Recombinant actin-depolymerizing factor of the apicomplexan Neospora caninum (NcADF) is susceptible to oxidation.

Neospora caninum is a member of Apicomplexa Phylum and the causative agent of neosporosis, a disease responsible for abortions in cattle. Apicomplexan parasites have a limited set of actin-binding proteins conducting the regulation of the dynamics of nonconventional actin. The parasite actin-based motility is implicated in the parasite invasion process in the host cell. Once no commercial strategy for the neosporosis control is available, the interference in the parasite actin function may result in novel drug targets. Actin-depolymerization factor (ADF) is a member of the ADF/cofilin family, primarily known for its function in actin severing and depolymerization. ADF/cofilins are versatile proteins modulated by different mechanisms, including reduction and oxidation. In apicomplexan parasites, the mechanisms involved in the modulation of ADF function are barely explored and the effects of oxidation in the protein are unknown so far. In this study, we used the oxidants N-chlorotaurine (NCT) and H2O2 to investigate the susceptibility of the recombinant N. caninum ADF (NcADF) to oxidation. After exposing the protein to either NCT or H2O2, the dimerization status and cysteine residue oxidation were determined. Also, the interference of NcADF oxidation in the interaction with actin was assessed. The treatment of the recombinant protein with oxidants reversibly induced the production of dimers, indicating that disulfide bonds between NcADF cysteine residues were formed. In addition, the exposure of NcADF to NCT resulted in more efficient oxidation of the cysteine residues compared to H2O2. Finally, the oxidation of NcADF by NCT reduced the ability of actin-binding and altered the function of NcADF in actin polymerization. Altogether, our results clearly show that recombinant NcADF is sensitive to redox conditions, indicating that the function of this protein in cellular processes involving actin dynamics may be modulated by oxidation.

Interleukin-38 interacts with destrin/actin-depolymerizing factor in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 family of cytokines, which was proposed to exert anti-inflammatory effects. IL-38 is constitutively expressed in the skin, where keratinocytes are the main producing cells. Little information is currently available concerning IL-38 biology. Here, we investigated the subcellular localization and interaction partners of the IL-38 protein in human keratinocytes. IL-38 expression was reduced in primary keratinocytes grown in monolayer (2D) cultures. We thus used IL-38 overexpressing immortalized normal human keratinocytes (NHK/38) to study this cytokine in cell monolayers. In parallel, differentiation of primary human keratinocytes in an in vitro reconstructed human epidermis (RHE) 3D model allowed us to restore endogenous IL-38 expression. In NHK/38 cells and in RHE, IL-38 was mainly cell-associated, rather than released into culture supernatants. Intracellular IL-38 was preferentially, although not exclusively, cytoplasmic. Similarly, in normal human skin sections, IL-38 was predominantly cytoplasmic in the epidermis and essentially excluded from keratinocyte nuclei. A yeast two-hybrid screen identified destrin/actin-depolymerizing factor (DSTN) as a potential IL-38-interacting molecule. Co-immunoprecipitation and proximity ligation assay confirmed this interaction. We further observed partial co-localization of IL-38 and DSTN in NHK/38 cells. Endogenous IL-38 and DSTN were also co-expressed in all epidermal layers in RHE and in normal human skin. Finally, IL-38 partially co-localized with F-actin in NHK/38 cells, in particular along the cortical actin network and in filopodia. In conclusion, IL-38 is found predominantly in the cytoplasm of human keratinocytes, where it interacts with DSTN. The functional relevance of this interaction remains to be investigated.

Functional characterisation of the actin-depolymerising factor from the apicomplexan Neospora caninum (NcADF).

Neospora caninum is an apicomplexan parasite that causes infectious abortion in cows. As an obligate intracellular parasite, N. caninum requires a host cell environment to survive and replicate. The locomotion and invasion mechanisms of apicomplexan parasites are centred on the actin-myosin system to propel the parasite forwards and into the host cell. The functions of actin, an intrinsically dynamic protein, are modulated by actin-binding proteins (ABPs). Actin-depolymerising factor (ADF) is a ubiquitous ABP responsible for accelerating actin turnover in eukaryotic cells and is one of the few known conserved ABPs from apicomplexan parasites. Apicomplexan ADFs have nonconventional properties compared with ADF/cofilins from higher eukaryotes. In the present paper, we characterised the ADF from N. caninum (NcADF) using computational and in vitro biochemical approaches to investigate its function in rabbit muscle actin dynamics. Our predicted computational tertiary structure of NcADF demonstrated a conserved structure and phylogeny with respect to other ADF/cofilins, although certain differences in filamentous actin (F-actin) binding sites were present. The activity of recombinant NcADF on heterologous actin was regulated in part by pH and the presence of inorganic phosphate. In addition, our data suggest a comparatively weak disassembly of F-actin by NcADF. Taken together, the data presented herein represent a contribution to the field towards the understanding of the role of ADF in N. caninum and a comparative analysis of ABPs in the phylum Apicomplexa.

Species-Specific Functions of Twinfilin in Actin Filament Depolymerization.

Twinfilin is a highly conserved member of the actin depolymerization factor homology (ADF-H) protein superfamily, which also includes ADF/Cofilin, Abp1/Drebrin, GMF, and Coactosin. Twinfilin has a unique molecular architecture consisting of two ADF-H domains joined by a linker and followed by a C-terminal tail. Yeast Twinfilin, in conjunction with yeast cyclase-associated protein (Srv2/CAP), increases the rate of depolymerization at both the barbed and pointed ends of actin filaments. However, it has remained unclear whether these activities extend to Twinfilin homologs in other species. To address this, we purified the three mouse Twinfilin isoforms (mTwf1, mTwf2a, mTwf2b) and mouse CAP1, and used total internal reflection fluorescence microscopy assays to study their effects on filament disassembly. Our results show that all three mouse Twinfilin isoforms accelerate barbed end depolymerization similar to yeast Twinfilin, suggesting that this activity is evolutionarily conserved. In striking contrast, mouse Twinfilin isoforms and CAP1 failed to induce rapid pointed end depolymerization. Using chimeras, we show that the yeast-specific pointed end depolymerization activity is specified by the C-terminal ADF-H domain of yeast Twinfilin. In addition, Tropomyosin decoration of filaments failed to impede depolymerization by yeast and mouse Twinfilin and Srv2/CAP, but inhibited Cofilin severing. Together, our results indicate that Twinfilin has conserved functions in regulating barbed end dynamics, although its ability to drive rapid pointed end depolymerization appears to be species-specific. We discuss the implications of this work, including that pointed end depolymerization may be catalyzed by different ADF-H family members in different species.

TIRF microscopy analysis of human Cof1, Cof2, and ADF effects on actin filament severing and turnover.

Dynamic remodeling and turnover of cellular actin networks requires actin filament severing by actin-depolymerizing factor (ADF)/Cofilin proteins. Mammals express three different ADF/Cofilins (Cof1, Cof2, and ADF), and genetic studies suggest that in vivo they perform both overlapping and unique functions. To gain mechanistic insights into their different roles, we directly compared their G-actin and F-actin binding affinities, and quantified the actin filament severing activities of human Cof1, Cof2, and ADF using in vitro total internal reflection fluorescence microscopy. All three ADF/Cofilins had similar affinities for G-actin and F-actin. However, Cof2 and ADF severed filaments much more efficiently than Cof1 at both lower and higher concentrations and using either muscle or platelet actin. Furthermore, Cof2 and ADF were more effective than Cof1 in promoting "enhanced disassembly" when combined with actin disassembly co-factors Coronin-1B and actin-interacting protein 1 (AIP1), and these differences were observed on both preformed and actively growing filaments. To probe the mechanism underlying these differences, we used multi-wavelength total internal reflection fluorescence microscopy to directly observe Cy3-Cof1 and Cy3-Cof2 interacting with actin filaments in real time during severing. Cof1 and Cof2 each bound to filaments with similar kinetics, yet Cof2 induced severing much more rapidly than Cof1, decreasing the time interval between initial binding on a filament and severing at the same location. These differences in ADF/Cofilin activities and mechanisms may be used in cells to tune filament turnover rates, which can vary widely for different actin structures.

NMR assignments of actin depolymerizing factor (ADF) like UNC-60A and cofilin like UNC-60B proteins of Caenorhabditis elegans.

The actin filament dynamics in nematode, Caenorhabditis elegans, is regulated by differential activity of two proteins UNC-60A and UNC-60B. UNC-60A exhibits strong pointed end depolymerization on C. elegans actin (Ce-actin), strong inhibition of polymerization, strong monomer sequestering activity, weak severing activity, and low affinity for F-actin binding, while UNC-60B exhibits strong pointed end depolymerization on rabbit muscle actin, strong severing activity, and high affinity for F-actin binding. Structural characterization of these proteins will help to understand (1) molecular mechanism of actin dynamics regulation and (2) the differential activity of these proteins. Here, we report (1)H, (13)C, and (15)N chemical shift assignments of these two proteins as determined by heteronuclear NMR experiments (at pH 6.5 and temperature 298 K).

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand ß6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.

Reduced dimensionality (4,3)D-hnCOCANH experiment: an efficient backbone assignment tool for NMR studies of proteins.

Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone ((1)H, (15)N, (13)C(α) and (13)C') resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment--(4,3)D-hnCOCANH and exploits the linear combinations of backbone ((13)C(α) and (13)C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks--which is different in sum (CA + CO) and difference (CA - CO) frequency regions--greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide (15)N and backbone (13)C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (i→i + 1) (15)N and (13)C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F2-F3 planes of the spectrum corresponding to sum (CA + CO) and difference (CA - CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.

Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite.

Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.

Solution structure and dynamics of ADF from Toxoplasma gondii.

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹5N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed ß-sheet comprised of six ß-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin.

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.

1H, 13C and 15N resonance assignments for a putative ADF/Cofilin from Trypanosoma brucei.

Actin-depolymerizing factor (ADF)/cofilin proteins are a family of actin-binding proteins expressed in almost all eukaryotic cells, and play a significant role in regulating actin-filament dynamics. Here we report the resonance assignments of a putative ADF/cofilin from Trypanosoma brucei for further understanding of the relationship between its structure and function.

Actin filament bundling and different nucleating effects of mouse Diaphanous-related formin FH2 domains on actin/ADF and actin/cofilin complexes.

Mouse Diaphanous-related formins (mDias) are members of the formin protein family that nucleate actin polymerization and subsequently promote filamentous actin (F-actin) elongation by monomer addition to fast-growing barbed ends. It has been suggested that mDias preferentially recruit actin complexed to profilin due to their proline-rich FH1 domains. During filament elongation, dimeric mDias remain attached to the barbed ends by their FH2 domains, which form an anti-parallel ring-like structure enclosing the filament barbed ends. Dimer formation of mDia-FH2 domains is dependent on their N-terminal lasso and linker subdomains (connector). Here, we investigated the effect of isolated FH2 domains on actin polymerization using mDia1-FH2 domain plus connector, as well as core mDia1, mDia2, and mDia3 missing the connector, by cosedimentation and electron microscopy after negative staining. Analytical ultracentrifugation showed that core FH2 domains of mDia1 and mDia2 exhibited a low degree of dimer formation, whereas mDia3-FH2 minus connector and mDia1-FH2 plus connector readily dimerized. Only core mDia3-FH2 was able to nucleate actin polymerization. However, all tested core FH2 domains decorated and bundled F-actin, as demonstrated by electron microscopy after negative staining. Bundling activity was highest for mDia3-FH2, decreased for mDia2-FH2, and further decreased for mDia1-FH2. The mDia1-FH2 domain plus connector induced actin polymerization also in the absence of profilin, but failed to induce F-actin deformation and bundling. We also tested whether mDia1-FH2 was able to repolymerize actin in complex with different proteins that stabilize globular actin. The data obtained demonstrated that mDia1-FH2 induced actin repolymerization only from the actin/cofilin-1 complex, but not when complexed to actin depolymerizing factor, gelsolin segment 1, vitamin D binding protein, or deoxyribonuclease I.

Modeling actin dynamics.

Actin monomers assemble into filaments that structurally support cells as well as drive membrane protrusion for cell movement. Within cells, some actin structures are very dynamic and turn over rapidly, while others are very stable. Even purified actin filament dynamics are complex, and researchers have often turned to mathematical models in order to interpret data, test hypotheses, make predictions, and deepen understanding. Models of actin dynamics can be broadly divided into time-dependent models and time-independent models. Most commonly, time-independent models use numerical solutions of sets of differential equations to explore the effects of key parameters on the actin cycle at steady state. Recent examples have been used to predict the nucleotide profile of steady-state filaments and to illuminate the mechanisms behind profilin's effects on actin dynamics. Time-dependent models of actin dynamics have been either Monte Carlo simulations, which track individual filaments at various levels of detail or less commonly stochastic models, which have been explored and solved analytically. These Monte Carlo and stochastic models have recently been used to investigate filament length diffusion, filament length distributions, annealing and fragmentation, and pyrene fluorescence overshoots. We do not review force production/protrusion models as they tend to reduce the complexity of actin dynamics to a single 'elongation rate' and because these models have been recently well reviewed.

ADF/cofilin: a functional node in cell biology.

Recent findings have significantly expanded our understanding of the regulation of actin-depolymerizing factor (ADF)/cofilin proteins and the profound multifaceted impact that these well-established regulators of actin dynamics have on cell biology. In this review we discuss new aspects of previously documented regulation, such as phosphorylation, but also cover novel recently established modes of regulation and functions of ADF (also known as destrin)/cofilin. We now understand that their activity responds to a vast array of inputs far greater than previously appreciated and that these proteins not only feed back to the crucially important dynamics of actin, but also to apoptosis cascades, phospholipid metabolism, and gene expression. We argue that this ability to respond to physiological changes by modulating those same changes makes the ADF/cofilin protein family a homeostatic regulator or 'functional node' in cell biology.

Isolation and characterization of cytoplasmic cofilin-actin rods.

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca(2+), and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

Structure of the actin-depolymerizing factor homology domain in complex with actin.

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.

Characterization and expression of an actin-depolymerizing factor from Eimeria tenella.

Actin-depolymerizing factor (ADF) plays an important role in remodeling the actin cytoskeleton which contributes much to the invasion of host cells by the apicomplexan parasite. The gene encoding for Eimeria tenella ADF with one intron was cloned and identified by the E. tenella genome raw sequence data ( http://www.sanger.ac.uk/projects/E.tenella/ ). The deduced polypeptide sequence was only composed of 118 amino acids (13.14 kDa) without signal peptide and nuclear localization sequence. The amino acid sequence was most similar to the ADF of Toxoplasma gondii, 69.1%. Compared the putative three-dimensional structures between E. tenella and yeast, the actin filament binding sites on the segment from the alpha4-helices to the C-terminal were mostly missed in E. tenella. Real-time RT-PCR and dot blot both revealed that ADF expression was relatively stronger in the sporozoites and merozoites than in sporulated and unsporulated oocysts in both mRNA and protein levels. Northern blot analysis suggested that there was only one form of ADF transcripts in all different life stages of E. tenella. Actin-binding experiment showed that the recombinant fusion ADF protein could bind with actin, which indicated that ADF probably plays an important role in the invasion host of E. tenella.

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime.

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, L, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: -2/3). We also showed that the average subunit residence time inside the filament, T, depends on the actin monomer (power law: -1/6) and ADF/cofilin (power law: -2/3) concentrations. In addition, filament length fluctuations are approximately 20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P(i) versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.