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At least 595 records · Page 33Linked to original sources
PubMed1985

Hormone-induced actin polymerization in rat hepatoma cells and human leucocytes.

Treatment of rat hepatoma cells with insulin, glucagon, thyroxine (T4) and triiodothyronine (T3) caused a concentration-dependent decrease in the monomeric actin content as measured by the deoxyribonuclease-I inhibition assay. Similarly, human peripheral blood neutrophils responded with a decrease in monomeric actin content when stimulated with T4, T3 and the adrenergic agonists phenylephrine and isoprenaline. The effect of phenylephrine could be blocked by phentolamine, demonstrating the specificity of the interaction. These observations suggest that hormone-induced actin changes might be an important event in response to both cell-surface-reactive hormones, such as insulin, glucagon and adrenergic agents, and those hormones that act through intracellular receptors, such as thyroid hormones. It is suggested that changes in actin state may have a role in metabolic regulation and cell growth.

Actins↗
PubMed1997

Inhibitors of actin polymerization and calmodulin binding enhance protein kinase C-induced translocation of MARCKS in C6 glioma cells.

MARCKS (myristoylated alanine-rich C-kinase substrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC alpha to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC alpha in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.

Actins↗
PubMed1975

Preparation and purification of polymerized actin from sea urchin egg extracts.

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

Actins↗
PubMed1996

Reconstitution of chemokine-induced actin polymerization in undifferentiated human leukemia cells (HL-60) by heterologous expression of interleukin-8 receptors.

The chemokines interleukin-8 (IL-8) and GRO alpha bind in neutrophils to the interleukin-8 receptor alpha and beta (IL-8R alpha and beta) triggering reorganization of the actin cytoskeleton and activation of phospholipase C (PLC). Reconstitution of chemokine-induced activation of PLC indicated coupling of IL-8R alpha and beta to pertussis toxin-insensitive G alpha 16-proteins as well as to pertussis toxin-sensitive G alpha i2- or G alpha i3-proteins. To identify the signal transduction mechanisms of chemokine-induced actin response, undifferentiated human leukemia cells (HL-60 cells) constitutively expressing G alpha 16-, G alpha i2- and G alpha i3-proteins were chosen for reconstitution studies. Expression of recombinant receptors after transfection of the cells with the cDNA of IL-8R alpha and beta was confirmed by binding studies with radiolabeled ligands. IL-8R alpha bound IL-8 with high affinity (Kd approximately 1 nM) and GRO alpha with low affinity (Kd approximately 1 microM), whereas IL-8R beta bound both IL-8 and GRO alpha with high affinity (Kd approximately 1nM). Flow cytometric actin measurements indicated that high affinity ligand-receptor interactions in both receptor transfectants displayed inducible responses. Pretreatment of transfectants with pertussis toxin caused ADP-ribosylation of G-proteins and blocked chemokine-induced polymerization, indicating involvement of G alpha i2- or G alpha i3-proteins, but not G alpha 16-proteins in this response.

Actins↗
PubMed2004

Host cell actin polymerization is required for cellular retention of Trypanosoma cruzi and early association with endosomal/lysosomal compartments.

One of the hallmarks of Trypanosoma cruzi invasion of non-professional phagocytes is facilitation of the process by host cell actin depolymerization. Host cell entry by invasive T. cruzi trypomastigotes is accomplished by exploiting a cellular wound repair process involving Ca(2+)-regulated lysosome exocytosis (i.e. lysosome-dependent) or by engaging a recently recognized lysosome-independent pathway. It was originally postulated that cortical actin microfilaments present a barrier to lysosome-plasma membrane fusion and that transient actin depolymerization enhances T. cruzi entry by increasing access to the plasma membrane for lysosome fusion. Here we demonstrate that cytochalasin D treatment of host cells inhibits early lysosome association with invading T. cruzi trypomastigotes by uncoupling the cell penetration step from lysosome recruitment and/or fusion. These findings provide the first indication that lysosome-dependent T. cruzi entry is initiated by plasma membrane invagination similar to that observed for lysosome-independent entry. Furthermore, prolonged disruption of host cell actin microfilaments results in significant loss of internalized parasites from infected host cells. Thus, the ability of internalized trypomastigotes to remain cell-associated and to fuse with host cell lysosomes is critically dependent upon host cell actin reassembly, revealing an unanticipated role for cellular actin remodelling in the T. cruzi invasion process.

Actin Cytoskeleton↗
PubMed2002

Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae.

Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. We found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin "tail" formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1995

Selective responses (actin polymerization, shape changes, locomotion, pinocytosis) to the PKC inhibitor Ro 31-8220 suggest that PKC discriminately regulates functions of human blood lymphocytes.

The results suggest that protein kinase C (PKC) plays a pivotal role in the control of F-actin levels, locomotion, pinocytosis, and cell shape in lymphocytes. The PKC inhibitor Ro 31-8220 elicits a high proportion of polarized (ED50 = 1.5 x 10(-6) M) and locomoting cells and reduces the relative amount of F-actin (by 29% at 10(-5) M) in initially resting cells. Phorbol myristate acetate (PMA) counterbalances the polarizing effect of Ro 31-8220. This indicates that the spherical shape and the F-actin content of resting cells are maintained by constitutive PKC activity. PMA-induced increases in fluid pinocytosis, F-actin content, and formation of nonpolar cells with surface protrusion are suppressed by Ro 31-8220 (IC50 = 2-4 x 10(-7) M). Spherical cells and, at higher concentrations (ED50 = 3.3 x 10(-6) M), polarized cells are formed instead. As a result, lymphocyte function switches from fluid pinocytosis to cell polarity and locomotion. The data indicate that PKC is instrumental in selectively switching lymphocyte function between resting state, locomotor activity, and fluid pinocytosis. Ro 31-8220 is extremely potent in stimulating lymphocyte polarity and locomotion (B and T cells). It acts faster and/or produces a higher proportion of polarized lymphocytes than other available agonists. It may thus be used as a tool in further experiments requiring locomoting lymphocytes.

Actins↗
PubMed1983

Actin polymerization and synthesis in cultured neurones.

The protein content of sympathetic neurones explanted from 10-11-day old chick embryos into culture medium containing nerve growth factor (NGF) increases steadily from about 100 to about 400 pg/cell in 7 days. Actin remains at close to 5% of the total protein during this period, but the proportion of unpolymerized actin falls. As measured by the inhibition of DNase I activity, rounded neurones without neurites contain 70 +/- 7% of their total actin in monomeric form, whereas cells in mature, neurite-bearing cultures contain 39 +/- 7%. When allowance is made for the increase in size of the neuronal cell bodies, the actin present in the neurites ('axons') alone is found to be almost entirely in filamentous form. Cultures exposed to radioactive leucine rapidly incorporate radioactivity into both sedimentable and non-sedimentable forms of actin. Actin-specific activities in the two fractions--estimated after isolation of the actin on small DNase I--Sepharose affinity columns--are similar after labelling for less than 1 h. Direct incorporation of newly-synthesized actin into filaments is suggested from these results. Pulse-chase experiments show that non-sedimentable protein in cultured sympathetic neurones turns over more rapidly than sedimentable protein. However, this is not true for actin, which shows a similar specific activity in sedimentable and non-sedimentable forms--even after 6 days of cold chase. This anomalous behaviour is simply explained by an exchange of actin molecules between filamentous and non-filamentous forms. Control experiments indicate that exchange does not occur to this degree during preparation of subcellular fractions. It is consequently attributed to exchange processes in the living cell.

Actins↗
PubMed1997

Relationship of ligand-receptor dynamics to actin polymerization in RBL-2H3 cells transfected with the human formyl peptide receptor.

The human formyl peptide receptor (FPR) expressed in RBL-2H3 transfectants (RBL[FPR]) behaves qualitatively like the FPR expressed by neutrophils except that it causes sustained F-actin accumulation and cell shape change responses on formyl peptide stimulation. These sustained responses were not accounted for by changes in the transfected receptor's ability to interact with ligand or by receptor density. Signal transduction pathways of transfected and neutrophil FPRs are apparently similar. In transfected cells, dissociation of ligand is sensitive to guanine nucleotide, the G protein is pertussis toxin-sensitive, FPR and G protein appear to be precoupled, the F-actin response is stimulated with the same dose-response profile as in neutrophils, and the F-actin accumulation response is directly regulated by the FPR, even long after initial stimulation. Potentially significant differences between neutrophil and transfected FPR were found when receptor processing was measured. In neutrophils, practically 100% of the FPR is converted to forms that dissociate slowly from ligand and are inactive in signal transduction within 2 min of ligand stimulation. By contrast, 20% or more of transfected FPR remains rapidly dissociating even 5 min after stimulation. Although 80% of neutrophil FPR is internalized by 5 min after stimulation, transfected FPR appears to plateau at 50-60% internalized. Because actin responses in neutrophils are regulated by a small number of active receptors, the inefficiency of receptor inactivation in RBL(FPR) transfectants may account for the prolonged F-actin accumulation response.

Actins↗
PubMed2006

Host protein interactions with enteropathogenic Escherichia coli (EPEC): 14-3-3tau binds Tir and has a role in EPEC-induced actin polymerization.

Enteropathogenic Escherichia coli (EPEC) cause infantile diarrhoea and are characterized by their ability to produce attaching and effacing lesions on the surface of intestinal epithelial cells. EPEC employ a filamentous type III secretion system to deliver effector molecules that subvert mammalian cell function to generate actin- and cytokeratin-rich pedestals beneath adherent bacteria. Tir is a major effector protein that is delivered to the plasma membrane of the eukaryotic cell where it acts as the receptor for the bacterial adhesin intimin. Host cell proteins that are recruited to the site of intimate attachment include focal adhesion and cytoskeletal proteins that contribute to pedestal formation. We have used Tir as bait in a yeast two-hybrid screen to identify the protein 14-3-3tau as a binding partner. 14-3-3 proteins are a family of adaptor proteins that modulate protein function in all eukaryotic cells. Here we demonstrate that the tau isoform (also known as theta) of 14-3-3 can bind specifically to Tir in a phosphorylation-independent manner, and that the interaction occurs during the infection process by co-immunoprecipitation of the partners from infected HeLa cell extracts. 14-3-3tau is recruited to the site of the pedestal (3 h after infection) and can decorate attached EPEC in the later stages of the infection process (5-7 h). Pedestal formation can be impaired by depletion of cellular 14-3-3tau using small interfering RNAs. This study indicates a direct functional role for the 14-3-3tau:Tir interaction and is the first to demonstrate the association of a host protein with the surface of EPEC.

14-3-3 Proteins↗
PubMed1993

Signal pathway regulation of interleukin-8-induced actin polymerization in neutrophils.

Interleukin-8 (IL-8), a recently described peptide cytokine, is a neutrophil chemoattractant and activator that exerts effects similar to fMLP, yet their receptors and their roles in pathophysiology differ. The effect of IL-8 on the neutrophil cytoskeleton has not been well studied; therefore, we compared and contrasted the effects of IL-8 and fMLP on neutrophil actin conformation and on the signal pathway regulation of actin responses. IL-8 caused a rapid, dose-dependent increase in neutrophil F-actin content within 30 seconds. The maximum increase was twofold. These changes were accompanied by the development of F-actin-rich pseudopods, as noted with fluorescence microscopy and scanning electron microscopy. Selected biochemical inhibitors were used to study the regulation of the IL-8-induced actin changes. Incubation of neutrophils with 2 micrograms/mL pertussis toxin resulted in a 67% inhibition of the IL-8-induced F-actin increase. The protein kinase C (PKC) inhibitors, staurosporine and H7, did not inhibit the increase in F-actin caused by IL-8. IL-8 caused a rapid increase in neutrophil intracellular calcium that could be completely inhibited by the chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPTA). However, BAPTA-treated neutrophils retained the ability to increase F-actin in response to IL-8. Similar results were seen with fMLP, indicating that, similar to fMLP, the IL-8-induced actin response is mediated through pertussis-toxin-sensitive G-proteins but is neither dependent on PKC nor increases in cytosolic calcium. Thus, although IL-8 and fMLP exert their effects on neutrophils through different receptors, the signal transduction pathways used and the effects on actin conformation and pseudopod formation are similar.

Actins↗