Regulation of actin cytoskeleton architecture by Eps8 and Abi1

Background  

The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive.     

Small Rho GTPases are important for acinus formation in a human salivary gland cell line

Rho GTPases participate in a wide variety of signal transduction pathways regulating the actin cytoskeleton, gene expression, cellular migration and proliferation. The aim of this study was to evaluate the role of Rho GTPases in signal transduction pathways during acinus formation in a human salivary gland (HSG) cell line initiated by extracellular matrix (ECM; Matrigel) alone or in combination with epidermal growth factor, basic fibroblast growth factor and lysophosphatidic acid (LPA). Immunohistochemical and Western blotting analyses showed that HSG cells contained RhoA, RhoB, Rac1 and Cdc42 proteins. All growth factors enhanced the effects of ECM on acinus formation, in a pathway dependent on PI3-kinase and Rho GTPases. The role of ROCK, a major RhoA effector, seemed limited to cortical actin polymerization. LPA stimulated cell migration and acinus formation in a PI3-kinase-independent pathway. The results suggest that Rho proteins are important for epithelial-mesenchymal interactions during salivary gland development.This work was supported by FAPESP (grant numbers: 97/09507-6, 01/09047-2).     

Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network

Dynamic rearrangements of the actin cytoskeleton, following T-cell antigen receptor (TCR) engagement, provide the structural matrix and flexibility to enable intracellular signal transduction, cellular and subcellular remodeling, and driving effector functions. Recently developed cutting-edge imaging technologies have facilitated the study of TCR signaling and its role in actin-dependent processes. In this review, we describe how TCR signaling cascades induce the activation of actin regulatory proteins and the formation of actin networks, and how actin dynamics is important for T-cell homeostasis, activation, migration, and other effector functions.     

Direct interaction of p21-activated kinase 4 with PDZ-RhoGEF, a G protein-linked Rho guanine exchange factor

Rho GTPases regulate a wide variety of cellular processes, ranging from actin cytoskeleton remodeling to cell cycle progression and gene expression. Cell surface receptors act through a complex regulatory molecular network that includes guanine exchange factors (GEFs), GTPase activating proteins, and guanine dissociation inhibitors to achieve the coordinated activation and deactivation of Rho proteins, thereby controlling cell motility and ultimately cell fate. Here we found that a member of the RGL-containing family of Rho guanine exchange factors, PDZ RhoGEF, which, together with LARG and p115RhoGEF, links the G(12/13) family of heterotrimeric G proteins to Rho activation, binds through its C-terminal region to the serine-threonine kinase p21-activated kinase 4 (PAK4), an effector for Cdc42. This interaction results in the phosphorylation of PDZ RhoGEF and abolishes its ability to mediate the accumulation of Rho-GTP by Galpha13. Moreover, when overexpressed, active PAK4 was able to dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation. Together, these results provide evidence that PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs, thus providing a novel potential mechanism for cross-talk among Rho GTPases.     

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization.MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells.ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation.ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers.General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.     

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.Download video file.^{(136M, mov)}     

Rho-associated coiled-coil kinase (ROCK) signaling and disease

Abstract

The small Rho GTPase family of proteins, encompassing the three major G-protein classes Rho, Rac and cell division control protein 42, are key mitogenic signaling molecules that regulate multiple cancer-associated cellular phenotypes including cell proliferation and motility. These proteins are known for their role in the regulation of actin cytoskeletal dynamics, which is achieved through modulating the activity of their downstream effector molecules. The Rho-associated coiled-coil kinase 1 and 2 (ROCK1 and ROCK2) proteins were the first discovered Rho effectors that were primarily established as players in RhoA-mediated stress fiber formation and focal adhesion assembly. It has since been discovered that the ROCK kinases actively phosphorylate a large cohort of actin-binding proteins and intermediate filament proteins to modulate their functions. It is well established that global cellular morphology, as modulated by the three cytoskeletal networks: actin filaments, intermediate filaments and microtubules, is regulated by a variety of accessory proteins whose activities are dependent on their phosphorylation by the Rho-kinases. As a consequence, they regulate many key cellular functions associated with malignancy, including cell proliferation, motility and viability. In this current review, we focus on the role of the ROCK-signaling pathways in disease including cancer.     

Deciphering actin remodelling in immune cells through the prism of actin-related inborn errors of immunity

Actin cytoskeleton remodelling drives cell motility, cell to cell contacts, as well as membrane and organelle dynamics. Those cellular activities operate at a particularly high pace in immune cells since these cells migrate through various tissues, interact with multiple cellular partners, ingest microorganisms and secrete effector molecules. The central and multifaceted role of actin cytoskeleton remodelling in sustaining immune cell tasks in humans is highlighted by rare inborn errors of immunity due to mutations in genes encoding proximal and distal actin regulators. In line with the specificity of some of the actin-based processes at work in immune cells, the expression of some of the affected genes, such as WAS, ARPC1B and HEM1 is restricted to the hematopoietic compartment. Exploration of these natural deficiencies highlights the fact that the molecular control of actin remodelling is tuned distinctly in the various subsets of myeloid and lymphoid immune cells and sustains different networks associated with a vast array of specialized tasks. Furthermore, defects in individual actin remodelling proteins are usually associated with partial cellular impairments highlighting the plasticity of actin cytoskeleton remodelling. This review covers the roles of disease-associated actin regulators in promoting the actin-based processes of immune cells. It focuses on the specific molecular function of those regulators across various immune cell subsets and in response to different stimuli. Given the fact that numerous immune-related actin defects have only been characterized recently, we further discuss the challenges lying ahead to decipher the underlying patho-mechanisms.     

Oxidation and reduction of actin: Origin,impact in vitro and functional consequences in vivo

Actin is among the most abundant proteins in eukaryotic cells and assembles into dynamic filamentous networks regulated by many actin binding proteins. The actin cytoskeleton must be finely tuned, both in space and time, to fulfill key cellular functions such as cell division, cell shape changes, phagocytosis and cell migration. While actin oxidation by reactive oxygen species (ROS) at non-physiological levels are known for long to impact on actin polymerization and on the cellular actin cytoskeleton, growing evidence shows that direct and reversible oxidation/reduction of specific actin amino acids plays an important and physiological role in regulating the actin cytoskeleton. In this review, we describe which actin amino acid residues can be selectively oxidized and reduced in many different ways (e.g. disulfide bond formation, glutathionylation, carbonylation, nitration, nitrosylation and other oxidations), the cellular enzymes at the origin of these post-translational modifications, and the impact of actin redox modifications both in vitro and in vivo. We show that the regulated balance of oxidation and reduction of key actin amino acid residues contributes to the control of actin filament polymerization and disassembly at the subcellular scale and highlight how improper redox modifications of actin can lead to pathological conditions.     

Cortactin: A multifunctional regulator of cellular invasiveness

Branched actin assembly is critical for a variety of cellular processes that underlie cell motility and invasion, including cellular protrusion formation and membrane trafficking. Activation of branched actin assembly occurs at various subcellular locations via site-specific activation of distinct WASp family proteins and the Arp2/3 complex. A key branched actin regulator that promotes cell motility and links signaling, cytoskeletal and membrane trafficking proteins is the Src kinase substrate and Arp2/3 binding protein cortactin. Due to its frequent overexpression in advanced, invasive cancers and its general role in regulating branched actin assembly at multiple cellular locations, cortactin has been the subject of intense study. Recent studies suggest that cortactin has a complex role in cellular migration and invasion, promoting both on-site actin polymerization and modulation of autocrine secretion. Diverse cellular activities may derive from the interaction of cortactin with site-specific binding partners.Key words: cortactin, migration, invasion, lamellipodia, invadopodia, cancer, actin, actin assembly, scaffold, membrane trafficking, secretion     

Remodeling of actin cytoskeleton in mouse periosteal cells under mechanical loading induces periosteal cell proliferation during bone formation

Background

The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo.

Principal Findings

We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells.

Conclusions

These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.     

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis,motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA₁, LPA₂, and LPA₃ on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.     

Morphological and oxidative alterations on Sertoli cells cytoskeleton due to retinol-induced reactive oxygen species

Retinol (vitamin A) is involved in several cellular processes, like cell division, differentiation, transformation and apoptosis. Although it has been shown that retinol is a limitant factor for all these processes, the precise mechanisms by which retinol acts are still unknown. In the present study we hypothesised that alterations in the cytoskeleton of Sertoli cells induced by retinol supplementation could indicate an adaptive maintenance of its functions, since it plays an important role in the transformation process that we observed. Previous results demonstrated that Sertoli cells treated with retinol showed an oxidative imbalance, that leads the cell to two phenotypes: apoptosis or transformation. Our group has identified characteristics of Sertoli cells transformed by retinol which results in normal cell functions modification. In the present study the actin filament fluorescence assay and the deformation coefficient showed a modification in the morphology induced by retinol. We also observed an oxidative alteration in isolated cytoskeleton proteins and did not show alterations when these proteins are analyzed by electrophoreses. Our results showed an increase in mitochondria superoxide production and a decrease in nitric oxide levels. All results were partially or completely reverted by co-treatment of the antioxidant Trolox®. These findings suggest that the cytoskeleton components suffer individual alterations in different levels and that these alterations generate a global phenotype modification and that these processes are probably ROS dependent. We believe that the results from this study indicate an adaptation of the cytoskeleton to oxidative imbalance since there was not a loss of its function. (Mol Cell Biochem 271: 189–196, 2005)     

The epithelial cell cytoskeleton and intracellular trafficking. III. How is villin involved in the actin cytoskeleton dynamics in intestinal cells?

Villin plays a key role in the maintenance of the brush border organization by bundling F-actin into a network of parallel filaments. Our previous in vivo data on villin knockout mice showed that, although this protein is not necessary for the bundling of F-actin, it is important for the reorganization of the actin cytoskeleton elicited by stress conditions. We further investigated villin property to initiate actin remodeling in cellular processes such as hepatocyte growth factor-induced motility, morphogenesis, and bacterial infection. Our data suggest that villin is involved in actin remodeling necessary for many cellular processes requiring the actin cytoskeleton plasticity.     

Actin,Membrane Trafficking and the Control of Prion Induction,Propagation and Transmission in Yeast

The model eukaryotic yeast Saccharomyces cerevisiae has proven a useful model system in which prion biogenesis and elimination are studied. Several yeast prions exist in budding yeast and a number of studies now suggest that these alternate protein conformations may play important roles in the cell. During the last few years cellular factors affecting prion induction, propagation and elimination have been identified. Amongst these, proteins involved in the regulation of the actin cytoskeleton and dynamic membrane processes such as endocytosis have been found to play a critical role not only in facilitating de novo prion formation but also in prion propagation. Here we briefly review prion formation and maintenance with special attention given to the cellular processes that require the functionality of the actin cytoskeleton.      

Lysophosphatidic acid (LPA) and its receptors: Role in airway inflammation and remodeling

Lysophosphatidic acid (LPA), a simple bioactive phospholipid, is present in biological fluids such as plasma and bronchoalveolar lavage (BAL). It appears to have both pro- and anti-inflammatory roles in inflammatory lung diseases. Exogenous LPA promotes inflammatory responses by regulating the expression of chemokines, cytokines, and cytokine receptors in lung epithelial cells. In addition to the modulation of inflammatory responses, LPA regulates cytoskeleton rearrangement and confers protection against lung injury by enhancing lung epithelial cell barrier integrity and remodeling. The biological effects of LPA are mediated through its cell surface G-protein coupled LPA_1–7 receptors. The roles of LPA receptors in lung fibrosis, asthma, and acute lung injury have been investigated using genetically engineered LPA receptor deficient mice and there appears to be a definitive role for endogenous LPA and its receptors in the pathogenesis of pulmonary inflammatory diseases. This review summarizes recent reports on the role of LPA and its receptors in the regulation of lung epithelial inflammatory responses and remodeling. This article is part of a Special Issue entitled: Advances in Lysophospholipid Research.     

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.     

Sphingosine 1-phosphate-mediated activation of ezrin-radixin-moesin proteins contributes to cytoskeletal remodeling and changes of membrane properties in epithelial otic vesicle progenitors

Hearing loss is among the most prevalent sensory impairments in humans. Cochlear implantable devices represent the current therapies for hearing loss but have various shortcomings. ERM (ezrin- radixin -moesin) are a family of adaptor proteins that link plasma membrane with actin cytoskeleton, playing a crucial role in cell morphology and in the formation of membrane protrusions. Recently, bioactive sphingolipids have emerged as regulators of ERM proteins. Sphingosine 1-phosphate (S1P) is a pleiotropic sphingolipid which regulates fundamental cellular functions such as proliferation, survival, migration as well as processes such as development and inflammation mainly via ligation to its specific receptors S1PR (S1P_1–5). Experimental findings demonstrate a key role for S1P signaling axis in the maintenance of auditory function. Preservation of cellular junctions is a fundamental function both for S1P and ERM proteins, crucial for the maintenance of cochlear integrity. In the present work, S1P was found to activate ERM in a S1P₂-dependent manner in murine auditory epithelial progenitors US/VOT-E36. S1P-induced ERM activation potently contributed to actin cytoskeletal remodeling and to the appearance of ionic currents and membrane passive properties changes typical of more differentiated cells. Moreover, PKC and Akt activation was found to mediate S1P-induced ERM phosphorylation. The obtained findings contribute to demonstrate the role of S1P signaling pathway in inner ear biology and to disclose potential innovative therapeutical approaches in the field of hearing loss prevention and treatment.     

Dynamin Rings: Not Just for Fission

The GTPase dynamin has captivated researchers for over two decades, even managing to establish its own research field. Dynamin's allure is partly due to its unusual biochemical properties as well as its essential role in multiple cellular processes, which include the regulation of clathrin‐mediated endocytosis and of actin cytoskeleton. On the basis of the classic model, dynamin oligomerization into higher order oligomers such as rings and helices directly executes the final fission reaction in endocytosis, which results in the generation of clathrin‐coated vesicles. Dynamin's role in the regulation of actin cytoskeleton is mostly explained by its interactions with a number of actin‐binding and ‐regulating proteins; however, the molecular mechanism of dynamin's action continues to elude us. Recent insights into the mechanism and role of dynamin oligomerization in the regulation of actin polymerization point to a novel role for dynamin oligomerization in the cell .