Actin cytoskeleton remodelling in the anterior pituitary folliculostellate cell line TtT/GF: participation of the actin-binding protein cortactin

We have previously shown that the folliculostellate (FS) cells of the anterior pituitary change their shape from stellate (type I) to polygonal (type II) coincidently with variations in the secretory activity of the pituitary. To elucidate the mechanisms involved in this switch in phenotypes, here we studied the impact of serum factors on the morphology of the FS cell line TtT/GF. TtT/GF cells cultured in serum-containing medium displayed elongated shapes and membrane ruffles similarly to type I cells. Serum deprivation caused the loss of plasma membrane activity and the acquisition by the cells of a sedentary phenotype and of a polygonal shape typical of type II FS cells. Addition of serum to the starved cells induced the reappearance of membrane raffles and lamellipodia. The switch in phenotypes and the maintenance of a motile phenotype depended on tyrosine kinase but not on Erk activity. Because the transition between phenotypes involved the tyrosine kinase-dependent reorganization of cortical actin filaments, we studied the participation of the actin-binding protein, cortactin, a tyrosine kinase substrate. Cortactin and its tyrosine-phosphorylated form, pY421-cortactin, localized to membrane ruffles and lamellipodia in serum-cultured TtT/GF cells, while they were evenly distributed over the whole cell cortex in serum-starved cells. Serum treatment of starved cells induced a transient increase in pY421-cortactin levels and the clustering of pY421-cortactin in membrane regions where protrusions were developing. Both serum responses were blocked by a tyrosine kinase inhibitor. Together, the results indicate that the transition from a polygonal to an elongated shape entails the acquisition of a dynamic cortical actin cytoskeleton that involves the tyrosine kinase-dependent phosphorylation of cortactin and the translocation of cortical pY421-cortactin to sites of ruffle formation at the plasma membrane.     

Dynasore, a dynamin inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments

Dynamic remodeling of actin filaments are bases for a variety of cellular events including cell motility and cancer invasion, and the regulation of actin dynamics implies dynamin, well characterized endocytotic protein. Here we report that dynasore, a inhibitor of dynamin GTPase, potently destabilizes F-actin in vitro, and it severely inhibits the formation of pseudopodia and cancer cell invasion, both of which are supported by active F-actin formation. Dynasore rapidly disrupted F-actin formed in brain cytosol in vitro, and the dynasore’s effect on F-actin was indirect. Dynasore significantly suppressed serum-induced lamellipodia formation in U2OS cell. Dynasore also destabilized F-actin in resting cells, which caused the retraction of the plasma membrane. A certain amount of dynamin 2 in U2OS cells localized along F-actin, and co-localized with cortactin, a physiological binding partner of dynamin and F-actin. However, these associations of dynamin were partially disrupted by dynasore treatment. Furthermore, invasion activity of H1080 cell, a lung cancer cell line, was suppressed by approximately 40% with dynasore treatment. These results strongly suggest that dynasore potently destabilizes F-actin, and the effect implies dynamin. Dynasore or its derivative would be suitable candidates as potent anti-cancer drugs.     

Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control

Cortactin is an important molecular scaffold for actin assembly and organization. Novel mechanistic functions of cortactin have emerged with more interacting partners identified, revealing its multifaceted roles in regulating actin cytoskeletal networks that are necessary for endocytosis, cell migration and invasion, adhesion, synaptic organization and cell morphogenesis. These processes are mediated by its multi-domains binding to F-actin and Arp2/3 complex and various SH3 targets. Furthermore, its role in actin remodeling is subjected to regulation by tyrosine and serine/threonine kinases. Elucidating the mechanisms underlying cortactin phosphorylation and its functional consequences would provide new insights to various aspects of cell dynamics control.     

A cloned rat thymic epithelial cell line established from serum-free selective culture

Summary A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic endocrine cells. This work was partly supported by grant PCM-834 0582 from the National Science Foundation, Washington, DC, and grant P01 CA 37589-2 from the National Cancer Institute, Bethesda, MD.     

Involvement of cortactin and phosphotyrosine proteins in cell–cell contact formation in cultured bovine corneal endothelial cells

Phosphotyrosine proteins involvement, particularly cortactin, was studied in cell–cell contacts of cultured bovine corneal endothelial (BCE) cells. These proteins, including α-catenin, vinculin and cortactin, are localized at cell–cell contacts separate from the cortical actin ring. Approximately 50% of cortactin isoforms p80 and p85 were associated with the Triton-insoluble fraction while phosphotyrosine proteins were in the soluble fraction. Disruption of cell–cell contacts by EDTA treatment was associated with a decrease in cortactin isoforms p80 (26%) and p85 (57%). Cortactin isoform p85 was phosphorylated at Y⁴⁶⁶, expressed in reattaching cells and associated with the Triton-soluble fraction, whereas cortactin isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction. In sub-confluent cultures, pY⁴²¹-cortactin was localized at the leading edge and pY⁴⁶⁶-cortactin at a perinuclear area. In confluent cultures both pY⁴⁶⁶- and pY⁴²¹-cortactin isoforms were localized at the cell–cell contacts. In conclusion, in BCE cells, the most prominent appearance of cortactin was at the cell–cell contacts separate from the cortical actin ring. Isoform p80 was phosphorylated at Y⁴²¹ and associated with the Triton-insoluble fraction and isoform p85 was phosphorylated at Y⁴⁶⁶ and associated with the Triton-insoluble fraction.     

Interaction of cortactin and Arp2/3 complex is required for sphingosine-1-phosphate-induced endothelial cell remodeling

Sphingosine-1-phosphate (S1P) induces capillary formation of endothelial cells on Matrigel in accompany with actin assembly and accumulation of cortactin and Arp2/3 complex at the cell-leading edge. Suppression of cortactin expression with a cortactin antisense oligo significantly impaired S1P-induced capillary formation, migration of endothelial cells, and actin assembly at the cell periphery. Overexpression of wild-type cortactin tagged by green fluorescent protein (GFP) increased the S1P-induced tube formation and cell motility, whereas the cells overexpressing the mutant formed poorly capillary network and became less motile in response to S1P. Analysis of distribution in Triton X-100 insoluble fractions demonstrated that the cortactin mutant inhibited the association of wild-type cortactin and Arp2/3 complex with the actin-enriched complex. Furthermore, actin polymerization at and distribution of Arp2/3 complex as well as endogenous cortactin into the cell-leading edge mediated by S1P was disturbed. These data suggest that the interaction between cortactin and Arp2/3 complex plays an important role in S1P-mediated remodeling of endothelial cells.     

Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.     

Variable association of DNA polymerase with the cytoskeletal fractions of resting and dividing cells

A DNA polymerase activity associated with the detergent insoluble cytoskeletal fraction has been identified in dividing and non-dividing rat hepatocytes and a hepatoma (the Zajdela Ascitic Hepatoma). About 35 % of the enzyme is found associated with the cytoskeletal fraction of non-dividing cells as compared to about 3–6 % of the enzyme in dividing cells even though the dividing cells contain larger amounts of the extranuclear enzyme. The properties of the enzyme are similar to those of DNA polymerase-v. It is suggested that the association of the enzyme with the cytoskeletal fraction has functional significance.     

Evolution of Chordate Actin Genes: Evidence from Genomic Organization and Amino Acid Sequences

The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome. Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates. Received: 20 June 1996 / Accepted: 16 October 1996     

Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing

Obscurin is a recently identified giant multidomain muscle protein (∼800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of α-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.     

The actin depolymerizing factor n-cofilin is essential for neural tube morphogenesis and neural crest cell migration

Cofilin/ADF proteins are a ubiquitously expressed family of F-actin depolymerizing factors found in eukaryotic cells including plants. In vitro, cofilin/ADF activity has been shown to be essential for actin driven motility, by accelerating actin filament turnover. Three actin depolymerizing factors (n-cofilin, m-cofilin, ADF) can be found in mouse and human. Here we show that in mouse the non-muscle-specific gene-n-cofilin-is essential for migration of neural crest cells as well as other cell types in the paraxial mesoderm. The main defects observed in n-cofilin mutant embryos are an impaired delamination and migration of neural crest cells, affecting the development of neural crest derived tissues. Neural crest cells lacking n-cofilin do not polarize, and F-actin bundles or fibers are not detectable. In addition, n-cofilin is required for neuronal precursor cell proliferation and scattering. These defects result in a complete lack of neural tube closure in n-cofilin mutant embryos. Although ADF is overexpressed in mutant embryos, this cannot compensate the lack of n-cofilin, suggesting that they might have a different function in embryonic development. Our data suggest that in mammalian development, regulation of the actin cytoskeleton by the F-actin depolymerizing factor n-cofilin is critical for epithelial-mesenchymal type of cell shape changes as well as cell proliferation.     

Regulation of TGF-beta1/MAPK-mediated PAI-1 gene expression by the actin cytoskeleton in human mesangial cells

The importance of transforming growth factor-beta1 (TGF-beta1) in plasminogen activator inhibitor-1 (PAI-1) gene expression has been established, but the precise intracellular mechanisms are not fully understood. Our hypothesis is that the actin cytoskeleton is involved in TGF-beta1/MAPK-mediated PAI-1 expression in human mesangial cells. Examination of the distributions of actin filaments (F-actin), alpha-actinin, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunofluorescence and immunoprecipitation revealed that ERK and JNK associate with alpha-actinin along F-actin and that TGF-beta1 stimulation promote the dissociation of ERK and JNK with F-actin. Disassembly of the actin cytoskeleton inhibited phosphorylation of ERK and JNK and modulated PAI-1 expression and promoter activity under both basal and TGF-beta1-stimulated conditions. Stabilizing actin prevented dephosphorylation of ERK and JNK. ERK and JNK inhibitors and overexpressed dominant negative mutants antagonized the ability of TGF-beta1 to increase PAI-1 expression and promoter activity. Disassembly of F-actin also inhibited AP-1 DNA binding activity as determined by electrophoretic mobility shift assay using AP-1 consensus oligonucleotides derived from human PAI-1 promoter. F-actin stabilization prevented loss of AP-1 DNA binding activity. Therefore, changes in actin cytoskeleton modulate the ability of TGF-beta1 to stimulate PAI-1 expression through a mechanism dependent on the activation of MAPK/AP-1 pathways.     

Deuterostomic Actin Genes and the Definition of the Chordates: cDNA Cloning and Gene Organization for Cephalochordates and Hemichordates

The evolutionary relationship of muscle and nonmuscle actin isoforms in deuterostomia was studied by the isolation and characterization of two actin genes from the cephalochordate Branchiostoma lanceolatum and two from the hemichordate Saccoglossus kowalevskii The Branchiostoma genes specify a muscle and a nonmuscle actin type, respectively. Together with earlier results on muscle actins from vertebrates and urochordates, a N-terminal sequence signature is defined for chordate muscle actins. These diagnostic amino acid residues separate the chordates from the echinoderms and other metazoa. Although the two Saccoglossus actins characterized so far lack the diagnostic residues, in line with the presumptive phylogenetic position of hemichordates outside the chordates, a definitive conclusion can only be expected once the full complement of actin genes of Saccoglossus is established. Comparison of the intron patterns of the various deuterostomic actin genes shows that intron 330-3, which is present in all vertebrate genes, is conspicuously absent from nonvertebrate genes. The possible origin of this intron is discussed. Received: 4 July 1997 / Accepted: 29 August 1997     

Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells

Actin in guard cells is assembled in a radial pattern when stomata are induced to open under light, but the filaments are disassembled when stomata are closed under darkness or by abscisic acid (S.-O. Eun and Y. Lee, 1997, Plant Physiol. 115: 1491–1498). To test if signals that open stomata commonly generate the polymerized form of actin in guard cells, leaves of Commelina communis L. were treated with a potent stomatal opening agent, fusicoccin, and the actin organization examined by immunolocalization techniques. When stomata were induced to open by fusicoccin, hardly any of the filamentous form of actin was detected; instead, the actin resembled that present in guard cells that had been treated with an antagonist to actin filaments, cytochalasin D, and showed a sharp contrast to the long filaments developed in illuminated guard cells. Furthermore, treatment of illuminated leaves with fusicoccin disintegrated actin filaments that had already been formed in the guard cells. Preincubation of leaves with phalloidin, which interferes with fusicoccin-induced actin depolymerization, delayed fusicoccin-induced opening during the early phase. These observations suggest that the prevention of actin filament formation and/or depolymerization of actin filaments may accelerate the stomatal opening process in response to fusicoccin. Received: 1 October 1999 / Accepted: 29 November 1999     

F-actin at Newly Invaginated Membrane in Neurons: Implications for Surface Area Regulation

Neuronal shape and volume changes require accompanying cell surface adjustments. In response to osmotic perturbations, neurons show evidence of surface area regulation; shrinking neurons invaginate membrane at the substratum, pinch off vacuoles, and lower their membrane capacitance. F-actin is implicated in reprocessing newly invaginated membrane because cytochalasin causes the transient shrinking-induced invaginations, vacuole-like dilations (VLDs), to persist indefinitely instead of undergoing recovery. To help determine if cortical F-actin indeed contributes to cell surface area regulation, we test, here, the following hypothesis: invaginating VLD membrane rapidly establishes an association with F-actin and this association contributes to VLD recovery. Cultured molluscan (Lymnaea) neurons, whose large size facilitates three-dimensional imaging, were used. In fixed neurons, fluorescent F-actin stains were imaged. In live neurons, VLD membrane was monitored by brightfield microscopies and actin was monitored via a fluorescent tag. VLD formation (unlike VLD recovery) is cytochalasin insensitive and consistent with this, VLDs formed readily in cytochalasin-treated neurons but showed no association with F-actin. Normally, however (i.e., no cytochalasin), VLDs were foci for rapid reorganization of F-actin. At earliest detection (1–2 min), nascent VLDs were entirely coated with F-actin and by 5 min, VLD mouths (i.e., at the substratum) had become annuli of F-actin-rich motile leading edge. Time lapse images from live neurons showed these rings to be motile filopodia and lamellipodia. The retrieval of VLD membrane (vacuolization) occurred via actin-associated constriction of VLD mouths. The interplay of surface membrane and cortical cytoskeleton in osmotically perturbed neurons suggests that cell surface area and volume adjustments are coordinated in part via mechanosensitive F-actin dynamics. Received: 25 March 1999/Revised: 15 June 1999     

Amoeboid Locomotion of Naegleria gruberi: the Effects of Cytochalasin B on Cell-Substratum Interactions and Motile Behaviour

The major manifestations of amoeboid locomotion in Naegleria—cytoplasmic streaming, pseudopod production, cell polarity and focal contact production—require that the actin-based cytoskeleton be extremely dynamic. Whether these features are causally linked is unclear. In an attempt to answer this question we have used the fungal product cytochalasin B (cyt B) to dissect the motility process. This drug can perturb the organisation of actin filaments both in vivo and in vitro. Essentially cyt B acts as a molecule which can cap the barbed ends of actin filaments. Not surprisingly therefore cyt B has an effect on rates of actin polymerization and the dynamic state of actin in the cytoplasm. We have found that cyt B has a profound effect on focal contact production and breakdown. Within minutes of addition of cyt B focal contact production ceases, existing focal contacts are stabilised but cytoplasmic streaming and pseudopod production are not blocked. In conclusion it is now clear that the state of actin required for focal contact production is different from that required for pseudopod extension and cytoplasmic streaming.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.     

Hepatocyte Growth Factor Enables Enhanced Integrin–Cytoskeleton Linkage by Affecting Integrin Expression in Subconfluent Epithelial Cells

Hepatocyte growth factor (HGF) exerts mitogenic and motogenic effects in different cell types. In the epithelial cell line mHepR1 we found that HGF induced pronounced alterations in cell morphology and promoted cell adhesion and spreading. To analyze the mechanisms how HGF affects these integrin mediated functions we studied the physical linkage of integrins with the cytoskeleton. First we found that HGF increased the expression of different integrin subunits in subconfluent cells and influenced the distribution of integrins on the cell surface. To address the physical association of integrins with the cytoskeleton we analyzed Triton X-100-extracted cell fractions using flow cytometry. Here we show that cultivation of the cells with HGF for 24 h prior to integrin cross-linking significantly enhanced the cytoskeletal anchorage of integrins. To further find out whether HGF directly induces an integrin–cytoskeleton link without subsequent cross-linking we added HGF to suspended cells but failed to detect cytoskeletally immobilized integrins in the detergent-insoluble cell fraction which could be related to the absence of a calcium response induced by HGF. Overall, the results indicate that HGF promotes the physical linkage of integrins to the cytoskeleton which requires additional stimulation of integrins.