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Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the mean length of individual actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different lengths using the same set of molecular building blocks. Here we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth rate modulation by actin binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneity. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning F-actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.SIGNIFICANCEActin filaments organize into different functional network architectures within eukaryotic cells. To maintain distinct actin network architectures, it is essential to regulate the lengths of actin filaments. While the mechanisms controlling the lengths of individual actin filaments have been extensively studied, the regulation of length heterogeneity in actin filament populations is not well understood. Here we show that the modulation of actin filament growth and nucleation rates by actin binding proteins can regulate actin length distribution and create distinct sub-populations with different lengths. In particular, by tuning concentrations of formin, profilin and capping proteins, various aspects of actin filament length distribution can be controlled. Insights gained from our results may have significant implications for the regulation of actin filament length heterogeneity and architecture within a cell.

Membrane-cytoskeleton interactions (MCIs) are mediated by actin-binding proteins (ABPs). Ezrin is a crucial ABP that links membranes to actin filaments during lamellipodia formation, cell polarization, and migration. We discuss the concept of MCI and the potential of ezrin as a druggable target for treating inflammatory diseases and cancers.

Drebrin E is a regulatory protein of intracellular force produced by actomyosin complexes, that is, myosin molecular motors interacting with actin filaments. The expression level of drebrin E in nerve cells decreases as the animal grows, suggesting its pivotal but unclarified role in neuronal development. Here, by applying the microscopic heat pulse method to actomyosin motility assay, the regulatory mechanism is examined from the room temperature up to 37 °C without a thermal denaturing of proteins. We show that the inhibition of actomyosin motility by drebrin E is eliminated immediately and reversibly during heating and depends on drebrin E concentration. The direct observation of quantum dot-labeled drebrin E implies its stable binding to actin filaments during the heat-induced sliding. Our results suggest that drebrin E allosterically modifies the actin filament structure to regulate cooperatively the actomyosin activity at the maintained in vivo body temperature., 胎児の神経を形作る仕組みは精密な温度センサー --母体の体温維持が神経の成熟に重要であることを示唆--. 京都大学プレスリリース. 2021-11-10.

In this work, a molecular analysis of some mechanisms of the actin cytoskeleton reorganization in the process of long-term cultivation of human mesenchymal stem cells was carried out. The distribution of actin-binding protein α-actinin-4 and small GTPase RhoA in mesenchymal stem cells of the ADH-MSC line isolated from adipose tissue of an adult human was studied using immunofluorescence methods and analysis of confocal images. It was found that in the process of replicative senescence during passages 8–17 in ADH-MSC cells, the redistribution of the studied proteins from the cytoplasm to the cell nuclei occurs, which is accompanied by changes in the organization of the actin cytoskeleton. To assess the organization of the actin cytoskeleton, we used the coefficient of local connected fractal dimension (LCFD), which characterizes local disturbances in the geometry of heterogeneous geometric objects and is an indirect measure for assessing the structural integrity of such a complex geometric object as the actin cytoskeleton of spread cells. By measuring the LCFD of confocal images of cells stained with rhodamine phalloidin, changes in the structural integrity of the actin cytoskeleton during replicative senescence were quantified. It was found that from passages 8 to 11, a partial reversible disassembly of the actin cytoskeleton occurs, followed by assembly by the passage 17.

The biological aggressiveness of a tumor is determined by the ability of tumor cells to invade and metastasize which is a consequence of their acquisition of a number of phenotypic characteristics. Remodeling of the actin cytoskeleton occurs during cell migration which is carried out by various groups of actin binding proteins in the regulation of which proteasomes and calpains play an important role. Therefore the study of the relationship of proteins associated with cell motility with the processes of lymphogenous metastasis as well as the assessment of the regulatory role of intracellular proteases in these processes is extremely important for fundamental oncology. This study demonstrates the associations of actin-binding proteins with the activity of proteasomes and calpain, which are specific for tumors and metastases of the mammary gland. We proposed a possible scheme of the relationship of intracellular systems with the actin-binding proteins. The results obtained expand the fundamental understanding of the processes of tumor progression and can also be used in the search for proteins-targets for therapeutic action in molecular targeted cancer therapy.

The Australian tree malletwood (Rhodamnia argentea) is unique. The genome of malletwood is the only known plant genome that contains a gene coding for an α‐actinin‐like protein. Several organisms predating the animal‐plant bifurcation express an α‐actinin or α‐actinin‐like protein. Therefore, it appears that plants in general, but not malletwood, have lost the α‐actinin or α‐actinin‐like gene during evolution. In order to characterize its structure and function, we synthesized the gene and expressed the recombinant R. argentea protein. The results clearly show that this protein has all properties of genuine α‐actinin. The N‐terminal actin‐binding domain (ABD), with two calponin homology motifs, is very similar to the ABD of any α‐actinin. The C‐terminal calmodulin‐like domain, as well as the intervening rod domain, are also similar to the corresponding regions in other α‐actinins. The R. argentea α‐actinin‐like protein dimerises in solution and thereby can cross‐link actin filaments. Based on these results, we believe the R. argentea protein represents a genuine α‐actinin, making R. argentea unique in the plant world., The Australian tree malletwood is so far the only known plant that expresses an alpha‐actinin or alpha‐actinin‐like protein. We show that the protein's N‐terminal actin‐binding domain (ABD) is functional and structurally very similar to other ABDs and that the C‐terminal calmodulin‐like domain contains two pairs of calcium‐insensitive EF‐hands. In the presence of filamentous, actin bundles or networks are formed.

The gene for galectin-13 (Gal-13, placental protein 13) is only present in primates, and its low expression level in maternal serum may promote preeclampsia. In the present study, we used pull-down experiments and biolayer interferometry to assess the interaction between Gal-13 and actin. These studies uncovered that human Gal-13 (hGal-13) and Saimiri boliviensis boliviensis (sGal-13) strongly bind to α- and β-/γ-actin, with Ca2+ and adenosine triphosphate, significantly enhancing the interactions. This in turn suggests that h/sGal-13 may inhibit myosin-induced contraction when vascular smooth muscle cells undergo polarization. Here, we solved the crystal structure of sGal-13 bound to lactose and found that it exists as a monomer in contrast to hGal-13 which is a dimer. The distribution of sGal-13 in HeLa cells is similar to that of hGal-13, indicating that monomeric Gal-13 is the primary form in cells. Even though sGal-13 binds to actin, hGal-13 ligand-binding site mutants do not influence hGal-13/actin binding, whereas the monomeric mutant C136S/C138S binds to actin more strongly than the wild-type hGal-13. Overall, our study demonstrates that monomeric Gal-13 binds to actin, an interaction that is independent of the galectin canonical ligand-binding site.

Background Lens morphogenesis, architecture, and clarity are known to be critically dependent on actin cytoskeleton organization and cell adhesive interactions. There is limited knowledge, however regarding the identity and role of key proteins regulating actin cytoskeletal organization in the lens. This study investigated the role of drebrin, a developmentally regulated actin-binding protein, in mouse lens development by generating and characterizing a conditional knockout (cKO) mouse model using the Cre-LoxP recombination approach. Results Drebrin E, a splice variant of DBN1 is a predominant isoform expressed in the mouse lens and exhibits a maturation-dependent downregulation. Drebrin co-distributes with actin in both epithelium and fibers. Conditional deficiency (both haploinsufficiency and complete absence) of drebrin results in disrupted lens morphogenesis leading to cataract and microphthalmia. The drebrin cKO lens reveals a dramatic decrease in epithelial height and width, E-cadherin, and proliferation, and increased apoptotic cell death and expression of α-smooth muscle actin, together with severely impaired fiber cell organization, polarity, and cell-cell adhesion. Conclusions This study demonstrates the requirement of drebrin in lens development and growth, with drebrin deficiency leading to impaired lens morphogenesis and microphthalmia. This article is protected by copyright. All rights reserved.

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. WH2 domains are short sequence motifs found in many different actin binding proteins including WASP family proteins which regulate the actin nucleating complex Arp2/3. In this study we reveal a phosphorylation site, Serine 554, within the WH2 domain of the yeast WASP homologue Las17. Both phosphorylation and a phospho-mimetic mutation reduce actin monomer binding affinity while an alanine mutation, generated to mimic the non-phosphorylated state, increases actin binding affinity. The effect of these mutations on the Las17-dependent process of endocytosis in vivo was analysed and leads us to propose that switching of Las17 phosphorylation states may allow progression through distinct phases of endocytosis from site assembly through to the final scission stage. While the study is focused on Las17, the sole WASP family protein in yeast, our results have broad implications for our understanding of how a key residue in this conserved motif can underpin the many different actin regulatory roles with which WH2 domains have been associated.

Through the process of chemotactic migration, neutrophils are able to recruit to an inflammatory site and eliminate pathogens, thus playing a vital role in host defense. The process of neutrophil chemotaxis is mediated by dynamic actin reorganization and polymerization. Adseverin, an actin-binding protein and member of the Gelsolin superfamily of proteins, has been hypothesized to regulate goal directed movement through the capping and severing of actin filaments, but has never been investigated in the context of neutrophil chemotaxis. Using an Adseverin knockout mouse model, we show that Adseverin plays a role in subcortical F-actin assembly at the leading edge during chemotaxis through the generation of free barbed ends on existing actin filaments. In addition, in the absence of Adseverin, neutrophils show reduced speed of migration in vitro and in vivo. This study indicates that Adseverin is a regulator of actin filament generation during neutrophil chemotaxis.

Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. The filamentous form of actin (F-actin) can be studied with help of natural products that specifically recognize it, as for example fluorophore-labeled probes of the bicyclic peptide phalloidin, but no synthetic probes exist for the monomeric form of actin (G-actin). Herein, we have panned a phage display library consisting of more than 10 billion bicyclic peptides against G-actin and isolated binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity to a region of thymosin-β4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between actin subdomains 1 and 3. Together with F-actin-specific peptides that we also isolated, we evaluated the G-actin peptides as probes in pull-down, imaging, and competition binding experiments. While the F-actin peptides were applied successfully for capturing actin in cell lysates and for imaging, the G-actin peptides did not bind in the cellular context, most likely due to competition with thymosin-β4 or related endogenous proteins for the same binding site.

Actin filament networks in eukaryotic cells are constantly remodeled through nucleotide state controlled interactions with actin binding proteins, leading to macroscopic structures such as bundled filaments, branched filaments, and so on. The nucleotide (ATP) hydrolysis, phosphate release, and polymerization/depolymerization reactions that lead to the formation of these structures are correlated with the conformational fluctuations of the actin subunits at the molecular scale. The resulting structures generate and experience varying levels of force and impart cells with several functionalities such as their ability to move, divide, transport cargo, etc. Models that explicitly connect the structure to reactions are essential to elucidate a fundamental level of understanding of these processes. In this regard, a bottom-up Ultra-Coarse-Grained (UCG) model of actin filaments that can simulate ATP hydrolysis, inorganic phosphate release (Pi), and depolymerization reactions is presented in this work. In this model, actin subunits are represented using coarse-grained particles that evolve in time and undergo reactions depending on the conformations sampled. The reactions are represented through state transitions, with each state represented by a unique effective coarse-grained potential. Effects of compressive and tensile strains on the rates of reactions are then analyzed. Compressive strains tend to unflatten the actin subunits, reduce the rate of ATP hydrolysis, and increase the Pi release rate. On the other hand, tensile strain flattens subunits, increases the rate of ATP hydrolysis, and decrease the Pi release rate. Incorporating these predictions into a Markov State Model highlighted that strains alter the steady-state distribution of subunits with ADPPi and ADP nucleotide, thus identifying possible additional factors underlying the cooperative binding of regulatory proteins to actin filaments.

The cellular cytoskeleton consisting of microtubules, intermediate filaments and the actin filaments is a dynamic structure providing shape and structural stability to cells. Particularly, the actin cytoskeleton formed by a combination of polymerized actin molecules and several other actin binding proteins including myosin is key to sensing and development of mechanical forces in cells. Given this and other features, the actin cytoskeleton has been implicated in a variety of cellular process including cellular motility and migration, cytokinesis, phagocytosis, cytoplasmic streaming, organelle transport, cellular transformation and metastasis, cellular metabolism, cell–matrix adhesion, and cell–cell adhesion. The latter is mediated by E-cadherin in the epithelial tissue and is fundamental to tissue morphogenesis and normal development. Here we discuss the role of the actin cytoskeleton in the assembly and maintenance of E-cadherin-based cell–cell adhesion through the formation of cellular appendages such as filopodia and lamellipodia and thus, impinging on one of the fundamental features of multicellular organisms.

Neural networks with precise connection are compulsory for learning and memory. Various cellular events occur during the genesis of dendritic spines to their maturation, synapse formation, stabilization of the synapse, and proper signal transmission. The cortical actin cytoskeleton and its multiple regulatory proteins are crucial for the above cellular events. The different types of ionotropic glutamate receptors (iGluRs) present on the postsynaptic density (PSD) are also essential for learning and memory. Interaction of the iGluRs in association of their auxiliary proteins with actin cytoskeleton regulated by actin-binding proteins (ABPs) are required for precise long-term potentiation (LTP) and long-term depression (LTD). There has been a quest to understand the mechanistic detail of synapse function involving these receptors with dynamic actin cytoskeleton. A major, emerging area of investigation is the relationship between ABPs and iGluRs in synapse development. In this review we have summarized the current understanding of iGluRs functioning with respect to the actin cytoskeleton, scaffolding proteins, and their regulators. The AMPA, NMDA, Delta and Kainate receptors need the stable underlying actin cytoskeleton to anchor through synaptic proteins for precise synapse formation. The different types of ABPs present in neurons play a critical role in dynamizing/stabilizing the actin cytoskeleton needed for iGluRs function., Graphical abstract Image 1, Highlights • Interaction of actin cytoskeleton with ionotropic glutamate receptors is prerequisite for memory formation. • Highly remodelled actin cytoskeleton is critical for AMPAR localization and functioning • Calcium influx and Rho GTPases modulate NMDAR activity through LIM kinases. • Delta receptors interacts with various actin-binding proteins like Delphilin, Spectrin, and nPIST. • iGluRs interacts with actin cytoskeleton through ABPs and auxiliary proteins.

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1‐Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three‐dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin‐binding sites of tropomodulin and generated a three‐dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self‐consistent three‐dimensional model of tropomodulin assembly at the pointed end. The model of the pointed‐end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

At the subcellular level, the cytoskeleton regulates cell structure, organelle movement, and cytoplasmic streaming. Autophagy is a process to remove unwanted biomaterials or damaged organelles through double membrane compartments known as autophagosomes. Autophagosome biogenesis requires vesicle trafficking between donor and acceptor compartments, membrane expansion, and fusion, which is very likely to be regulated by the cytoskeleton. Recent studies have demonstrated that by knocking out key actin-regulating proteins, autophagosome biogenesis is inhibited. However, the formation of ATG8 positive structures are not affected when the entire actin network is disrupted. Here, we discuss this paradox and propose the function of the actin cytoskeleton in plant autophagy.

Actin cytoskeleton is crucial to support spermatogenesis in the mammalian testis. However, the molecular mechanism(s) underlying changes of actin cytoskeletal organization in response to cellular events that take place across the seminiferous epithelium (e.g., self-renewal of spermatogonial stem cells, germ cell differentiation, meosis, spermiogenesis, spermiation) at specific stages of the epithelial cycle of spermatogenesis remain largely unexplored. This, at least in part, is due to the lack of suitable study models to identify the crucial regulatory proteins and to investigate how these proteins work in concert to support actin dynamics. Much of the information on the role of actin binding proteins in the literature, namely the actin bundling proteins, actin nucleation proteins and motor proteins, are either findings based on genetic models or morphological analyses. While this information is helpful to delineate the function of these proteins to support spermatogenesis, they are not helpful to identify the regulatory signaling proteins, the signaling pathways and the cascade of events to modulate actin cytoskeleton dynamics. Recent studies based on the use of toxicant models, both in vitro and in vivo, however, have bridged this gap by identifying putative regulatory and signaling proteins of actin cytoskeleton. Herein, we summarize and critically evaluate these findings. We also provide a hypothetical model by which actin cytoskeletal dynamics in Sertoli cells are regulated, which in turn supports spermatid transport across the seminiferous epithelium, and at the blood-testis barrier (BTB) during the epithelial cycle of spermatogenesis.

Tropomyosins are elongated alpha-helical proteins that form co-polymers with most actin filaments within a cell and play important roles in the structural and functional diversification of the actin cytoskeleton. How the assembly of tropomyosins along an actin filament is regulated and the kinetics of tropomyosin association with an actin filament is yet to be fully determined. A recent series of publications have used total internal reflection fluorescence (TIRF) microscopy in combination with advanced surface and protein chemistry to visualise the molecular assembly of actin/tropomyosin filaments in vitro. Here, we review the use of the in vitro TIRF assay in the determination of kinetic data on tropomyosin filament assembly. This sophisticated approach has enabled generation of real-time single-molecule data to fill the gap between in vitro bulk assays and in vivo assays of tropomyosin function. The in vitro TIRF assays provide a new foundation for future studies involving multiple actin-binding proteins that will more accurately reflect the physiological protein-protein interactions in cells.

Actin filaments (F-actin) are a key component of eukaryotic cells. Whether serving as a scaffold for myosin or using their polymerization to push onto cellular components, their function is always related to force generation. To control and fine-tune force production, cells have a large array of actin-binding proteins (ABPs) dedicated to control every aspect of actin polymerization, filament localization, and their overall mechanical properties. Although great advances have been made in our biochemical understanding of the remodeling of the actin cytoskeleton, the structural basis of this process is still being deciphered. In this review, we summarize our current understanding of this process. We outline how ABPs control the nucleation and disassembly, and how these processes are affected by the nucleotide state of the filaments. In addition, we highlight recent advances in the understanding of actomyosin force generation, and describe recent advances brought forward by the developments of electron cryomicroscopy.

Although best known from its functions in the cytoplasm, actin also localizes to the cell nucleus, where it has been linked to many essential functions from regulation of gene expression to maintenance of genomic integrity. While majority of cytoplasmic functions of actin depend on controlled actin polymerization, in the nucleus both actin monomers and filaments have their own specific roles. Actin monomers are core components of several chromatin remodeling and modifying complexes and can also regulate the activity of specific transcription factors, while actin filaments have been linked to DNA damage response and cell cycle progression. Consequently the balance between monomeric and filamentous actin must be precise controlled also in the nucleus, since their effects are dynamically coupled. In this review, we discuss the recent data on how actin dynamics is regulated within the nucleus and how this influences the different nuclear processes dependent on actin.

The dynamics of actin networks is modulated by a machinery consisting of actin binding proteins that control the turnover of filaments in space and time. To study this complex orchestration, in vitro reconstitution approaches strive to project actin dynamics in ideal, minimal systems. To this extent we reconstitute a self-supplying, dense network of globally treadmilling filaments. In this system we analyze growth and intrinsic turnover by means of FRAP measurements and thereby demonstrate how the depletion of monomers and actin binding partners modulate the dynamics in active actin networks. The described effects occur only in dense networks, as single filament dynamics are unable to produce depletion effects to this extent. Furthermore, we demonstrate a synergistic relationship between the nucleators formin and Arp2/3 when branched networks and formin-induced networks are colocalized. As a result, the formin-enhanced filament turnover depletes cofilin at the surface and thus protects the dense, Arp2/3 polymerized network from debranching. Ultimately, these results may be key for understanding the maintenance of the two contradicting requirements of network stability and dynamics in cells.

Synapses are enriched in the cytoskeletal protein actin, which determines the shape of the pre- and post-synaptic compartments, organizes the neurotransmitter release machinery, and provides a framework for trafficking of components. In the post-synaptic compartment, interactions with actin or its associated proteins are also critical for the localization and activity of synaptic neurotransmitter receptors and ion channels. Actin binding proteins, including spectrin and alpha-actinin, serve as molecular linkages between the actin cytoskeleton and a diverse collection of receptors, including the NMDA receptor (NMDAR) and voltage gated sodium channels. The actin cytoskeleton can regulate neurotransmitter receptors and ion channels by controlling their trafficking and localization at the synapse and by directly gating receptor channel opening. We highlight evidence that synaptic actin couples physically and functionally to NMDAR and supports its activity. The molecular mechanisms by which actin regulates NMDARs are only just emerging, and recent advancements in light and electron microscopy-based imaging techniques should aide in elucidating these mechanisms.

The actin-binding proteins profilin, fascin, and ezrin were tested for involvement in metastasis of non-small cell lung cancer (NSCLC). The levels of the PFN1, FSCN1, and EZR mRNAs and respective proteins were determined by real-time PCR and Western blotting; tumor and adjacent normal lung tissue samples were obtained from 46 NSCLC patients. Patients with lymphatic metastasis had higher expression levels of the profilin, fascin, and ezrin mRNAs and the profilin and fascin proteins. Both mRNA and protein expression levels increased in patients with distant metastasis. The molecules may serve as predictors to evaluate the prognosis in NSCLC.

Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the β-subunit is downregulated,. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.

Gelsolin, encoded by the Gsn gene, is a highly conserved actin-binding protein detected in all studied eukaryotic organisms. Gelsolin is the founding member of the gelsolin superfamily of proteins, whose main functions are binding, severing, and capping actin filaments. Moreover, gelsolin is engaged in apoptosis regulation, signal transduction, and embryonic development. Gelsolin is also implicated in some human diseases such as Alzheimer disease and familial amyloidosis of the Finnish type (FAF). Because of its multifunctionality, gelsolin is a potential candidate for diverse therapeutic applications. Most of our knowledge about gelsolin superfamily proteins comes from investigations conducted on vertebrate model organisms such as zebrafish (Danio rerio), African clawed frogs (Xenopus laevis), chickens (Gallus gallus), and mice (Mus musculus). Here, we present fundamental findings as well as new knowledge to indicate main research directions and future possibilities. Different model organisms provide specific possibilities and present particular advantages. However, all of them give useful data, which allow us to gain a better understanding of the function of gelsolin and related proteins.

Primary root growth is required by the plant to anchor in the soil and reach out for nutrients and water, while dealing with obstacles. Efficient root elongation and bending depends upon the coordinated action of environmental sensing, signal transduction, and growth responses. The actin cytoskeleton is a highly plastic network that constitutes a point of integration for environmental stimuli and hormonal pathways. In this review, we present a detailed compilation highlighting the importance of the actin cytoskeleton during primary root growth and we describe how actin-binding proteins, plant hormones, and actin-disrupting drugs affect root growth and root actin. We also discuss the feedback loop between actin and root responses to light and gravity. Actin affects cell division and elongation through the control of its own organization. We remark upon the importance of longitudinally oriented actin bundles as a hallmark of cell elongation as well as the role of the actin cytoskeleton in protein trafficking and vacuolar reshaping during this process. The actin network is shaped by a plethora of actin-binding proteins; however, there is still a large gap in connecting the molecular function of these proteins with their developmental effects. Here, we summarize their function and known effects on primary root growth with a focus on their high level of specialization. Light and gravity are key factors that help us understand root growth directionality. The response of the root to gravity relies on hormonal, particularly auxin, homeostasis, and the actin cytoskeleton. Actin is necessary for the perception of the gravity stimulus via the repositioning of sedimenting statoliths, but it is also involved in mediating the growth response via the trafficking of auxin transporters and cell elongation. Furthermore, auxin and auxin analogs can affect the composition of the actin network, indicating a potential feedback loop. Light, in its turn, affects actin organization and hence, root growth, although its precise role remains largely unknown. Recently, fundamental studies with the latest techniques have given us more in-depth knowledge of the role and organization of actin in the coordination of root growth; however, there remains a lot to discover, especially in how actin organization helps cell shaping, and therefore root growth.

Phagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. However, how CR3-mediated phagosome completion and closure are orchestrated is not known. Here, we reveal that dynamin-2 is recruited concomitantly with polymerised actin at the site of the nascent phagosomes and accumulates until membrane scission. Inhibition of dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. Acute inhibition of dynamin activity in living phagocytosing cells established that dynamin-2 plays a critical role in the effective scission of the CR3-phagosome from the plasma membrane. Thus, dynamin-2 has two distinct roles in CR3-mediated phagocytosis, in the assembly of the F-actin phagocytic cup and during phagosome scission.

Simple Summary Although actin is a highly conserved protein, it is involved in many diverse cellular processes. Actin owes its diversity of function to its ability to bind to a host of actin-binding proteins (ABPs) that localize across its surface. Among the most studied ABPs is the molecular motor, myosin. Myosin generates force on actin filaments by pairing ATP hydrolysis, product release, and actin-binding to the conformational changes that lead to movement. Central to this process is the progression of myosin binding to the actin surface as it moves through its ATPase cycle. During binding, actin acts as a myosin ATPase activator, catalyzing essential hydrolysis release steps. Here, we use the current model of actin-myosin binding as a roadmap to describe the portions of the actin-myosin interface that are sequentially formed throughout the motor cycle. At each step, we compare the interactions of a diverse set of high-resolution actin-myosin cryo-electron microscopy structures to define what portions of the interface are conserved and which are isoform-specific. Abstract Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

Actin is an important unit of the cytoskeletal system, involved in many cellular processes including cell motility, signaling, and intracellular trafficking. Various studies have been undertaken to understand the regulatory mechanisms pertaining actin functions, especially the ones controlled by actin-binding proteins. However, not much has been explored about the molecular aspects of these proteins implicated in various diseases. In this study, we aimed to demonstrate the molecular properties of gelsolin, an actin-severing protein on the disassembly of the aggregation of actin-rich intracellular inclusions, Hirano body. We observed a decreasing tendency of actin aggregation by co-sedimentation assay and transmission electron microscopy in the presence of gelsolin. Therefore, we provide suggestive evidence for the use of actin-severing protein in novel therapeutic strategies for neurodegenerative conditions.

Using Drosophila actin-binding protein Dunc-115 as model system, this chapter describes a MARCM (mosaic analysis with a repressible cell marker)-based method for analyzing cytoskeletal components for their functions in the nervous system. Following a concise description about the principle, a step-by-step protocol is provided for generating the needed stocks and for histological analysis. Additional details and explanations have been given in the accompanying notes. Together, this should form a practical and sufficient recipe for performing at the single-cell-level loss-of-function and gain-of-function analyses of proteins associated with the cytoskeleton.

Το ουροθηλιακό καρκίνωμα της ουροδόχου κύστης αποτελεί την ένατη σε συχνότητα κακοήθεια παγκοσμίως με κίνδυνο διπλάσιο για τους άνδρες συγκριτικά με τις γυναίκες. Στην παθογένειά του εμπλέκονται δύο διακριτά βιολογικά μονοπάτια: από το φυσιολογικό ουροθήλιο η νεοπλασματική διεργασία μέσω in situ καρκινώματος καταλήγει σε μυοδιηθητικό ουροθηλιακό καρκίνωμα, ενώ μέσω υπερπλασίας καταλήγει σε χαμηλού βαθμού κακοήθειας ουροθηλιακό νεόπλασμα, με διακριτά μοριακά χαρακτηριστικά για την κάθε οδό. Στην περίπτωση χαμηλού βαθμού κακοήθειας καρκινωμάτων εμπλέκονται μεταλλάξεις των HRAS και FGFR, ενώ στα μυοδιηθητικά καρκινώματα εμπλέκονται κυρίως μεταλλάξεις των ογκογονιδίων p53 και Rb. Η πρόγνωση είναι ανάλογη του βαθμού διαφοροποίησης και του σταδίου, με τα μυοδιηθητικά καρκινώματα να υποτροπιάζουν και να παρουσιάζουν χαμηλό ποσοστό πενταετούς επιβίωσης (5%).Ο κυτταροσκελετός ακτίνης αποτελεί μια δυναμική δομή αποτελούμενη από νημάτια πολυμερισμένης σφαιρικής ακτίνης καθώς και από πλήθος ρυθμιστικών πρωτεϊνών. Ο κυτταροσκελετός ακτίνης έχει σημαντικές λειτουργίες καθώς παρέχει στο κύτταρο δυνατότητα κίνησης, επικοινωνίας-πρόσφυσης στην εξωκυττάρια ουσία, διατήρησης του σχήματος και της πολικότητάς του, εξωκυττάρωσης και ενδοκυττάρωσης. Ο πολυμερισμός ακτίνης με τη βοήθεια των πρωτεϊνών που δεσμεύονται σε αυτή , έχει σαν αποτέλεσμα τη δημιουργία προεξεχόντων δομών της κυτταρικής μεμβράνης, όπως ελασματοπόδια και πόδια διήθησης, τα οποία προσδίδουν διηθητικό δυναμικό στο κύτταρο. Επιπλέον, η αναδιαμόρφωση του κυτταροσκελετού ακτίνης είναι απολύτως απαραίτητη για την απόκτηση διηθητικής συμπεριφοράς του κυττάρου και συμμετέχει στην επιθηλιομεσεγχυματική μετατροπή με το σχηματισμό ψευδοπόδιων.Σκοπός της παρούσας μελέτης είναι να εξετάσουμε την έκφραση πρωτεινών που αλληλεπιδρούν με τον κυτταροσκελετό ακτίνης στα ουροθηλιακά καρκινώματα της ουροδόχου κύστης σε σχέση με παθολογονατομικές παραμέτρους ενδεικτικές της βιολογικής συμπεριφοράς αυτών των νεοπλασμάτων. Συνεπώς, μελετήσαμε την έκφραση των πρωτεινών εζρίνης, παξιλλίνης και κορτακτίνης σε 104 ουροθηλιακά νεοπλάσματα ουροδόχου κύστης με την μέθοδο της ανοσοιστοχημείας και εξετάσαμε την πιθανή συσχέτιση των πρωτεϊνών αυτών με παθολογοανατομικές παραμέτρους της νόσου όπως η διήθηση του μυϊκού χιτώνα καθώς και την σχέση αυτών των πρωτεινών τόσο μεταξύ τους, όσο και με τους παράγοντες επιθήλιο-μεσεγχυματικής μετατροπής E-καντχερίνη, β-κατενίνη και τον προγνωστικό δείκτη p53. Η μειωμένη ανοσοϊστοχημική έκφραση της εζρίνης σχετίστηκε με επιθετική βιολογική συμπεριφορά και επιπλέον η πολυπαραγοντική ανάλυση έδειξε ότι η μεμβρανική ανοοσοϊστοχημική έκφρασή της αποτελεί ανεξάρτητο προβλεπτικό παράγοντα της διήθησης του μυϊκού χιτώνα στα ουροθηλιακά καρκινώματα της ουροδόχου κύστης. Η μειωμένη ανοσοϊστοχημική έκφραση της παξιλλίνης σχετίστηκε με διηθητική συμπεριφορά, ενώ η ανοσοϊστοχημική έκφραση της κορτακτίνης ήταν μεγαλύτερη στα ουροθηλιακά καρκινώματα της ουροδόχου κύστης και σχετίστηκε με επιθετική βιολογική συμπεριφορά. Επιπλέον, η πολυπαραγοντική ανάλυση έδειξε ότι η ότι η μεμβρανική και κυτταροπλασματική έκφραση της κορτακτίνης αποτελεί ανεξάρτητο προβλεπτικό παράγοντα τουβαθμού διαφοροποίησης. του σταδίου pT και της διήθησης. Επίσης, η στατιστική ανάλυση δεν ανέδειξε καμία στατιστικώς σημαντική διαφορά στην έκφραση της Ε-κατχερίνης σε σχέση με τις παθολογοανατομικές παραμέτρους που εξετάσθηκαν, ενώ ανέδειξε στατιστικώς σημαντική διαφορά στην μεμβρανική έκφραση της β-κατενίνης σε σχέση με το βαθμό διαφοροποίησης, την παρουσία διήθησης, την διήθηση του μυϊκού χιτώνα και το στάδιο της νόσου. Επίσης, παρατηρήθηκε στατιστικώς σημαντική διαφορά στην έκφραση του p53 σε σχέση με την παρουσία διήθησης καθώς και με το στάδιο της νόσου. Τέλος, η ανοσοϊστοχημική έκφραση της εζρίνης βρέθηκε να σχετίζεται στατιστικώς σημαντικά με την έκφραση της Ε-κατχερίνης και την έκφραση της παξιλλίνης, ενώ η ανοσοϊστοχημική έκφραση της παξιλλίνης και της κορτακτίνης βρέθηκε να σχετίζεται με την έκφραση της πρωτεΐνης p53.Συμπερασματικά, η μειωμένη έκφραση της εζρίνης και της παξιλλίνης και η αυξημένη έκφραση της κορτακτίνης σχετίζεται με επιθετικά χαρακτηριστικά των ουροθηλιακών καρκινωμάτων της ουροδόχου κύστης του ανθρώπου. Μία πιθανή εξήγηση της συμμετοχής της εζρίνης στην απόκτηση διηθητικού δυναμικού των ουροθηλιακών κακρινικών κυττάρων, είναι η μειωμένη κυτταρική συνοχή, όπως αποτυπώνεται από τη σχέση της χαμηλής έκφρασης της εζρίνης με τη χαμηλή επίσης έκφραση της E-καντχερίνης. Επιπλέον, η ισχυρή συσχέτιση της έκφρασης της εζρίνης με αυτή της παξιλλίνης στη μελέτη αυτή, προτείνει μια πιθανή σηματοδοτική συνεργασία μεταξύ των πρωτεϊνών που ρυθμίζουν την F-ακτίνη, κατά τη διαδικασία της διήθησης στον καρκίνο της ουροδόχου κύστης. Τέλος, τα παραπάνω μόρια, θα μπορούσαν ενδεχομένως να χρησιμοποιηθούν μελλοντικά ως βιοδείκτες στα ουροθηλιακά καρκινώματα, προσδιορίζοντας τους ασθενείς εκείνους που έχουν τάση υποτροπής, εξέλιξης και μετάστασης της νόσου.

A cell should deal with the changing external environment or the neighboring cells. Inevitably, the cell surface receives and transduces a number of signals to produce apt responses. Typically, cell surface receptors are activated, and during this process, the subplasmalemmal actin cytoskeleton is often rearranged. An intriguing point is that some signaling enzymes and ion channels are physically associated with the actin cytoskeleton, raising the possibility that the subtle changes of the local actin cytoskeleton can, in turn, modulate the activities of these proteins. In this study, we reviewed the early and new experimental evidence supporting the notion of actin-regulated enzyme and ion channel activities in various cell types including the cells of immune response, neurons, oocytes, hepatocytes, and epithelial cells, with a special emphasis on the Ca2+ signaling pathway that depends on the synthesis of inositol 1,4,5-trisphosphate. Some of the features that are commonly found in diverse cells from a wide spectrum of the animal species suggest that fine-tuning of the activities of the enzymes and ion channels by the actin cytoskeleton may be an important strategy to inhibit or enhance the function of these signaling proteins.

The blood–testis barrier (BTB) and apical ectoplasmic specialization (ES), which are synchronized through the crosstalk of Sertoli cells and Sertoli germ cells, are required for spermatogenesis and sperm release. Here, we show that Wnt5a, a noncanonical Wnt signaling pathway ligand, is predominately expressed in both the BTB and apical ES and has a specific expression pattern during the seminiferous epithelium cycle. We employed siRNA to knockdown Wnt5a expression in testis and Sertoli cells, and then identified elongated spermatids that lost their polarity and were embedded in the seminiferous epithelium. Moreover, phagosomes were found near the tubule lumen. These defects were due to BTB and apical ES disruption. We also verified that the expression level and/or location of BTB-associated proteins, actin binding proteins (ABPs), and F-actin was changed after Wnt5a knockdown in vivo and in vitro. Additionally, we demonstrated that Wnt5a regulated actin dynamics through Ror2-mediated mTORC1 and mTORC2. This study clarified the molecular mechanism of Wnt5a in Sertoli cell junctions through the planar cell polarity (PCP) signaling pathway. Our findings could provide an experimental basis for the clinical diagnosis and treatment of male infertility caused by Sertoli cell junction impairment.

Postsynaptic structures on excitatory neurons, dendritic spines, are actin-rich. It is well known that actin-binding proteins regulate actin dynamics and by this means orchestrate structural plasticity during the development of the brain, as well as synaptic plasticity mediating learning and memory processes. The actin-binding protein cortactin is localized to pre- and postsynaptic structures and translocates in a stimulus-dependent manner between spines and the dendritic compartment, thereby indicating a crucial role for synaptic plasticity and neuronal function. While it is known that cortactin directly binds F-actin, the Arp2/3 complex important for actin nucleation and branching as well as other factors involved in synaptic plasticity processes, its precise role in modulating actin remodeling in neurons needs to be deciphered. In this study, we characterized the general neuronal function of cortactin in knockout mice. Interestingly, we found that the loss of cortactin leads to deficits in hippocampus-dependent spatial memory formation. This impairment is correlated with a prominent dysregulation of functional and structural plasticity. Additional evidence shows impaired long-term potentiation in cortactin knockout mice together with a complete absence of structural spine plasticity. These phenotypes might at least in part be explained by alterations in the activity-dependent modulation of synaptic actin in cortactin-deficient neurons.

Highlights • Early aberrant expression of actin subunits can serve as a biomarker for neoplastic transformation. • Abnormal actin isoform expression can lead to increased migration, cell proliferation and drug resistance. • Actin can serve as a potential therapeutic target for chemotherapy and also as an indicator for the efficacy of currently used chemotherapeutic agents., Actin is a key structural protein that makes up the cytoskeleton of cells, and plays a role in functions such as division, migration, and vesicle trafficking. It comprises six different cell-type specific isoforms: ACTA1, ACTA2, ACTB, ACTC1, ACTG1, and ACTG2. Abnormal actin isoform expression has been reported in many cancers, which led us to hypothesize that it may serve as an early biomarker of cancer. We show an overview of the different actin isoforms and highlight mechanisms by which they may contribute to tumorigenicity. Furthermore, we suggest how the aberrant expression of actin subunits can confer cells with greater proliferation ability, increased migratory capability, and chemoresistance through incorporation into the normal cellular F-actin network and altered actin binding protein interaction. Studying this fundamental change that takes place within cancer cells can further our understanding of neoplastic transformation in multiple tissue types, which can ultimately aid in the early-detection, diagnosis and treatment of cancer., Graphical abstract Image, graphical abstract

Cytoskeleton in Eukaryotic cell is very important for cell to keep the exact shape and supports the plasma membrane and give the mechanical support to cell. Actin is a type of cytoskeleton that is involved in several functions. Actin filaments are made up of indistinguishable actin binding proteins which is set in a lengthy curved chain which performs important function of body. Expresion of some actin binding proteins are regulated during carcinogenesis and metastasis, these proteins include Arp2/3 (Actin related protein 2 and 3 complex), fascin and the tropomyosin’s, WASP/WAVE (Wiskott-Aldrich syndrome protein/WASP and Verprolin homologous protein) complexes. The expansion of cancer cells from the primary tumor to other tissues and the organs is called metastasis. The connection amongst the hyperactivated signaling pathways plays a very important role in the development of metastatic. GPCRs (G Protein Coupled Receptors) through β-arrs (β-arrestins) act as a intracellular signaling particles. In tumor cells the increased and decreased expression of these actin binding proteins are involved in progression of different types of cancer and metastasis. For further insight we have to understand what is actin and the function of actin binding proteins.

Palani et al. reveal a new mechanism by which the F-actin binding protein tropomyosin is regulated. They find that phosphorylation of tropomyosin reduces its affinity for F-actin, allowing the competing Adf1 to bind and sever actin filaments., Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin-severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.

Persistent transcriptional and morphological events in the nucleus accumbens (NAc) and other brain reward regions contribute to the long-lasting behavioral adaptations that characterize drug addiction. Opiate exposure reduces the density of dendritic spines on medium spiny neurons of the NAc; however, the underlying transcriptional and cellular events mediating this remain unknown. We show that heroin self-administration negatively regulates the actin-binding protein drebrin in the NAc. Using virus-mediated gene transfer, we show that drebrin overexpression in the NAc is sufficient to decrease drug seeking and increase dendritic spine density, whereas drebrin knockdown potentiates these effects. We demonstrate that drebrin is transcriptionally repressed by the histone modifier HDAC2, which is relieved by pharmacological inhibition of histone deacetylases. Importantly, we demonstrate that heroin-induced adaptations occur only in the D1+ subset of medium spiny neurons. These findings establish an essential role for drebrin, and upstream transcriptional regulator HDAC2, in opiate-induced plasticity in the NAc., The underlying transcriptional and cellular events mediating the reduction of dendritic spines on medium spiny neurons of the nucleus accumbens (NAc) remains unknown. Here, authors demonstrate that heroin self-administration negatively regulates the actin-binding protein drebrin in the NAc, which is shown to be transcriptionally repressed by the histone modifier HDAC2, and that overexpression of drebrin is sufficient to decrease drug seeking and increase dendritic spine density

Accreta and gestational trophoblastic disease (ie, choriocarcinoma) are placental pathologies characterized by hyperproliferative and invasive trophoblasts. Cellular proliferation, migration, and invasion are heavily controlled by actin-binding protein (ABP)-mediated actin dynamics. The ABP vasodilator-stimulated phosphoprotein (VASP) carries key regulatory role. Profilin-1, cofilin-1, and VASP phosphorylated at Ser157 (pVASP-S157) and Ser239 (pVASP-S239) are ABPs that regulate actin polymerization and stabilization and facilitate cell metastases. Docosahexaenoic acid (DHA) inhibits cancer cell migration and proliferation. We hypothesized that analogous to malignant cells, ABPs regulate these processes in extravillous trophoblasts (EVTs), which exhibit aberrant expression in placenta accreta. Placental–myometrial junction biopsies of histologically confirmed placenta accreta had significantly increased immunostaining levels of cofilin-1, VASP, pVASP-S239, and F-actin. Treatment of choriocarcinoma-derived trophoblast (BeWo) cells with DHA (30 µM) for 24 hours significantly suppressed proliferation, migration, and pVASP-S239 levels and altered protein profiles consistent with increased apoptosis. We concluded that in accreta changes in the ABP expression profile were a response to restore homeostasis by counteracting the hyperproliferative and invasive phenotype of the EVT. The observed association between VASP phosphorylation, apoptosis, and trophoblast proliferation and migration suggest that DHA may offer a therapeutic solution for conditions where EVT is hyperinvasive.

The interactions of cytoskeletal actin filaments with myosin family motors are essential for the integrity and function of eukaryotic cells. They support a wide range of force-dependent functions. These include mechano-transduction, directed transcellular transport processes, barrier functions, cytokinesis, and cell migration. Despite the indispensable role of tropomyosins in the generation and maintenance of discrete actomyosin-based structures, the contribution of individual cytoskeletal tropomyosin isoforms to the structural and functional diversification of the actin cytoskeleton remains a work in progress. Here, we review processes that contribute to the dynamic sorting and targeted distribution of tropomyosin isoforms in the formation of discrete actomyosin-based structures in animal cells and their effects on actin-based motility and contractility.

ObjectiveTo investigate the role of microRNA-486-5p (miR-486-5p) in nonsmall-cell lung cancer (NSCLC) resistance to cisplatin.MethodsThis retrospective study examined tumours and normal lung tissues from patients with NSCLC. The levels of miR-486-5p in NSCLC and normal tissues were determined using reverse transcription–polymerase chain reaction. The binding site of miR-486-5p on twinfilin actin binding protein 1 (TWF1) mRNA was predicted using TargetScan and investigated using a luciferase reporter gene assay. Cytotoxicity assays were used to measure the sensitivity of A549 cells to cisplatin. Western blotting was used to measure the levels of specific proteins. The role of miR-486-5p in the resistance of A549 to cisplatin was verified in vivo using a nude mouse tumorigenicity assay.ResultsMiR-486-5p levels were downregulated in NSCLC tissues compared with normal lung tissues. Lower levels of miR-486-5p were associated with reduced overall survival of patients with NSCLC. The cisplatin-resistant NSCLC cell line, A549/DDP, had lower miR-486-5p levels compared with A549 cells. Luciferase reporter gene assays confirmed that miR-486-5p bound to the 3ʹ untranslated region of TWF1 mRNA. In vivo experiments demonstrated the inhibitory effect of miR-486-5p on chemotherapy resistance.ConclusionMiR-486-5p appears to play an important role in improving chemotherapy sensitivity to cisplatin.

The genome of Trypanosoma cruzi encodes for an expanded number of actins, myosins and actin binding proteins compared to Trypanosoma brucei or Leishmania spp. In T. cruzi only the expression of actin 1 (i.e. conventional actin) and profilin, an actin binding protein, has been described. In this work, the expression of a kinetoplastid-specific actin, named actin 2 (TcAct2; TriTryp Gene ID: TcCLB.507129.10) was characterized in different developmental stages of T. cruzi. With the aid of a polyclonal antibody, we showed that TcAct2 is expressed throughout the life cycle of the parasite. Detergent fractionation of epimastigote extracts showed that this protein is cytosolic and is not associated with membrane or cytoskeletal fractions. The protein is localized along the cellular body and the flagellum in all parasite stages with a fine granular pattern and does not co-localize with actin 1. 2DE-immunoblotting studies demonstrated the presence of several variants of each actin. We also demonstrate that TcAct1 and TcAct2 have distinct subcellular distributions suggesting differential functions in this organism. The search of TcAct2 orthologues in the TriTrypDB, allowed the identification of this gene in other trypanosomatids, all of them restricted to the stercorarian clade. In addition, TcAct2 was also identified in the closely related non-trypanosomatid species Bodo saltans. Our findings are consistent with the appearance of a complex actin system early in the evolution of kinetoplastids.

Oury et al. show that the scaffolding protein MACF1 links Rapsyn, which binds acetylcholine receptors, to the microtubule- and actin-network at neuromuscular synapses. MACF1 thereby plays a role in synaptic maturation in mice, and mutations of MACF1 are associated with congenital myasthenia in humans., Complex mechanisms are required to form neuromuscular synapses, direct their subsequent maturation, and maintain the synapse throughout life. Transcriptional and post-translational pathways play important roles in synaptic differentiation and direct the accumulation of the neurotransmitter receptors, acetylcholine receptors (AChRs), to the postsynaptic membrane, ensuring for reliable synaptic transmission. Rapsyn, an intracellular peripheral membrane protein that binds AChRs, is essential for synaptic differentiation, but how Rapsyn acts is poorly understood. We screened for proteins that coisolate with AChRs in a Rapsyn-dependent manner and show that microtubule actin cross linking factor 1 (MACF1), a scaffolding protein with binding sites for microtubules (MT) and actin, is concentrated at neuromuscular synapses, where it binds Rapsyn and serves as a synaptic organizer for MT-associated proteins, EB1 and MAP1b, and the actin-associated protein, Vinculin. MACF1 plays an important role in maintaining synaptic differentiation and efficient synaptic transmission in mice, and variants in MACF1 are associated with congenital myasthenia in humans.

Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer’s disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions., Drebrin is an actin-binding protein known to play a role in neuronal dendritic spines but its precise regulation is unclear. Here, the authors report that DBN is activated by oxidative stress in an ATM-kinase dependent manner and increases resistance to oxidative stress in mice and in C. elegans.

Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin-binding protein, the state of the actin cytoskeleton and MF myosin activity.

The cytoskeletal protein actin is highly abundant and conserved in eukaryotic cells. It occurs in two different states- the globular (G-actin) form, which can polymerise into the filamentous (F-actin) form, fulfilling various critical functions including cytokinesis, cargo trafficking and cellular motility. In higher eukaryotes, there are several actin isoforms with nearly identical amino acid sequences. Despite the high level of amino acid identity, they display regulated expression patterns and unique non-redundant roles. The number of actin isoforms together with conserved sequences may reflect the selective pressure exerted by scores of actin binding proteins (ABPs) in higher eukaryotes. In contrast, in many protozoans such as apicomplexan parasites which possess only a few ABPs, the regulatory control of actin and its multiple functions are still obscure. Here, we provide a summary of the regulation and biological functions of actin in higher eukaryotes and compare it with the current knowledge in apicomplexans. We discuss future experiments that will help us understand the multiple, critical roles of this fascinating system in apicomplexans.

Pulmonary microvascular barrier dysfunction is a hallmark feature of acute lung injury (ALI). IQGAP1 is a ubiquitously expressed scaffolding protein known to regulate cancer metastasis, angiogenesis, and barrier stability. However, the function of IQGAP1 in lipopolysaccharide (LPS)-induced microvascular endothelial hyperpermeability remains poorly understood. In the present study, we demonstrated that IQGAP1 was markedly upregulated in LPS-induced ALI models and rat pulmonary microvascular endothelial cells (RPMVECs). Lentivirus-mediated knockdown of IQGAP1 significantly attenuated the formation of actin stress fibers, phosphorylation of myosin light chain (MLC), and disruption of VE-cadherin, thereby protecting the RPMVECs barrier failure from LPS damage. In addition, IQGAP1 depletion reduced the reactive oxygen species (ROS)-mediated increase in intracellular adhesion molecule-1 (ICAM-1) in RPMVECs stimulated with LPS. Mechanistically, we found that the upregulation of IQGAP1 affected the activity of Rap1 and the downstream phosphorylation of Src. In conclusion, these findings reveal an essential mechanism by which increased IQGAP1 in LPS-treated RPMVECs promotes barrier dysfunction and ICAM-1 upregulation, at least in part by regulating Rap1/Src signalling, indicating that IQGAP1 may be a potential therapeutic target to prevent endothelial hyperpermeability and inflammation in ALI.

Thymosin β4 (Tβ4) is a highly conserved actin binding protein associated with the metastatic potential of tumor cells by stimulating cell migration. The role of Tβ4 and its derived fragment peptides in migration of ovarian cancer cells has not been studied. To analyze the effects of Tβ4 and its derived fragment peptides on ovarian cancer cell migration and invasion, we applied Tβ4 and three Tβ4-derived synthetic peptides to SKOV3 ovarian cancer cells. The migration and invasion of SKOV3 cells treated with Tβ4(1–43), Tβ4(1–15), Tβ4(12–26), Tβ4(23–), and untreated control were analyzed by in vitro migration and invasion assay with transwell plate. Cell proliferation assay was conducted to identify the effect of Tβ4 and its derived peptide on SKOV3 cell proliferation. The expression of Tβ4 related proteins related with cell proliferation was analyzed by Western blot after treatment with Tβ4 and its derived peptides. Cell migration and invasion were significantly increased in Tβ4 peptide-treated SKOV3 cells compared with untreated control. All three Tβ4-derived fragment peptides including those without an actin binding site significantly stimulated migration and invasion of SKOV3 cells. Tβ4 and its derived peptide significantly stimulated SKOV3 cell proliferation and up-regulated the expression of RACK-1 protein. The Tβ4 peptide and all of its derived fragment peptides including those without an actin binding motif stimulate migration and invasion of SKOV3 ovarian cancer cells. All peptides significantly increased RACK-1 expression and cell proliferation of SKOV3 cells. These results suggest that Tβ4 stimulates migration and invasion of SKOV3 cells by stimulation of cell proliferation through up-regulation of RACK-1 protein.