Stabilization Of F-actin Research Articles

Actin Dynamics Regulates Voltage‐Dependent Calcium‐Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca(2+) ([Ca(2+)](cyt)) is an ubiquitous second messenger in plant cell signaling, and [Ca(2+)](cyt) elevation is associated with Ca(2+)-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca(2+) channels and their regulation remains limited in planta. A type of voltage-dependent Ca(2+)-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba(2+) and Ca(2+), and their activities can be inhibited by micromolar Gd(3+). The unitary conductance and the reversal potential of the channels depend on the Ca(2+) or Ba(2+) gradients across the plasma membrane. The inward whole-cell Ca(2+) (Ba(2+)) current, as well as the unitary current amplitude and NP(o) of the single Ca(2+) channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.

Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens

Hexagonal packing geometry is a hallmark of close-packed epithelial cells in metazoans. Here, we used fiber cells of the vertebrate eye lens as a model system to determine how the membrane skeleton controls hexagonal packing of post-mitotic cells. The membrane skeleton consists of spectrin tetramers linked to actin filaments (F-actin), which are capped by tropomodulin1 (Tmod1) and stabilized by tropomyosin (TM). In mouse lenses lacking Tmod1, initial fiber cell morphogenesis is normal, but fiber cell hexagonal shapes and packing geometry are not maintained as fiber cells mature. Absence of Tmod1 leads to decreased gammaTM levels, loss of F-actin from membranes, and disrupted distribution of beta2-spectrin along fiber cell membranes. Regular interlocking membrane protrusions on fiber cells are replaced by irregularly spaced and misshapen protrusions. We conclude that Tmod1 and gammaTM regulation of F-actin stability on fiber cell membranes is critical for the long-range connectivity of the spectrin-actin network, which functions to maintain regular fiber cell hexagonal morphology and packing geometry.

IgE Receptor-Mediated Alteration of Membrane−Cytoskeleton Interactions Revealed by Mass Spectrometric Analysis of Detergent-Resistant Membranes

We use electrospray ionization mass spectrometry to quantify >100 phospholipid (PL) components in detergent-resistant membrane (DRM) domains that are related to ordered membrane compartments commonly known as lipid rafts. We previously compared PL compositions of DRMs with plasma membrane vesicles and whole cell lipid extracts from RBL mast cells, and we made the initial observation that antigen stimulation of IgE receptors (FcepsilonRI) causes a significant change in the PL composition of DRMs [Fridriksson, E. K., et al. (1999) Biochemistry 38, 8056-8063]. We now characterize the signaling requirements and time course for this change, which is manifested as an increase in the recovery of polyunsaturated PL in DRM, particularly in phosphatidylinositol species. We find that this change is largely independent of tyrosine phosphorylation, stimulated by engagement of FcepsilonRI, and can be activated by Ca(2+) ionophore in a manner independent of antigen stimulation. Unexpectedly, we found that inhibitors of actin polymerization (cytochalasin D and latrunculin A) cause a similar, but more rapid, change in the PL composition of DRMs in the absence of FcepsilonRI activation, indicating that perturbations in the actin cytoskeleton affect the organization of plasma membrane domains. Consistent with this interpretation, a membrane-permeable stabilizer of F-actin, jasplakinolide, prevents antigen-stimulated changes in DRM PL composition. These results are confirmed by a detailed analysis of multiple experiments, showing that receptor and cytochalasin D-stimulated changes in DRM lipid composition follow first-order kinetics. Analysis in terms of the number of double bonds in the fatty acid chains is valid for total PL of the major headgroups and for headgroups individually. In this manner, we show that, on average, concentrations of saturated or monounsaturated PL decrease in the DRM, whereas concentrations of PL with two or more double bonds (polyunsaturated PL) increase due to cytoskeletal perturbation. We find that these changes are independent of fatty acid chain length. Our mass spectrometric analyses provide a detailed accounting of receptor-activated alterations in the plasma membrane that are regulated by the actin cytoskeleton.

γ-Actin is required for cytoskeletal maintenance but not development

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.

Manipulating Actin Dynamics Affects Human In Vitro Decidualization1

The differentiation of uterine stromal fibroblasts into decidual cells is critical for establishing pregnancy. This process, called decidualization, requires the reorganization of the actin cytoskeleton, which mainly depends on actin dynamics and the phosphorylation status of the myosin light chain. We manipulated actin dynamics with jasplakinolide (100 nM) and latrunculin B (1 microM), both of which significantly inhibited the synthesis of decidualization markers induced by 6 days of treatment with embryo-mimicking stimulus interleukin 1beta (IL1B) and steroid hormones (SHs; 17beta-estradiol and medroxyprogesterone acetate) in the human uterine fibroblast (HuF) in vitro model. However, only jasplakinolide had long-lasting effects on the G-actin:F-actin ratio and prevented decidualization induced by the artificial stimulus cAMP (and SHs). Actin-binding protein cofilin mainly colocalized with G-actin in the nucleus as well as the cytoplasm. Only some spots of colocalization between cofilin and F-actin were detected in the cytoplasm. Brief extraction of cytosolic proteins from living cells revealed that in cells treated with IL1B or cAMP (and SHs) for 6 days, cofilin was mainly detected in the nucleus. The translocation of cofilin from cytosol to nucleus was also detected in HuFs treated for 12 days with SHs, IL1B and SHs, and cAMP and SHs. The same significant translocation was confirmed in primary baboon stromal uterine fibroblasts. We conclude that changes in actin dynamics, particularly the stabilization of F-actin, have a significant negative impact on decidualization, and the translocation of cofilin to the nucleus is a key feature of this process in the primate.

HS1 and Itk cooperate to regulate actin remodeling and Ca2+ signaling in T cells (35.28)

Abstract Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We have shown that the actin regulatory protein HS1 is required for the stabilization of F-actin and Vav1 at the immunological synapse (IS) and for efficient Ca2+ responses. The Tec family kinase Itk also regulates actin responses, in part by facilitating the localization of Vav1 at the IS, suggesting that these proteins act in the same pathway. Using video microscopy, we found that T cells lacking Itk or HS1 behaved similarly, extending unstable actin-rich protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's SH2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, HS1 failed to localize to the IS. Since Itk is required for PLCγ1 phosphorylation and Ca2+ store release, we examined the Ca2+ signaling pathway in HS1 knockout T cells in greater detail. T cells lacking HS1 exhibited abnormal Ca2+ store release. These cells showed normal phosphorylation but altered localization of PLCγ1. We conclude that Itk-dependent recruitment of HS1 is required for actin stabilization at the IS, and directs the localization of PLCγ1. Supported by NIH F31-AI071385 to EC and NIH R01-A1065644 to JKB

The cytoprotective role of Ras in complement-mediated glomerular epithelial cell injury

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to loss of glomerular permselectivity and proteinuria. Incubation of cultured GEC with antibody and serially-increasing concentrations of complement induced cytotoxicity in a dose-dependent manner. Stable expression of constitutively-active Ras (V 12Ras) in GEC attenuated injury significantly. In the V 12Ras-expressing GEC, disruption of the F-actin cytoskeleton with latrunculin B or swinholide A, or stabilization of F-actin with jasplakinolide reversed the cytoprotective effect of V 12Ras. GEC displayed cortical F-actin; V 12Ras-expressing GEC showed smaller and more rounded morphology, and decreased activity of the Rho GTPase, Rac1, compared with control GEC. Thus, the protective effect of V 12Ras is dependent on remodeling of the actin cytoskeleton, and may be associated with a reduction in Rac activity, thereby altering the equilibrium in the activities of Rho GTPases. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury.

Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

Cofilin translocation to the nucleus is a key feature of decidualization

Decidualization (D), the differentiation of stromal fibroblasts (SF) into decidual cells, is critical for pregnancy establishment. D requires the reorganization of the actin cytoskeleton that is governed by phosphorylation (P) of myosin light chain and actin dynamics. Manipulation of actin dynamics by jasplakinolide prevents D of human SF. Actin dynamics are regulated by actin‐binding proteins, one of which is cofilin. The activity of cofilin depends on its P, which is regulated by LIMK1 and LIMK2. D of human SF is associated with changes in the distribution and activity of the proteins of the LIMK‐cofilin pathway. D induced by a stimulus of embryonic origin, interleukin‐1β (IL‐1β) and steroid hormones (H), is accompanied by an upregulation of LIMK1, a downregulation of LIMK2 and the translocation of inactivated (phosphorylated) cofilin from the nucleus into the cytosol. On the other hand, D induced by an artificial stimulus cAMP (with H) is characterized by a significant decrease in phosphorylated cofilin and unchanged amounts of LIMK1 and LIMK2. Despite the differences in molecular mechanisms underlying the D induced by H alone, IL‐1β+H or cAMP+H, the most striking observation during D is the translocation of cofilin from the cytosol to the nucleus. Taken together, the stabilization of F‐actin significantly inhibits D; and the translocation of cofilin to the nucleus is a key feature of D. Supported by HD044713

Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia–reperfusion-induced endothelial cell apoptosis

Activation of cytoskeleton regulator Rho-kinase during ischemia–reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R2 = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.

Coronin-1A Stabilizes F-Actin by Bridging Adjacent Actin Protomers and Stapling Opposite Strands of the Actin Filament

Coronins are F-actin-binding proteins that are involved, in concert with Arp2/3, Aip1, and ADF/cofilin, in rearrangements of the actin cytoskeleton. An understanding of coronin function has been hampered by the absence of any structural data on its interaction with actin. Using electron microscopy and three-dimensional reconstruction, we show that coronin-1A binds to three protomers in F-actin simultaneously: it bridges subdomain 1 and subdomain 2 of two adjacent actin subunits along the same long-pitch strand, and it staples subdomain 1 and subdomain 4 of two actin protomers on different strands. Such a mode of binding explains how coronin can stabilize actin filaments in vitro. In addition, we show which residues of F-actin may participate in the interaction with coronin-1A. Human nebulin and Xin, as well as Salmonella invasion protein A, use a similar mechanism to stabilize actin filaments. We suggest that the stapling of subdomain 1 and subdomain 4 of two actin protomers on different strands is a common mechanism for F-actin stabilization utilized by many actin-binding proteins that have no homology.

Integrin-linked Kinase Regulates N-WASp-mediated Actin Polymerization and Tension Development in Tracheal Smooth Muscle

The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a beta-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and alpha-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and alpha-parvin form a stable cytosolic complex and that the ILK.PINCH.alpha-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with beta-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK.PINCH.alpha-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.

Tmod3 regulates polarized epithelial cell morphology

Although the role of the actin cytoskeleton in morphogenesis of polarized epithelial sheets is generally accepted as centrally important, the regulation of actin dynamics in this process remains unclear. Here, we show that the pointed-end capping protein Tmod3 contributes to epithelial cell shape within confluent monolayers of polarized epithelial cells. Tmod3 localizes to lateral cell membranes in polarized epithelia of several cell types. Reduction of Tmod3 levels by shRNA leads to a loss of F-actin and tropomyosins from lateral cell membranes, and a decrease in epithelial cell height, without effects on localisation of tight junction or adherens junction proteins, or any apparent changes in cell-cell adhesion. Instead, distribution of alphaII-spectrin on lateral membranes is disrupted upon reduction of Tmod3 levels, suggesting that loss of Tmod3 function leads to destabilization and disassembly of tropomyosin-coated actin filaments followed by disorganization of the spectrin-based membrane skeleton on lateral membranes. These data demonstrate for the first time a role for pointed-end capping in morphology regulation of polarized epithelial cells through stabilization of F-actin on lateral membranes. We propose that Tmod3-capped tropomyosin-actin filaments provide crucial links in the spectrin membrane skeleton of polarized epithelial cells, enabling the membrane skeleton to maintain cell shape.

Role of Actin Cytoskeletal Dynamics in Activation of the Cyclic AMP Pathway and HWP1 Gene Expression in Candida albicans

Changes in gene expression during reversible bud-hypha transitions of the opportunistic fungal pathogen Candida albicans permit adaptation to environmental conditions that are critical for proliferation in host tissues. Our previous work has shown that the hypha-specific adhesin gene HWP1 is up-regulated by the cyclic AMP (cAMP) signaling pathway. However, little is known about the potential influences of determinants of cell morphology on HWP1 gene expression. We found that blocking hypha formation with cytochalasin A, which destabilizes actin filaments, and with latrunculin A, which sequesters actin monomers, led to a loss of HWP1 gene expression. In contrast, high levels of HWP1 gene expression were observed when the F-actin stabilizer jasplakinolide was used to block hypha formation, suggesting that HWP1 expression could be regulated by actin structures. Mutants defective in formin-mediated nucleation of F-actin were reduced in HWP1 gene expression, providing genetic support for the importance of actin structures. Kinetic experiments with wild-type and actin-deficient cells revealed two distinct phases of HWP1 gene expression, with a slow, actin-independent phase preceding a fast, actin-dependent phase. Low levels of HWP1 gene expression that appeared to be independent of stabilized actin and cAMP signaling were detected using indirect immunofluorescence. A connection between actin structures and the cAMP signaling pathway was shown using hyper- and hypomorphic cAMP mutants, providing a possible mechanism for up-regulation of HWP1 gene expression by stabilized actin. The results reveal a new role for F-actin as a regulatory agent of hypha-specific gene expression at the bud-hypha transition.

The flare Gene, Which Encodes the AIP1 Protein of Drosophila, Functions to Regulate F-Actin Disassembly in Pupal Epidermal Cells

Adult Drosophila are decorated with several types of polarized cuticular structures, such as hairs and bristles. The morphogenesis of these takes place in pupal cells and is mediated by the actin and microtubule cytoskeletons. Mutations in flare (flr) result in grossly abnormal epidermal hairs. We report here that flr encodes the Drosophila actin interacting protein 1 (AIP1). In other systems this protein has been found to promote cofilin-mediated F-actin disassembly. In Drosophila cofilin is encoded by twinstar (tsr). We show that flr mutations result in increased levels of F-actin accumulation and increased F-actin stability in vivo. Further, flr is essential for cell proliferation and viability and for the function of the frizzled planar cell polarity system. All of these phenotypes are similar to those seen for tsr mutations. This differs from the situation in yeast where cofilin is essential while aip1 mutations result in only subtle defects in the actin cytoskeleton. Surprisingly, we found that mutations in flr and tsr also result in greatly increased tubulin staining, suggesting a tight linkage between the actin and microtubule cytoskeleton in these cells.

Repeated Allergen Inhalation Induces Cytoskeletal Remodeling in Smooth Muscle from Rat Bronchioles

Airway hyperresponsiveness (AHR) is associated with airway wall structural remodeling and alterations in airway smooth muscle (ASM) function. Previously, in bronchioles from Brown Norway rats challenged by repeated ovalbumin (OVA) inhalation, we have reported increased force generation and depletion of smooth muscle contractile proteins. Here, we investigated if cytoskeletal changes in smooth muscle could account for this paradox. Sensitized rats (n = 5/group) were repeatedly challenged with OVA or saline, and the lungs were removed 24 h after the last challenge. Levels of globular (G) and filamentous (F) actin in bronchioles were determined by DNase I inhibition and contraction assessed in intact small bronchioles using a myograph. DNase I inhibition assays showed that G-actin monomers were more abundant ( approximately 1F:2G) in extracts from resting small bronchioles from OVA- or saline-challenged animals. However, while contractile protein levels in bronchioles were reduced by OVA (P < 0.05), the proportion of F:G actin was 1.8-fold greater compared with saline challenge (P < 0.05). Consistent with induction of F-actin after OVA challenge, increases in maximum tension development to carbachol or KCl in small bronchioles from OVA-challenged animals were abrogated (P < 0.01) by actin cytoskeleton disruption with 0.5 microM latrunculin A. Cytoskeletal stabilization of F-actin with 0.1 microM jasplakinolide potentiated maximum contractions to carbachol or KCl (P < 0.05) in bronchioles from OVA- but not saline-treated rats. We conclude that alterations in the composition and/or arrangement of the contractile apparatus after OVA exposure confer enhanced contractile responses, possibly as a result of increased F-actin content. Such a mechanism may have relevance for AHR found in allergic asthma.

Regulation of TGF-β1/MAPK-mediated PAI-1 gene expression by the actin cytoskeleton in human mesangial cells

The importance of transforming growth factor-β1 (TGF-β1) 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-β1/MAPK-mediated PAI-1 expression in human mesangial cells. Examination of the distributions of actin filaments (F-actin), α-actinin, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunofluorescence and immunoprecipitation revealed that ERK and JNK associate with α-actinin along F-actin and that TGF-β1 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-β1-stimulated conditions. Stabilizing actin prevented dephosphorylation of ERK and JNK. ERK and JNK inhibitors and overexpressed dominant negative mutants antagonized the ability of TGF-β1 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-β1 to stimulate PAI-1 expression through a mechanism dependent on the activation of MAPK/AP-1 pathways.

Antagonistic Effects of Cofilin, Beryllium Fluoride Complex, and Phalloidin on Subdomain 2 and Nucleotide-Binding Cleft in F-Actin

Cofilin/ADF, beryllium fluoride complex (BeFx), and phalloidin have opposing effects on actin filament structure and dynamics. Cofilin/ADF decreases the stability of F-actin by enhancing disorder in subdomain 2, and by severing and accelerating the depolymerization of the filament. BeFx and phalloidin stabilize the subdomain 2 structure and decrease the critical concentration of actin, slowing the dissociation of monomers. Yeast cofilin, unlike some other members of the cofilin/ADF family, binds to F-actin in the presence of BeFx; however, the rate of its binding is strongly inhibited by BeFx and decreases with increasing pH. The inhibition of the cofilin binding rate increases with the time of BeFx incubation with F-actin, indicating the existence of two BeFx-F-actin complexes. Cofilin dissociates BeFx from the filament, while BeFx does not bind to F-actin saturated with cofilin, presumably because of the cofilin-induced changes in the nucleotide-binding cleft of F-actin. These changes are apparent from the increase in the fluorescence intensity of F-actin bound ɛ-ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of ɛ-ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2.