may be isoform-specific functions. In mammals, profilin-1 (PFN1) is ubiquitously expressed and engaged in the regulation of various motility processes in all cell types. By contrast, profilin-2a (PFN2a) is mainly restricted to neuronal cells and there is evidence that it is involved in neuronal plasticity and membrane trafficking. However, the PFN2a sequence is much better conserved than PFN1 throughout different phyla, indicating that its restricted expression and specialized function in mammals might be unique. Using isoform-specific antibodies, we show that the situation is different in birds. PFN2a is ubiquitously expressed in embryonic and adult chicken tissues at equal and frequently higher amounts than in mammals. Together with PFN1, it is present in cultivated chicken fibroblasts, but differentially localized. Knockdown experiments with miRNA reveal that PFN2a is involved in cell adhesion, spreading and locomotion, and silencing this isoform has pronounced consequences on these processes. Our results indicate profilin isoform expression is differentially regulated among vertebrates.

The role of actin in transcription and RNA processing is now widely accepted but the form of nuclear actin remains enigmatic. Monomeric, oligomeric or polymeric forms of actin seem to be involved in nuclear functions. Moreover, uncommon forms of actin such as the "lower dimer" have been observed in vitro. Antibodies have been pivotal in revealing the presence and distribution of different forms of actin in different cellular locations. Because of its high degree of conservation, actin is a poor immunogen and only few specific actin antibodies are available. To unravel the mystery of less common forms of actin, in particular those in the nucleus, we chose to tailor monoclonal antibodies to recognize distinct forms of actin. To increase the immune response, we used a new approach based on peptide nanoparticles, which are designed to mimic an icosahedral virus capsid and allow the repetitive, ordered display of a specific epitope on their surface. Actin sequences representing the highly conserved "hydrophobic loop," which is buried in the filamentous actin filament, were grafted onto the surface of nanoparticles by genetic engineering. After immunization with "loop nanoparticles," a number of monoclonal antibodies were established that bind to the hydrophobic loop both in vitro and in situ. Immunofluorescence studies on cells revealed that filamentous actin filaments were only labeled once the epitope had been exposed. Our studies indicate that self-assembling peptide nanoparticles represent a versatile platform that can easily be customized to present antigenic determinants in repetitive, ordered arrays and elicit an immune response against poor antigens.

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days' knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.

Raver1 is an hnRNP protein that interacts with the ubiquitous splicing regulator PTB and binds to cytoskeletal components like alpha-actinin and vinculin/metavinculin. Cell culture experiments suggested that raver1 functions as corepressor in PTB-regulated splicing reactions and may thereby increase proteome complexity. To determine the role of raver1 in vivo, we inactivated the gene by targeted disruption in the mouse. Here we report that raver1-deficient mice develop regularly to adulthood and show no obvious anatomical or behavioral defects. In keeping with this notion, cells from raver1-null mice were indistinguishable from wild type cells and displayed normal growth, motility, and cytoskeletal architecture in culture. Moreover, alternative splicing of exons, including the model exon 3 of alpha-tropomyosin, was not markedly changed in mutant mice, suggesting that the role of raver1 for PTB-mediated exon repression is not absolutely required to generate splice variants during mouse development. Interestingly however, loss of raver1 caused significantly reduced plasticity of synapses on acute hippocampal slices, as elicited by electrophysiological measurements of markedly lower LTP and LTD in mutant neurons. Our results provide evidence that raver1 may play an important role for the regulation of neuronal synaptic plasticity, possibly by controlling especially the late LTP via posttranscriptional mechanisms.

Raver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere. In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band. A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.

Profilins are small proteins involved in actin dynamics. In accordance with this function, they are found in all eukaryotes and are structurally highly conserved. However, their precise role in regulating actin-related functions is just beginning to emerge. This article recapitulates the wealth of information on structure, expression and functions accumulated on profilins from many different organisms in the 30 years after their discovery as actin-binding proteins. Emphasis is given to their interaction with a plethora of many different ligands in the cytoplasm as well as in the nucleus, which is considered the basis for their various activities and the significance of the tissue-specific expression of profilin isoforms.

Actin is a rather uncommitted protein with a high degree of structural plasticity: it can adopt a variety of structural states, depending on the specific ionic conditions or the interaction with ligand proteins. These interactions lock actin into a distinct conformation, which specifies the oligomeric or polymeric form it can assume. The interplay between monomeric, oligomeric and polymeric forms is used by the cell to execute an enormous variety of motility processes, such as lamellipodium formation during locomotion or intracellular transport of vesicles. In these cytoplasmic events, monomeric G-actin and filamentous F-actin are the prevalent forms. However, there might be other structural states of actin in cells that have so far not received the attention they deserve. Here, we propose that specific, "unconventional" actin conformations might contribute especially to the multitude of functions executed by actin in the nucleus. We present evidence for the existence of different forms of nuclear actin, taken from studies with selected antibodies.

Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), alpha-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.

For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked 'lower dimer' (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.

Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo-cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB-mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio-temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.

The dynamics of cell adhesion sites control cell morphology and motility. Adhesion-site turnover is thought to depend on the local availability of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) can bind to many cell adhesion proteins such as vinculin and talin, but the consequences of this interaction are poorly understood. To study the significance of phospholipid binding to vinculin for adhesion-site turnover and cell motility, we constructed a mutant, vinculin-LD, deficient in acidic phospholipid binding yet with functional actin-binding sites. When expressed in cells, vinculin-LD was readily recruited to adhesion sites, as judged by fluorescence recovery after photobleaching (FRAP) analysis, but cell spreading and migration were strongly impaired, and PIP(2)-dependent disassembly of adhesions was suppressed. Thus, PIP(2) binding is not essential for vinculin activation and recruitment, as previously suggested. Instead, we propose that PIP(2) levels can regulate the uncoupling of adhesion sites from the actin cytoskeleton, with vinculin functioning as a sensor.

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

Profilins, regulators of cytoplasmic actin dynamics, also bind to several nuclear proteins but the significance of these interactions is mostly unclear. Here, we describe a novel Myb-related transcription factor, p42POP, as a new ligand for profilin and show that profilin regulates its activity. p42POP comprises a unique combination of domains and is widely expressed in mouse tissues. In contrast to many other Myb proteins, it contains only one functional tryptophan-cluster motif. This is followed by an acidic domain, a leucine zipper that mediates dimerization and functional nuclear import and export signals that can direct p42POP to either the nuclear or the cytoplasmic compartment. Binding to profilins is mediated by a proline-rich cluster. p42POP-profilin complexes can be precipitated from cell lysates. In transfected cells displaying p42POP in the nucleus, nuclear profilin is markedly increased. When p42POP is anchored at mitochondrial membranes, profilin is targeted to this location. Hence, in a cellular environment, p42POP and profilin are found in the same protein complex. In luciferase assays, p42POP acts as repressor and this activity is substantially reduced by profilins, indicating that profilin can regulate p42POP activity and is therefore involved in gene regulation.

Metavinculin, the muscle-specific splice variant of the cell adhesion protein vinculin, is characterized by a 68-amino acid insert within the C-terminal tail domain. The findings that mutations within this region correlate with hereditary idiopathic dilated cardiomyopathy in man suggest a specific contribution of metavinculin to the molecular architecture of muscular actin-membrane attachment sites, the nature of which, however, is still unknown. In mice, metavinculin is expressed in smooth and skeletal muscle, where it co-localizes with vinculin in dense plaques and costameres, respectively, but is of conspicuously low abundance in the heart. Immunoprecipitates suggest that both isoforms are present in the same complex. On the molecular level, both vinculin isoforms are regulated via an intramolecular head-tail interaction, with the metavinculin tail domain having a lower affinity for the head as compared with the vinculin tail. In addition, metavinculin displays impaired binding to acidic phospholipids and reduced homodimerization. Only in the presence of phospholipid-activated vinculin tail, the metavinculin tail domain is readily incorporated into heterodimers. Mutational analysis revealed that the metavinculin insert significantly alters binding of the C-terminal hairpin loop to acidic phospholipids. In summary, our data lead to a model in which unfurling of the metavinculin tail domain is impaired by the negative charges of the 68-amino acid insert, thus requiring vinculin to fully activate the metavinculin molecule. As a consequence, microfilament anchorage may be modulated at muscular adhesion sites through heterodimer formation.

Cytoskeletal elements, in particular microtubules and microfilaments, are essential players in a large variety of phenomena requiring cellular and intracellular motility. To name but a few, they are intimately involved in determining cell shape and adhesion, establishment and maintenance of polarity, locomotion and organelle transport in all eukaryotic cells, including neurons. Here, we would like to focus on the synapse in the vertebrate central nervous system, proposing a model for a specific dialogue between neuronal microfilaments and other protein components in neurotransmission and synaptic plasticity.

Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1.

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.

Filamin A is an established structural component of cell-matrix adhesion sites. In addition, it serves as a scaffold for the subcellular targeting of different signaling molecules. Protein kinase C (PKC) has been found associated with filamin; however, details about this interaction and its significance for cell-matrix adhesion-dependent signaling have remained elusive. We performed a yeast two-hybrid analysis using protein kinase Calpha as a bait and identified filamin as a direct binding partner. The interaction was confirmed in transfected HeLa cells, and serial truncation fragments of filamin A were employed to identify two binding sites on filamin. In vitro ligand binding assays revealed a Ca2+ and phospholipid-dependent association of the regulatory domain of protein kinase C with these sites. Phosphorylation of filamin was found to be isoform-restricted, leading to phosphate incorporation in the C termini of filamin A and C, but not B. PKC-dependent phosphorylation of filamin was also detected in cells. Our data suggest an intimate interaction between filamin and PKC in cell signaling.

RNA processing, directed transport along cytoskeletal tracks, and site-specific translation of mRNA at the cell periphery are considered discrete steps in the generation of microfilament-membrane adhesion complexes. A recently identified member of the heterogeneous nuclear ribonucleoprotein family, raver1, may couple these steps and contribute to the assembly and maintenance of these structures.

The vasodilator-stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain. Point mutations within this motif drastically weaken VASP/G-actin interactions, thereby abolishing any actin-nucleating activity of VASP. Additionally, actin nucleation was found to depend on VASP oligomerisation since VASP monomers fail to induce the formation of actin filaments. Phosphorylation negatively affects VASP/G-actin interactions preventing VASP-induced actin filament formation.

During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.

Background: Vinculin and its isoform metavinculin are protein components of intercalated discs, structures that anchor thin filaments and transmit contractile force between cardiac myocytes. We tested the hypothesis that heritable dysfunction of metavinculin may contribute to the pathogenesis of dilated cardiomyopathy (DCM)., Methods and Results: We performed mutational analyses of the metavinculin-specific exon of vinculin in 350 unrelated patients with DCM. One missense mutation (Arg975Trp) and one 3-bp deletion (Leu954del) were identified. These mutations involved conserved amino acids, were absent in 500 control individuals, and significantly altered metavinculin-mediated cross-linking of actin filaments in an in vitro assay. Ultrastructural examination was performed in one patient (Arg975Trp), revealing grossly abnormal intercalated discs. A potential risk-conferring polymorphism (Ala934Val), identified in one DCM patient and one control individual, had a less pronounced effect on actin filament cross-linking., Conclusions: These data provide genetic and functional evidence for vinculin as a DCM gene and suggest that metavinculin plays a critical role in cardiac structure and function. Disruption of force transmission at the thin filament-intercalated disc interface is the likely mechanism by which mutations in metavinculin may lead to DCM.

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

The widely expressed diaphanous proteins, a subclass of formins, comprise links between the Rho GTPases and the actin-based cytoskeleton. They contain several functional domains that are thought to be responsible for interaction with different ligands: the FH1 domain for binding the actin-associated protein profilin; the RBD for targeting activated Rho; and the C-terminal CIID module for autoregulation of the overall diaphanous activity. Using deletion constructs of the murine mDia1, we have analyzed the functional properties of these three domains separately in in vitro assays and in transiently and stably transfected cell lines. We show that the proline-rich FH1 domain effectively binds to profilins in vitro as well as in cells, that the RBD complexes with the CIID in a species-restricted manner and that overexpression of RBD causes spontaneous ruffling and loss of stress fibers, together with loss of directional motility. Supertransfection of cells stably expressing the RBD with dominant negative Rac effectively suppresses ruffling. Our data contribute to the understanding of the function of these domains in linking the actin cytoskeleton with the Rho-signaling cascade. Furthermore, they suggest that inactivation of Rho by exogenous RBD causes upregulation of Rac activity in the transfected cells.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

The cytoskeleton-associated protein vinculin is composed of a globular head and an elongated tail domain. The protein can be cleaved by V8 protease treatment into two fragments with apparent molecular masses of 90 and 29/27 kDa, respectively. So far, no high-resolution data on the tertiary structure of the N-terminal 90-kDa fragment are available. We analyzed the 90-kDa fragment in detail, using electron spectroscopic imaging in conjunction with modelling experiments. The front view projection of this fragment appears roughly rhomboidal, with 4 intensity maxima arranged at the vertices and a stain-filled region in the center. Based on a detailed examination of different particle projections, a 3-dimensional model was constructed which appears as a flattened tetrahedron. A comparison of the 90-kDa fragment with the intact protein allows for a correlation between the subdomain organization of the vinculin head and the biochemically defined V8 protease cleavage sites (aa 851 and 857).

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of alpha-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.

Shigella flexneri, the causative agent of bacillary dysentery, enters into epithelial cells by a macropinocytic process. IpaA, a Shigella protein secreted upon cell contact, binds to the focal adhesion protein vinculin and is required for efficient bacterial uptake. IpaA was shown here to bind with high affinity to the N-terminal residues 1-265 of vinculin. Using co-sedimentation and solid-phase assays, we demonstrated that binding of IpaA to vinculin strongly increases the association of vinculin with F-actin. We also characterized a depolymerizing activity on actin filaments associated with the vinculin-IpaA complex both in vitro and in microinjected cells. We propose that the conformational change of vinculin induced by IpaA binding allows interaction of the vinculin-IpaA complex with F-actin and subsequent depolymerization of actin filaments.

The vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.

Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles.

For a detailed analysis of the profilin-actin interaction, we designed several point mutations in bovine profilin I by computer modeling. The recombinant proteins were analyzed in vitro for their actin-binding properties. Mutant proteins with a putatively higher affinity for actin were produced by attempting to introduce an additional bond to actin. However, these mutants displayed a lower affinity for actin than wild-type profilin, suggesting that additional putative bonds created this way cannot increase profilin's affinity for actin. In contrast, mutants designed to have a reduced affinity for actin by eliminating profilin-actin bonds displayed the desired properties in viscosity assays, while their binding sites for poly(L)proline were still intact. The profilin mutant F59A, with an affinity for actin reduced by one order of magnitude as compared to wild-type profilin, was analyzed further in cells. When microinjected into fibroblasts, F59A colocalized with the endogenous profilin and actin in ruffling areas, suggesting that profilins are targeted to and tethered at these sites by ligands other than actin. Profilin null cells of Dictyostelium were transfected with bovine wild-type profilin I and F59A. Bovine profilin I, although expressed to only approximately 10% of the endogenous profilin level determined for wild-type Dictyostelium, caused a substantial rescue of the defects observed in profilin null amoebae, as seen by measuring the growth of colony surface areas and the percentage of polynucleated cells. The mutant protein was much less effective. These results emphasize the highly conserved biological function of profilins with low sequence homology, and correlate specifically their actin-binding capacity with cell motility and proliferation.