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PubMed2006

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B.

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.

Actin Cytoskeleton↗

PubMed2003

Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs.

The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.

Actins↗

PubMed1999

Protrusive growth from giant liposomes driven by actin polymerization.

Development of protrusions in the cell is indispensable in the process of cell motility. Membrane protrusion has long been suggested to occur as a result of actin polymerization immediately beneath the cell membrane at the leading edge, but elucidation of the mechanism is insufficient because of the complexity of the cell. To study the mechanism, we prepared giant liposomes containing monomeric actin (100 or 200 microM) and introduced KCl into individual liposomes by an electroporation technique. On the electroporation, the giant liposomes deformed. Most importantly, protrusive structure grew from the liposomes containing 200 microM actin at rates (ranging from 0.3 to 0.7 micrometer/s) similar to those obtained in the cell. The deformation occurred in a time range (30 approximately 100 s) similar to that of actin polymerization monitored in a cuvette (ca. 50 s). Concomitant with deformation, Brownian motion of micron-sized particles entrapped in the liposomes almost ceased. From these observations, we conclude that actin polymerization in the liposomes caused the protrusive formation.

Actins↗

PubMed1999

Heterogeneity in F-actin polymerization of cord blood polymorphonuclear leukocytes stimulated by N-formyl-methionyl-leucyl-phenylalanine.

BACKGROUND: To elucidate the mechanism responsible for defects of polymorphonuclear leukocyte (PMNL) chemotaxis of neonates, we determined actin polymerization of NBD (7-nitrobenz-2-oxa-diazol)-phallacidin-stained PMNL following stimulation with either N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA) in cord blood and adult controls. METHODS: We measured F-actin content in PMNL stained with NBD-phallacidin using flow cytometry. RESULTS: Relative F-actin polymerization, that is, a ratio of stimulated F-actin to basal F-actin, was significantly decreased in cord blood PMNL when compared with that of adult PMNL. Distribution of fMLP-stimulated F-actin showed a bimodal pattern, while adult PMNL disclosed a single pattern. Following stimulation with PMA, however, F-actin levels were equal in both cord and adult PMNL. A fluorescein isothiocyanate-conjugated fMLP receptor assay showed no significant difference in binding capacity of fMLP receptors between adult and cord PMNL. CONCLUSION: These results indicate that a deficiency of PMNL chemotaxis in neonates may be due, in part, to decreased relative F-actin polymerization, which may be caused by functional heterogeneity in cord blood PMNL.

Actins↗

PubMed2001

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.

Actins↗

PubMed1988

Actin polymerization and pseudopod extension during amoeboid chemotaxis.

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.

Actins↗

PubMed2004

Cortactin and Crk cooperate to trigger actin polymerization during Shigella invasion of epithelial cells.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation-dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding-deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk-cortactin complex in actin polymerization downstream of tyrosine kinase signaling.

Actins↗

PubMed2001

The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia.

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2-4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility.

Actins↗

PubMed1994

ADP-ribosylation of the GTP-binding protein Rho by Clostridium limosum exoenzyme affects basal, but not N-formyl-peptide-stimulated, actin polymerization in human myeloid leukaemic (HL60) cells.

Treatment of human myeloid leukaemic (HL60) cells with Clostridium limosum exoenzyme, which inactivates the small GTP-binding protein Rho by ADP-ribosylation, decreased the basal F-actin content. Inhibition of F-actin occurred after long-term treatment (24 h) of intact HL60 cells or after introduction of the toxin by electropermeabilization in a toxin-concentration-dependent manner. Concomitantly with the decrease in the basal F-actin content, the GTP-binding protein Rho was ADP-ribosylated in intact cells. However, Clostridium limosum toxin had no inhibitory effect on N-formyl-peptide-induced actin polymerization. Moreover, the relative N-formyl-peptide-stimulated polymerization was substantially enhanced in cells treated with Clostridium limosum transferase. In contrast with Clostridium limosum exoenzyme, component C21 of the Clostridium botulinum C2 toxin, which ADP-ribosylates G-actin, depolymerized basal F-actin and inhibited N-formyl-peptide-induced actin polymerization in electropermeabilized HL60 cells. These findings indicate that Rho proteins are involved in the basal, but not the ligand-evoked, actin polymerization in HL60 cells.

ADP Ribose Transferases↗

PubMed1992

Actin polymerization contributes to neutrophil chemotactic dysfunction following thermal injury.

The agent(s) and mechanism(s) responsible for suppression of neutrophil chemotaxis in association with major thermal injury have not been identified. We have proposed that the reduced random motility characterizing patients' cells may contribute to their generalized chemotactic dysfunction. Here we report that actin polymerization may be responsible for the loss of neutrophil motility associated with major thermal injury. Using a fluorescent ligand specific for polymerized or filamentous actin (NBD-phallacidin) in conjunction with flow cytometry, we have discovered that peripheral blood and exudate neutrophils from patients with major thermal injury contain increased levels of actin in a stably polymerized form. Because cyclic polymerization and depolymerization of actin is essential to cell motility, we suggest that actin polymerization may contribute in a major way to the attenuation of neutrophil random and chemotactic functions induced by major thermal injury.

Actin Cytoskeleton↗

PubMed1984

Angiotensin II stimulation of granuloma macrophage phagocytosis and actin polymerization in murine schistosomiasis mansoni.

In murine schistosomiasis, components of the renin-angiotensin system are present in isolated liver granulomas. Furthermore, granuloma macrophages have receptors for angiotensin II (AII). These observations with other evidence suggest that angiotensins function in immunoregulation. Since AII can stimulate actin-myosin interactions and since macrophages have contractile proteins, it was determined whether AII could alter the phagocytic activity of granuloma macrophages. AII, at concentrations which saturate AII receptors, enhanced phagocytic activity for both unopsonized and opsonized sheep red blood cells (RBC). Using a DNase inhibitory assay, it was also shown that similar concentrations of AII stimulated intracellular actin polymerization. Cytochalasin B, which impedes actin filament interactions, inhibited AII-stimulated phagocytosis. AII analogs, with little-known agonist activity, blocked AII-stimulated actin polymerization and phagocytosis of RBC and RBC opsonized with IgG. These data support the contention that AII binds to AII receptors on the macrophage and stimulates phagocytosis possibly through polymerization of intracellular actin.

Actins↗

PubMed1981

Change of reactivity of lysine residues upon actin polymerization.

The reactivity of lysine residues of actin was measured by a surface labeling method--limited reductive methylation. After labeling, actin was subjected to CNBr and enzymatic cleavage, and all lysines were obtained either singly in a peptide or as a free residue. The specific activity of each lysine was taken as the measure of its reactivity. In actin denatured in 8 M urea, the reactivity of each lysine residue is approximately equal whereas those in G-actin fall into three categories: Lys-61 and Lys-113 are the most reactive ones; Lys-18, -213, -215, -314, and -358 are hardly reactive; the remainder, including Lys-50, -68, -84, -118, -191, -237, -283, -290, -325, -327, -335, and -372, are moderately reactive. The least reactive ones are probably buried in the native G-actin and all the others are most likely on the surface. Upon actin polymerization the reactivities of Lys-61, -68, -113, and -283 are significantly reduced while that of Lys-335 is strikingly enhanced. The decrease in reactivity could be readily explained if these residues were located in the monomer-monomer contact area although a polymerization-induced conformational change cannot be excluded. Such a conformational change may be invoked to explain the increase in the reactivity of Lys-335. Alternatively, the latter may be interacting with the bound ATP of G-actin, and the increased reactivity might be directly attributable to the loss of gamma-P for ATP accompanying polymerization.

Actins↗

PubMed1991

Actin polymerization promoted by a heptapeptide, an analog of the actin-binding S site on myosin head.

Polymerization of G-actin to F-actin was indicated by an increase in light-scattering intensity after the addition of a heptapeptide (Ile-Arg-Ile-Cys(MT)-Arg-Lys-Gly-OEt), an analog of the actin-binding S-site on S-1 heavy chain. The half-maximal concentration of the heptapeptide which induced an increase in the light-scattering intensity at 25 degrees C was about 110 microM, which was in the range of the dissociation constant of this peptide with F-actin. The polymerization of G-actin to F-actin by binding of the heptapeptide was further demonstrated by ultracentrifugal separation, Pi liberation, and electron microscopy. The polymerization of G-actin was induced only by the heptapeptide, but not by fragments of the heptapeptide. The well known acceleration of polymerization of G-actin by the myosin head may be due to the binding of G-actin with the S-site on the myosin head.

Actins↗

PubMed2003

Two phases of actin polymerization display different dependencies on PI(3,4,5)P3 accumulation and have unique roles during chemotaxis.

The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of Dictyostelium discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P3/PI(3,4)P2 reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild-type cells but partially restore polarity and chemotactic response in pten- cells. Surprisingly, the fast phase of PI(3,4,5)P3 accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis.

Actins↗

PubMed1993

Inhibitory effects of azelastine hydrochloride on Ca2+ influx, actin polymerization and release of eosinophil cationic protein of an eosinophilic leukaemia cell line EoL-1.

The inhibitory effects of azelastine hydrochloride on PAF-induced and fMLP-induced Ca2+ influx, actin polymerization and calcium ionophore A23187-induced and aggregated IgG-induced release of eosinophil cationic protein (ECP) of an eosinophilic leukaemia cell line, EoL-1, were examined. EoL-1 cells cultured with 0.2 mM dibutyryladenosine-cyclic monophosphate for 48 hours showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) and actin polymerization when stimulated by PAF and fMLP. Azelastine hydrochloride inhibited PAF-induced and fMLP-induced Ca2+ influx ([Ca2+]i) in a dose-dependent manner with an IC50 of 1 x 10(-8) M and 1 x 10(-7) M, respectively. It also inhibited PAF-induced and fMLP-induced actin polymerization in a dose-dependent manner up to 40% and 30%, respectively. EoL-1 cells were differentiated to contain ECP in their eosinophilic granules when cultured for 9 days with supernatants of a human adult T cell leukaemia cell line, HIL-3 (HIL-3 sup). Calcium ionophore A23187 and aggregated IgG induced the secretion of ECP by EoL-1 cells. Azelastine hydrochloride inhibited the secretion of ECP in a dose-dependent manner. These inhibitory effects were seen even at therapeutic concentrations of 10(-8) M to 10(-9) M. These results indicate that the therapeutic effects of azelastine hydrochloride as an anti-allergic agent may include inhibition of the accumulation of eosinophils into the locus of allergic inflammation and of the release of cytotoxic granules from eosinophils.

Actins↗

PubMed2003

Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses.

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite-mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN-1) modified actin in a concentration-dependent manner, resulting in an inhibition of globular-actin polymerization and filamentous-actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU-101033E, which has been shown previously to specifically block peroxynitrite-mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite-treated actin demonstrated a concentration-dependent increase in nitrotyrosine, which was also blocked by PNU-101033E. Activation of neutrophils in the presence of a nitric oxide donor (S-nitroso-N-acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite-treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin-dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN-1 treatment in a concentration-dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin-dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Actins↗

PubMed2000

Influence of the effector peptide of MARCKS-related protein on actin polymerization: a kinetic analysis

The members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with membranes, protein kinase C, and calmodulin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. This domain is also involved in the interaction between MARCKS/MRP and actin. In this article we show that a peptide corresponding to the effector domain of MRP, the effector peptide, strongly influences the dynamics of actin polymerization. Depending on the stoichiometric ratio of effector peptide to actin the peptide either accelerates or retards the actin polymerization process, which takes place in the presence of near-physiological salt concentrations. A model is developed in which this phenomenon is explained by two independent nucleation processes involving free actin monomers and peptide-bound actin monomers, respectively. As a control, a possible regulatory mechanism has been investigated: we show that calmodulin inhibits the actin polymerizing activity of the MRP effector peptide, thereby validating our model approach.

Journal Article↗

PubMed1991

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin.

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.

Actin Depolymerizing Factors↗