Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

Summary The distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S₁. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S₁. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.     

Dynamic motion of paxillin on actin filaments in living endothelial cells

Our three-dimensional (3-D) images showed that paxillin co-localized on actin filaments as fibrous structures, as well as clusters, in endothelial cells (ECs). In living ECs under flow condition, we monitored concurrently the intracellular dynamics of DsRed2-paxillin and GFP-actin by time-lapse video recording and dual-color fluorescence imaging. The results showed that the dynamic motion of paxillin as fibrous structures was associated with actin filaments, but not with microtubules. Our findings suggest that the actin network plays an important role not only in the assembly/disassembly of paxillin at focal adhesions, but also as a track for the intracellular transport of paxillin, which is involved in signaling pathway.     

The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock

Human heat shock 27-kDa protein 1 (HSPB1)/heat shock protein (Hsp) 27 is a small heat shock protein which is thought to have several roles within the cell. One of these roles includes regulating actin filament dynamics in cell movement, since Hsp27 has previously been found to inhibit actin polymerization in vitro. In this study, the role of Hsp27 in regulating actin filament dynamics is further investigated. Hsp27 protein levels were reduced using siRNA in SW480 cells, a human colon cancer cell line. An in vitro wound closure assay showed that cells with knocked down Hsp27 levels were unable to close wounds, indicating that this protein is involved in regulating cell motility. Immunoprecipitation pull down assays were done, to observe if and when Hsp27 and actin are in the same complex within the cell, before and after heat shock. At all time points tested, Hsp27 and actin were present in the same cell lysate fraction. Lastly, indirect immunostaining was done before and after heat shock to evaluate Hsp27 and actin interaction in cells. Hsp27 and actin showed colocalization before heat shock, little association 3 h after heat shock, and increased association 24 h after heat shock. Cytoprotection was observed as early as 3 h after heat shock, yet cells were still able to move. These results show that Hsp27 and actin are in the same complex in cells and that Hsp27 is important for cell motility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells

Summary. Complete depolymerization of actin filaments (AFs) at low temperature (0 °C) is followed by the formation of transient actin structures at 25 °C in tobacco BY-2 cells (Nicotiana tabacum L.). Using antibodies against fission yeast actin-related proteins (ARP2 and ARP3), we show here that transient actin structures (dots, dotted filaments, rods) colocalize with epitopes stained by these antibodies and thus are likely to represent sites of actin filament nucleation (SANs). In contrast to the cold-induced disassembly of AFs, no transient actin structures were detectable during recovery of AFs from latrunculin B-induced depolymerization. However, the staining pattern obtained with ARP antibodies in latrunculin B-treated cells was similar to that in controls and cold-treated cells. This suggests that, in addition to the complete depolymerization of AFs, disruption of other cellular structures is needed for the formation of transient actin structures during the early phase of recovery from cold treatment. Correspondence and reprints: Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic.     

Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins

We previously found that capsaicin induces tight-junction (TJ) opening accompanied with cofilin dephosphorylation/activation in intestinal Caco-2 cells. Here, we examined the role of cofilin in TJ regulation, and analyzed the structural events that lead to TJ opening. We transfected Caco-2 cells with wild-type cofilin [cofilin(wt)] or its constitutively active mutant cofilin(S3A). We found that the decreases in transepithelial electrical resistance (TER) was slower in cofilin(wt) transfectants and faster in cofilin(S3A) mutants than in vector controls. Moreover, cofilin dephosphorylation corresponded to the rate of TER decrease. Capsaicin treatment changed the localization of TJ proteins and altered the F-actin structure, but in a manner different from those depend on myosin light chain kinase (MLCK). These results strongly support the importance of cofilin in TJ opening, suggesting cofilin as a target for TJ permeability regulation in epithelial cells.     

Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells

The blood-brain barrier (BBB) serves as a critical organ in the maintenance of central nervous system homeostasis and is disrupted in a number of neurological disorders, including stroke. We examined the effects of hypoxia/aglycemia on the expression and localization of tight junction proteins, and on the function of the BBB in an in vitro model system. A receptor-operated/store-operated calcium channel blocker, SKF 96365, was used to determine if calcium flux was important in mediating hypoxia/aglycemia effects on the BBB. Expression of the tight junction protein occludin increased after hypoxic/aglycemic stress when cells were exposed to SKF 96365; this was correlated with partial protection of membrane localization of occludin and inhibition of the hypoxia-induced increase in permeability. Actin expression was dramatically reduced by hypoxia/aglycemia. Treatment with SKF 96365 during hypoxic stress protected monolayer permeability of sucrose, but transendothelial electrical resistances decreased with exposure to hypoxic stress regardless of treatment. Therefore, the presence of occludin at the membrane is dependent in part on calcium-sensitive signaling cascades; this provides a target for therapeutic intervention to minimize BBB disruption after stroke.     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

A teamwork promotion of formin-mediated actin nucleation by Bud6 and Aip5 in Saccharomyces cerevisiae

Actin nucleation is achieved by collaborative teamwork of actin nucleator factors (NFs) and nucleation-promoting factors (NPFs) into functional protein complexes. Selective inter- and intramolecular interactions between the nucleation complex constituents enable diverse modes of complex assembly in initiating actin polymerization on demand. Budding yeast has two formins, Bni1 and Bnr1, which are teamed up with different NPFs. However, the selective pairing between formin NFs and NPFs into the nucleation core for actin polymerization is not completely understood. By examining the functions and interactions of NPFs and NFs via biochemistry, genetics, and mathematical modeling approaches, we found that two NPFs, Aip5 and Bud6, showed joint teamwork effort with Bni1 and Bnr1, respectively, by interacting with the C-terminal intrinsically disordered region (IDR) of formin, in which two NPFs work together to promote formin-mediated actin nucleation. Although the C-terminal IDRs of Bni1 and Bnr1 are distinct in length, each formin IDR orchestrates the recruitment of Bud6 and Aip5 cooperatively by different positioning strategies to form a functional complex. Our study demonstrated the dynamic assembly of the actin nucleation complex by recruiting multiple partners in budding yeast, which may be a general feature for effective actin nucleation by formins.     

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.     

Distribution of actin and myosin in muscle and non-muscle cells

Summary Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in nonmuscle cells.The authors thank Dr. M. Owen, National Institute of Medical Research, Mill Hill, for the gift of rabbit anti-actin antibodyOn sabbatical leave from Monash University, and supported by a Commonwealth Medical FellowshipThe Brompton Hospital, London     

Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.     

Localization of myosin and actin in ocular nonmuscle cells

Summary Myosin and actin were localized by indirect immunofluorescence microscopy using specific antibodies prepared in rabbits against highly purified gizzard myosin and actin. A strong fluorescence staining with both antibodies was observed in rat corneal epithelial cells, anterior lens epithelial cells, rod inner segments, and in rat and frog pigment epithelial cells. The immunohistochemical localization of myosin in corneal epithelial cells was further supported by the electrophoretic and immunological identification of smooth muscle type myosin heavy chain in pure corneal epithelial abrasions. Electron-microscopic observations revealed a clear correlation between staining with actin antibodies and the presence of numerous thin cytoplasmic filaments (50–80 Å in diameter). The functional and biochemical nature of 90–110 Å filaments occurring in corneal and lens epithelial cells, as well as the ultrastructural localization of myosin in ocular nonmuscle cells under study remains obscure.     

Membrane-deforming proteins play distinct roles in actin pedestal biogenesis by enterohemorrhagic Escherichia coli

Many bacterial pathogens reorganize the host actin cytoskeleton during the course of infection, including enterohemorrhagic Escherichia coli (EHEC), which utilizes the effector protein EspF(U) to assemble actin filaments within plasma membrane protrusions called pedestals. EspF(U) activates N-WASP, a host actin nucleation-promoting factor that is normally auto-inhibited and found in a complex with the actin-binding protein WIP. Under native conditions, this N-WASP/WIP complex is activated by the small GTPase Cdc42 in concert with several different SH3 (Src-homology-3) domain-containing proteins. In the current study, we tested whether SH3 domains from the F-BAR (FCH-Bin-Amphiphysin-Rvs) subfamily of membrane-deforming proteins are involved in actin pedestal formation. We found that three F-BAR proteins: CIP4, FBP17, and TOCA1 (transducer of Cdc42-dependent actin assembly), play different roles during actin pedestal biogenesis. Whereas CIP4 and FBP17 inhibited actin pedestal assembly, TOCA1 stimulated this process. TOCA1 was recruited to pedestals by its SH3 domain, which bound directly to proline-rich sequences within EspF(U). Moreover, EspF(U) and TOCA1 activated the N-WASP/WIP complex in an additive fashion in vitro, suggesting that TOCA1 can augment actin assembly within pedestals. These results reveal that EspF(U) acts as a scaffold to recruit multiple actin assembly factors whose functions are normally regulated by Cdc42.     

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.     

Bending over backwards: BAR proteins and the actin cytoskeleton in mammalian receptor-mediated endocytosis

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer.     

Cortical actin filaments fragment and aggregate to form chloroplast-associated and free F-actin rings in mechanically isolatedZinnia mesophyll cells

Summary Changes in F-actin organization following mechanical isolation ofZinnia mesophyll cells were documented by rhodamine-phalloidin staining. Immediately after isolation, most cells contained irregular cortical actin fragments of varying lengths, and less than 5% of cells contained intact cortical filaments. During the first 8 h of culture, filament fragments were replaced by actin rings, stellate actin aggregates, and bundled filament fragments. Some of these aggregates had no association with organelles (free actin aggregates). Other aggregates were associated with chloroplasts, which changed in shape and location at the same time actin aggregates appeared. F-actin was concentrated within or around the nucleus in a small percentage of cells. After 12 h in culture, the percentage of cells with free actin rings and chloroplast-associated actin aggregates began to decline and the percentage of cells having intact cortical actin filaments increased greatly. Intermediate images were recorded that strongly indicate that free actin rings, chloroplast-associated actin rings, and other actin aggregates self-assemble by successive bundling of actin filament fragments. The fragmentation and bundling of F-actin observed in mechanically isolatedZinnia cells resembles changes in F-actin distribution reported after diverse forms of cell disturbance and appears to be an example of a generalized response of the actin cytoskeleton to cell stress.Abbreviations FITC fluorescein isothiocyanate - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - RhPh tetramethylrhodamine isothiocyanate-phalloidin     

Evolutionarily conserved modules in actin nucleation: lessons from Dictyostelium discoideum and plants

Summary. The actin cytoskeleton plays a central part in the dynamic organization of eukaryotic cell structure. Nucleation of actin filaments is a crucial step in the establishment of new cytoskeletal structures or modification of existing ones, providing abundant targets for regulatory processes. A substantial part of our understanding of actin nucleation derives from studies on yeast and metazoan cells. However, recent advances in structural and functional genome analysis in less traditional models, such as plants or Dictyostelium discoideum, provide an emerging picture of an evolutionarily conserved core of at least two actin nucleation mechanisms, one mediated by the Arp2/3 complex and the other one by the formin-based module. A considerable degree of conservation is found also in the systems controlling the balance between filamentous and globular actin (profilin, actin-depolymerizing factor/cofilin) and even in certain regulatory aspects, such as the involvement of Rho-related small GTPases. Identification of such conserved elements provides a prerequisite for the characterization of evolutionarily variable aspects of actin regulation which may be responsible for the rich morphological diversity of eukaryotic cells.Correspondence and reprints: Department of Plant Physiology, Faculty of Sciences, Charles University, Vininá 5, 128 44 Praha 2, Czech Republic.     

Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Three-dimensional organization of actin cytoskeleton and podosomal contact structures in neoplastic cells in vitro

In spontaneously metastasizing rat RPS sarcoma cells, a 3D structure of oblique F-actin cables was observed which was associated with active cell migration in vitro. This led us to further comparative investigations of several other neoplastic and normal cell populations in vitro for F-actin structures using confocal laser scanning microscopy (CLSM). Various forms of F-actin cytoskeleton were observed and the incidence of podosome-related contact structures appeared to be associated with malignancy, interpreted as metastatic capacity.