Inhibition of Arp2/3 Complex after ADP-Ribosylation of Arp2 by Binary Clostridioides Toxins.

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures.

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.

Deoxyribonuclease 1-Mediated Clearance of Circulating Chromatin Prevents From Immune Cell Activation and Pro-inflammatory Cytokine Production, a Phenomenon Amplified by Low Trap1 Activity: Consequences for Systemic Lupus Erythematosus.

Increased concentrations of circulating chromatin, especially oligo-nucleosomes, are observed in sepsis, cancer and some inflammatory autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, circulating nucleosomes mainly result from increased apoptosis and decreased clearance of apoptotic cells. Once released, nucleosomes behave both as an autoantigen and as a damage-associated molecular pattern (DAMP) by activating several immune cells, especially pro-inflammatory cells. Deoxyribonuclease 1 (DNase1) is a major serum nuclease whose activity is decreased in mouse and human lupus. Likewise, the mitochondrial chaperone tumor necrosis factor (TNF) receptor-associated protein-1 (Trap1) protects against oxidative stress, which is increased in SLE. Here, using wild type, DNase1-deficient and DNase1/Trap1-deficient mice, we demonstrate that DNase1 is a major serum nuclease involved in chromatin degradation, especially when the plasminogen system is activated. In vitro degradation assays show that chromatin digestion is strongly impaired in serum from DNase1/Trap1-deficient mice as compared to wild type mice. In vivo, after injection of purified chromatin, clearance of circulating chromatin is delayed in DNase1/Trap1-deficient mice in comparison to wild type mice. Since defective chromatin clearance may lead to chromatin deposition in tissues and subsequent immune cell activation, spleen cells were stimulated in vitro with chromatin. Splenocytes were activated by chromatin, as shown by interleukin (IL)-12 secretion and CD69 up-regulation. Moreover, cell activation was exacerbated when Trap1 is deficient. Importantly, we also show that cytokines involved in lupus pathogenesis down-regulate Trap1 expression in splenocytes. Therefore, combined low activities of both DNase1 and Trap1 lead to an impaired degradation of chromatin in vitro, delayed chromatin clearance in vivo and enhanced activation of immune cells. This situation may be encountered especially, but not exclusively, in SLE by the negative action of cytokines on Trap1 expression.

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.

VEGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone.

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott-Aldrich-Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP.

Actin ADP-ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F-actin.

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3-intoxicated HeLa cells transfected with AcGFP-actin shows condensation of F-actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F-actin stress fibres. Recombinant TccC3 toxin ADP-ribosylates purified skeletal and non-muscle actin at threonine148 leading to a strong propensity to polymerize and F-actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148-ADP-ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1-3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP-ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148-ADP-ribosylated actin to curled F-actin bundles even in the presence of thymosin ß4, gelsolin or G1-3. Thr148-ADP-ribosylated F-actin cannot be depolymerized by gelsolin or G1-3 as verified by TIRF, co-sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin-1.

Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy.

Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures.

Gelsolin interacts with LamR, hnRNP U, nestin, Arp3 and ß-tubulin in human melanoma cells as revealed by immunoprecipitation and mass spectrometry.

Gelsolin, a multifunctional actin binding protein, plays a not yet fully understood role in tumorigenesis. Therefore the goal of this study was to identify additional molecular partners of gelsolin in human melanoma cells, separately in the cytoplasmic compartment and cell nuclei. For this purpose we performed immunoprecipitation experiments based on a modified protocol followed by mass spectrometry. The obtained results were confirmed by Western blot analysis, proximity ligation assays and confocal microscopy. As expected gelsolin interacted with actin, in particular we demonstrate its interaction with cytoplasmic ß and γ actins, and a newly discovered actin isoform, actbl2. As new gelsolin-interacting partners we identified the ribosomal protein Rpsa, also known as a non-integrin laminin receptor (LamR), and the heterogeneous nuclear ribonucleoprotein hnRNP U. Our data furthermore indicate that gelsolin interacts with particular components of the three cytoskeleton systems: nestin (intermediate filaments), Arp3 (actin cytoskeleton) and ß-tubulin (microtubules). We also report for the first time that gelsolin is a constituent of midbodies, a tubulin containing structure formed at the end of cytokinesis.

VEGF Signaling Regulates Cofilin and the Arp2/3-complex within the Axonal Growth Cone.

Over the last decade, our understanding of the vascular endothelial growth factor (VEGF) has rapidly increased, becoming the focus of many investigations the world over. Besides its classical role in the vascular system, VEGF was also identified as a factor affecting the nervous system. One structure that responds to VEGF-signaling is the axonal growth cone, the correct behavior of which is essential for the development of a properly working neuronal network. It navigates growing axons to their final destination and helps to create proper synapses at predetermined locations. Recent data concerning the impact of VEGF on the actin cytoskeleton of neuronal growth cones are discussed and new findings of VEGF-signaling in regard to actin dynamics are specified. Overall, we describe the role of VEGF regulation of cofilin and the Arp2/3-complex in axonal growth cones.

Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tß4) 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 Tß4 decreased treadmilling, presumably by sequestering monomers. Tß4 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 Tß4 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 Tß4 in HeLa cells showed that an increase in Tß4 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 Tß4 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.

Structure of the rigor actin-tropomyosin-myosin complex.

Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.

Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells.

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation "status" of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac ß-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells.

AIMS: Formation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells. MAIN METHODS: Colon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization. KEY FINDINGS: Increased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential. SIGNIFICANCE: Our experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.

An antiparallel actin dimer is associated with the endocytic pathway in mammalian cells.

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.

Modulation of actin filament dynamics by actin-binding proteins residing in lamellipodia.

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.

Methotrexate induces apoptosis in CaSki and NRK cells and influences the organization of their actin cytoskeleton.

Methotrexate is a widely used drug in treatments of various types of malignancies and in the therapy of rheumatoid arthritis. The goal of our study was to look at the effect of this dihydrofolate reductase inhibitor on the actin cytoskeleton, since actin plays an important role in cancer transformation and metastasis. For this reason we compared results obtained from experiments on CaSki (human uterine cervix cancer) and NRK (normal fibroblastic rat kidney) cells treated with methotrexate. It has been shown previously that methotrexate can induce apoptosis. Therefore we first examined whether methotrexate induces apoptosis in our model cells. For this aim we applied several assays like Caspase Glo 3/7, DNA fragmentation and binding of phosphatidylserine by annexin V-fluorescein. The data obtained indicated that methotrexate induces programmed cell death in CaSki and NRK cells. However, differences between CaSki and NRK cells were observed in the morphological alterations and dynamics of apoptosis induced by methotrexate. It seemed that cancer cells were more sensitive towards the cell death inducing activity at lower concentrations of methotrexate. Analysis by confocal microscopy of methotrexate-treated cells demonstrated that treatment with this folate antagonist affected the actin cytoskeleton, although the dis-organization of the actin cytoskeleton after treatment with methotrexate differed between cancer and normal cells.

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.