Function Of Actin-binding Proteins Research Articles

Functions of actin-binding proteins in cilia structure remodeling and signaling.

Cilia are microtubule-based organelles found on the surfaces of many types of cells, including cardiac fibroblasts, vascular endothelial cells, human retinal pigmented epithelial-1 (RPE-1) cells, and alveolar epithelial cells. These organelles can be classified as immotile cilia, referred to as primary cilia in mammalian cells, and motile cilia. Primary cilia are cellular sensors that detect extracellular signals; this is a critical function associated with ciliopathies, which are characterized by the typical clinical features of developmental disorders. Cilia are extensively studied organelles of the microtubule cytoskeleton. However, the ciliary actin cytoskeleton has rarely been studied. Clear evidence has shown that highly regulated actin cytoskeleton dynamics contribute to normal ciliary function. Actin-binding proteins (ABPs) play vital roles in filamentous actin (F-actin) morphology. Here, we discuss recent progress in understanding the roles of ABPs in ciliary structural remodeling and further downstream ciliary signaling with a focus on the molecular mechanisms underlying actin cytoskeleton-related ciliopathies.

Actin's C-terminus coordinates actin structural changes and functions.

Actin is essential to eukaryotic cellular processes. Actin's C-terminus appears to play a direct role in modulating actin's structure and properties, facilitating the binding and function of actin-binding proteins (ABPs). The structural and functional characterization of filamentous actin's C-terminus has been impeded by its inherent flexibility, as well as actin's resistance to crystallization for x-ray diffraction and the historical resolution constraints associated with electron microscopy. Many biochemical studies have established that actin's C-terminus must retain its flexibility and structural integrity to modulate actin's structure and functions. For example, C-terminal structural changes are known to affect nucleotide binding and exchange, as well as propagate actin structural changes throughout extensive allosteric networks, facilitating the binding and function of ABPs. Advances in electron microscopy have resulted in high-resolution structures of filamentous actin, providing insights into subtle structural changes that are mediated by actin's C-terminus. Here, we review existing knowledge establishing the importance of actin's C-terminus within actin structural changes and functions and discuss how modern structural characterization techniques provide the tools to understand the role of actin's C-terminus in cellular processes.

A Ciliary Branched Actin Network Drives Photoreceptor Disc Morphogenesis.

The light-detecting organelle of the photoreceptor cell is a modified primary cilium, called the outer segment. The outer segment houses hundreds of light-sensitive membrane, "discs," that are continuously renewed by the constant formation of new discs at the outer segment base and the phagocytosis of old ones from outer segment tips by the retinal pigment epithelium. In this chapter, we describe how an actin cytoskeleton network, residing precisely at the site of disc formation, provides the driving force that pushes out the ciliary plasma membrane to form each disc evagination that subsequently can mature into a bona fide disc. We highlight the functions of actin-binding proteins, particularly PCARE and Arp2/3, that are known to participate in disc formation. Finally, we describe a working model of disc formation built upon the many studies focusing on the role of actin during disc morphogenesis.

Actin-Binding Proteins as Potential Biomarkers for Chronic Inflammation-Induced Cancer Diagnosis and Therapy.

Actin-binding proteins (ABPs), by interacting with actin, regulate the polymerization, depolymerization, bundling, and cross-linking of actin filaments, directly or indirectly, thereby mediating the maintenance of cell morphology, cell movement, and many other biological functions. Consequently, these functions of ABPs help regulate cancer cell invasion and metastasis when cancer occurs. In recent years, a variety of ABPs have been found to be abnormally expressed in various cancers, indicating that the detection and interventions of unusual ABP expression to alter this are available for the treatment of cancer. The early stages of most cancer development involve long-term chronic inflammation or repeated stimulation. This is the case for breast cancer, gastric cancer, lung cancer, prostate cancer, liver cancer, esophageal cancer, pancreatic cancer, melanoma, and colorectal cancer. This article discusses the relationship between chronic inflammation and the above-mentioned cancers, emphatically introduces relevant research on the abnormal expression of ABPs in chronic inflammatory diseases, and reviews research on the expression of different ABPs in the above-mentioned cancers. Furthermore, there is a close relationship between ABP-induced inflammation and cancer. In simple terms, abnormal expression of ABPs contributes to the chronic inflammation developing into cancer. Finally, we provide our viewpoint regarding these unusual ABPs serving as potential biomarkers for chronic inflammation-induced cancer diagnosis and therapy, and interventions to reverse the abnormal expression of ABPs represent a potential approach to preventing or treating the corresponding cancers.

A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells.

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP–actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP–actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.

LAMC2 modulates the acidity of microenvironments to promote invasion and migration of pancreatic cancer cells via regulating AKT-dependent NHE1 activity

LAMC2, as a unique chain in the Laminin 5 molecule, has been found to be associated with malignant metastases in some cancers. However, the roles and mechanisms by which LAMC2 affects the migration and invasion of pancreatic cancer cells remain unclear. First, we found that laminin 5/LAMC2 and its receptors were highly expressed in pancreatic cancer tissues and cells. Then, we investigated the effects of LAMC2 on pancreatic cancer cell migration/invasion and extracellular (pHe). We also demonstrated that LAMC2 phosphorylated Akt-Ser473 to promote the expression, activity and cell membrane accumulation of NHE1 within pancreatic cancer cells. So we speculated that LAMC2 modulated the pHe to promote migration and invasion of pancreatic cancer cells. Additionally, our data also showed that LAMC2/NHE1 resulted in altered cell morphology and aberrant expression of mesenchymal markers. The function of actin-binding proteins (ABPs) were affected by LAMC2/NHE1 signaling. LAMC2/NHE1 signaling generated extracellular acidification to induce dynamic actin-dependent pseudopodial formation and EMT programs that promote tumor cell invasion in pancreatic cancer cells. Therefore, we found that LAMC2 was responsible for generating the extracellular acidic conditions that mediated invasion of pancreatic cancer cells by activating Akt/NHE1 signaling. LAMC2 is a characteristic prognostic and therapeutic agent of PDCA.

The dynamics and regulation of microfilament during spermatogenesis.

Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future.

Expression of Cytoskeleton Actin in Cancer and Metastasis

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.

Mechanical Roles of F-Actin in the Differentiation of Stem Cells: A Review.

In the development and differentiation of stem cells, mechanical forces associated with filamentous actin (F-actin) play a crucial role. The present review aims to reveal the relationship among the chemical components, microscopic structures, mechanical properties, and biological functions of F-actin. Particular attention is given to the functions of the cytoplasmic and nuclear microfilament cytoskeleton and their regulation mechanisms in the differentiation of stem cells. The distributions of different types of actin monomers in mammal cells and the functions of actin-binding proteins are summarized. We discuss how the fate of stem cells is regulated by intra/extracellular mechanical and chemical cues associated with microfilament-related proteins, intercellular adhesion molecules, etc. In addition, we also address the differentiation-induced variation in the stiffness of stem cells and the correlation between the fate and geometric shape change of stem cells. This review not only deepens our understanding of the biophysical mechanisms underlying the fates of stem cells under different culture conditions but also provides inspirations for the tissue engineering of stem cells.

Novel actin-binding-proteins in plants

The actin cytoskeleton participates in numerous intracellular and physiological processes, and is present in all eukaryote. Tight regulation of actin cytoskeleton organization and dynamics is regulated by a plethora of actin-binding proteins (ABPs), which is the foundation of cellular processes. Although actin is highly conserved and the mechanisms underlying the regulation of actin dynamics are similar in eukaryotic cells, there are some differences between animal and plant cells owing to the diversity of cell type and living habits. ABPs coevolved with actin leading to the diverse biochemical functions of ABPs in eukaryote, such as several ABPs disappearing but conserved in animal and yeast, novel plants ABPs arising, typical ABPs and non-ABPs evolving into new actin-regulating function and activity. This review summarizes the latest progress in the function of novel plants ABPs and the methods for searching new ABPs in plants. Finally, we discuss the future studies and potential research hotspots in plant actin cytoskeleton.

The neuronal and actin commitment: Why do neurons need rings?

The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc.

Integrating actin dynamics, mechanotransduction and integrin activation: The multiple functions of actin binding proteins in focal adhesions

Focal adhesions are clusters of integrin transmembrane receptors that mechanically couple the extracellular matrix to the actin cytoskeleton during cell migration. Focal adhesions sense and respond to variations in force transmission along a chain of protein-protein interactions linking successively actin filaments, actin binding proteins, integrins and the extracellular matrix to adapt cell-matrix adhesion to the composition and mechanical properties of the extracellular matrix. This review focuses on the molecular mechanisms by which actin binding proteins integrate actin dynamics, mechanotransduction and integrin activation to control force transmission in focal adhesions.

Actin depolymerizing factors ADF7 and ADF10 play distinct roles during pollen development and pollen tube growth

An important player in actin remodeling is the actin depolymerizing factor (ADF) which increases actin filament treadmilling rates. Previously, we had prepared fluorescent protein fusions of two Arabidopsis pollen specific ADFs, ADF7 and ADF10. These had enabled us to determine the temporal expression patterns and subcellular localization of these proteins during male gametophyte development. Here we generated stable transformants containing both chimeric genes allowing for simultaneous imaging and direct comparison. One of the striking differences between the two proteins was the localization profile in the growing pollen tube apex. Whereas ADF10 was associated with the filamentous actin array forming the subapical actin fringe, ADF7 was present in the same cytoplasmic region, but in diffuse form. This suggests that ADF7 is involved in the high actin turnover that is likely to occur in the fringe by continuously and efficiently depolymerizing filamentous actin and supplying monomeric actin to the advancing end of the fringe. The possibility to visualize both of these pollen-specific ADFs simultaneously opens avenues for future research into the regulatory function of actin binding proteins in pollen.

Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has long been a useful model organism for muscle research. Its body wall muscle is obliquely striated muscle and exhibits structural similarities with vertebrate striated muscle. Actin is the core component of the muscle thin filaments, which are highly ordered in sarcomeric structures in striated muscle. Genetic studies have identified genes that regulate proper organization and function of actin filaments in C. elegans muscle, and sequence of the worm genome has revealed a number of conserved candidate genes that may regulate actin. To precisely understand the functions of actin-binding proteins, such genetic and genomic studies need to be complemented by biochemical characterization of these actin-binding proteins in vitro. This article describes methods for purification and biochemical characterization of actin from C. elegans. Although rabbit muscle actin is commonly used to characterize actin-binding proteins from many eukaryotic organisms, we detect several quantitative differences between C. elegans actin and rabbit muscle actin, highlighting that use of actin from an appropriate source is important in some cases. Additionally, we describe probes for cell biological analysis of actin in C. elegans.

Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.

Actin Branching Is Affected by Local Bending of the Mother Filament

Actin filaments serve as structural elements of the cytoskeleton subject to mechanical forces and provide binding sites for actin-binding proteins (ABPs). Structural studies have established that actin filaments can adopt several different twist structures that are stabilized by ABPs such as ADF/cofilin or scruin. We sought to investigate whether strain on the actin filament due to bending affects the binding or function of ABPs and chose the Arp2/3 complex, which is involved in the assembly of force-generating actin networks, as our initial target. The Arp2/3 complex nucleates the growth of actin branches from pre-existing filaments, making it easy to detect where on the mother filament it has bound. To test the effect of bending on branching, biotinylated and rhodamine-phalloidin-stabilized actin filaments were bound to a streptavidin-coated glass surface, immobilized in a distribution of bent conformations. These tethered mother filaments were then incubated with actin, Arp2/3, and its activator. Growth of branches was stabilized with green fluorescent phalloidin. Branches originating from highly curved sections (radius of curvature < 1.5um) of actin filaments were more likely to grow from the extended side of the filament (positive strain) than from the compressed side (negative strain), with a statistically significant (P < 0.05) difference. To elucidate structural distortions that may give rise to this effect, we used Monte Carlo simulations based on a coarse-grained model of the actin filament to estimate the changes in inter-monomer spacing that occur for the experimentally probed range of filament curvatures. We conclude that mother filament bend strain is sufficient to alter actin branching and may play an important role in the organization of actin networks growing under load. Similar bend-induced effects may be important for other ABPs and provide a mechanism for mechanotransduction in cells.

The function of actin-binding proteins in pollen tube growth

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. It is well known that actin-binding proteins are responsible for the direct control of dynamic actin filaments and serve as a link between signal transduction pathways and dynamic actin changes in determining cellular architecture. Several of these classes have been identified in pollen tubes and their detailed characterisation is progressing rapidly. Here, we aim to survey what is known about the major actin-binding proteins that affect actin assembly and dynamics, and their higher-order organisation in pollen tube growth.

Phosphoinositide metabolism during membrane ruffling and macropinosome formation in EGF-stimulated A431 cells

Inhibitors of phosphoinositide 3-kinase (PI3K) were found to perturb macropinosome formation without affecting the membrane ruffling and actin polymerization in epidermal growth factor-stimulated A431 cells. Live-cell imaging and quantitative image analysis of the fluorescence intensity ratio of the YFP-tagged phospholipase Cδ1-pleckstrin homology domain (YFP-PLC-PH) relative to membrane-targeted CFP (CFP-Mem) demonstrated that the concentration of PI(4,5)P 2 in the membrane ruffles forming macropinocytic cups increased to more than double that in planar plasma membranes. The PI(4,5)P 2 level in the membrane reached its maximum just before macropinosome closure and rapidly fell as the macropinocytic cups closed. In contrast, the PI(3,4,5)P 3 concentrations visualized based on the YFP-Akt-PH or YFP-Bruton's tyrosine kinase (Btk)-PH/CFP-Mem ratio increased locally at the site of macropinosome formation and peaked at the time of macropinosome closure. The kinetics of PI(4,5)P 2 and PI(3,4,5)P 3 appeared to be mechanistically linked to actin remodeling during macropinocytosis. From the pharmacological data using inhibitors and synthetic phosphoinositides and other data, it could be concluded that both PI(4,5)P 2 elimination and PI(3,4,5)P 3 production by PI3K might be crucial for macropinosome formation from membrane ruffles. This study emphasizes that locally controlled levels of phosphoinositides are important for regulating the function of actin-binding proteins which effect changes in the membrane architecture.

Cloning and developmental expression of Xenopus Enabled (Xena)

Regulation of actin dynamics, organization, and interaction with cell surface adhesion proteins is critical for tissue morphogenesis during development. The Ena/VASP family of actin-binding proteins function in several cellular processes that involve dynamic regulation of the actin cytoskeleton, including axon guidance, platelet aggregation, cell migration, and cell adhesion. The vertebrate Ena/VASP family is composed of three genes: Ena (Enabled), VASP (Vasodilator Stimulated Phosphoprotein), and Evl (Ena/VASP-Like). To better understand the role of Ena/VASP proteins during vertebrate development, we have cloned and characterized the developmental expression of Ena in Xenopus laevis. Analysis of the temporal expression of Xenopus Ena (Xena) demonstrates that multiple isoforms of Xena are detected throughout embryogenesis and that the presence of different isoforms is developmentally regulated. In situ hybridization analyses reveal that Xena is broadly expressed throughout development. During gastrulation and neurulation, Xena is detected in the neuroepithelium, notochord, and somites. In tadpoles, Xena expression is restricted to dorsal regions of the brain, whereas it is expressed at lower levels throughout the spinal cord. Xena expression is also detected in the notochord, myotome, heart, pronephros, and cranial placodes, including the olfactory and otic placodes. Analysis of the subcellular localization of Xena using a GFP fusion protein revealed that Xena localizes to adherens junctions and focal adhesions in Xenopus animal caps and NIH3T3 fibroblasts, respectively. These results define spatiotemporal windows in which Xena may function during early Xenopus development to modulate actin-dependent processes such as cell adhesion and migration.