Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.     

Early events related with the behaviour of Trypanosoma cruzi within an endocytic vacuole in mouse peritoneal macrophages

Transmission electron microscopy was used to analyse the process of interaction of Trypanosoma cruzi with resident and activated mouse peritoneal macrophages. Initially, the parasites are located within a membrane-bounded endocytic vacuole. Lysosomes from the host cell fuse and discharge their content into the parasite-containing vacuole, as visualized by localization of horseradish peroxidase and acid phosphatase activity. Acridine orange was used to label secondary lysosomes in order to quantify the process of lysosome-phagosome fusion by fluorescence microscopy. The fusion index was higher for amastigote than for epimastigote and trypomastigote forms. Images were obtained showing that a few hours after ingestion of trypomastigote forms by the macrophages there is progressive disruption of the membrane lining the vacuole, until its complete disappearance.     

Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.     

Inhibition of liver mitochondrial monoamine oxidase activity by alkaloids isolated from Chelidonium and Macleaya and by their derivative drugs

It has been shown that the major alkaloids from plants Chelidonium majus L. and Macleaya (Bocconia) cordata and microcarpa, namely, berberine, sanguinarine, chelidonine, and drugs "Ukrain" (thiophosphoric acid derivative of a sum of the alkaloids isolated from Ch. majus L.) and "Sanguirythrine" (a mixture of the alkaloids sanguinarine and chelerythrine, w/w 3:7, isolated from Macleaya), are irreversible inhibitors of oxidative deamination reaction of serotonin and tyramine as substrates, catalyzed by rat liver mitochondrial monoamine oxidase (MAO). At the same time these substances do not influence the oxidative deamination reaction of benzylamine as substrate (in concentration 1 mM or less). The substrate specificity of this inhibition manifests that mainly the oxidative deamination reactions catalyzed by MAO form A are inhibited by the agents studied. Among the examined agents, alkaloid chelidonine and drug "Ukrain" are the strongest inhibitors of the reaction. Alkaloids berberine and sanguinarine and drug "Sanguirythrine" exhibit a weaker action. Judging from the data obtained, sanguinarine and chelerythrine appear to exert similar inhibitory effects in this reaction, since sanguinarine and "Sanguirythrine" have similar values of bimolecular rate constants of their interaction with mitochondrial MAO. As it is well known, the MAO inhibitors appear to be, as a rule, pronounced antidepressants. The combination of malignotoxicity and antidepressive activity in drug "Ukrain" seems to be favourable for its clinical applications.     

Effects of forskolin and cilostazol on clot retraction

We examined the effects of two inhibitors of aggregation of human platelets the, Forskolin and Cilostazol on clot retraction. Both substances suppressed clot retraction in a dose-dependent way. Both suppress platelet aggregation because of an increase in intercellular cyclic AMP, but there was no close correlations were shown between suppression rate for clot retraction and cyclic-AMP content in platelets in the clot in each substance. Furthermore, although it has been considered that actomyosin in platelets is a major contractile source for clot retraction and that failure of actin polymerization results suppression of clot retraction. As it was difficult to obtain active actin from platelets of the reagents on the polymerization. Cilostazol accelerated actin polymerization, whereas Forskolin did not. From these findings, it was considered that the effects of both substances on clot retraction could not be interpreted directly just by the increasing effect for intracellular cyclic-AMP. Clot retraction is consider to be a in vitro model of hemostasis and its contractile force is supplied from platelets (1,2,3). Experiments used prostaglandin E-1 revealed that elevation of cyclic-AMP (c-AMP) would regulate the clot retraction, and experiments used cytochalasin B demonstrated that actomyosin is responsible to the retraction (4,5). Many date demonstrate that elevation of c-AMP level suppresses platelet aggregation (6,7). c-AMP, therefore, should play a key role on platelet activation. Forskolin and Cilostazol are newly-developed reagents as a suppress for platelet functions. Pharmacological action of these substances have been interpreted to process increase effect for intracellular c-AMP of platelets(8,9). If so, both substances should show some effect on clot retraction. Under this assumption, we examined the effects.     

Effect of cAMP-elevating drugs on Ca2+ efflux and actin polymerization in peritoneal macrophages stimulated with N-formyl chemotactic peptide

To investigate intracellular cAMP inhibitory mechanisms related to migration of guinea-pig peritoneal macrophages, we examined the effects of cAMP-elevating drugs on the Ca²⁺ efflux and actin polymerization in macrophages stimulated with fMet-Leu-Phe, a chemotactic peptide. The stimulation with 1·10^?8 M fMet-Leu-Phe enhanced the Ca²⁺ efflux, and induced actin polymerization. Dibutyryl cAMP, theophylline and papaverine, which continuously increased the levels of intracellular cAMP, inhibited the enhancement of Ca²⁺ efflux and induction of actin polymerization by fMet-Leu-Phe. On the other hand, isoproterenol, which transiently increased the cAMP level, inhibited only the early phase of Ca²⁺ efflux and not the actin polymerization. As additions of both cAMP and cAMP-dependent protein kinase did not modify the Ca²⁺ uptake of phagocytic vesicles, the inhibition of Ca²⁺ efflux by these drugs may be due to the inhibition of the Ca²⁺ release from the intracellular store site(s). The cAMP-elevating drugs increased the monomeric actin content without change in the total actin content, indicating an induction of the depolymerization of filamentous actin. From these findings, we conclude that the inhibition of macrophage migration induced by cAMP may be due to the inhibition of both the increase of intracellular Ca²⁺ concentration and actin polymerization. Furthermore, the intracellular levels of cAMP probably play a role in regulating actin states in the macrophages.     

The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain

The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.     

12/15-lipoxygenase translocation enhances site-specific actin polymerization in macrophages phagocytosing apoptotic cells

The enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells.     

The effect of polyamines on lysosome fusion with phagosomes in mouse peritoneal macrophages

The influence of polyamines on the phagosome-lysosome fusion in murine peritoneal macrophages and on polymerization of G-actin from the rabbit muscle in vitro has been studied. Both natural polyamines (spermin, spermidin, putrescin) and synthetic phenyl derivates of polyamines (3,3'-diaminobensidin, 1,5-naphtalin diamine, 4,4'-diaminodiphenilmetan, dancylcadaverin) were used. Unlike the phenyl derivates of polyamines and putrescin, spermin and spermidin stimulate the phagosome-lysosome fusion to induce G-actin polymerization. Possible mechanisms of action of the above polyamines are discussed.     

A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion

Myoblast fusion is an essential step during muscle differentiation. Previous studies in Drosophila have revealed a signaling pathway that relays the fusion signal from the plasma membrane to the actin cytoskeleton. However, the function for the actin cytoskeleton in myoblast fusion remains unclear. Here we describe the characterization of solitary (sltr), a component of the myoblast fusion signaling cascade. sltr encodes the Drosophila ortholog of the mammalian WASP-interacting protein. Sltr is recruited to sites of fusion by the fusion-competent cell-specific receptor Sns and acts as a positive regulator for actin polymerization at these sites. Electron microscopy analysis suggests that formation of F-actin-enriched foci at sites of fusion is involved in the proper targeting and coating of prefusion vesicles. These studies reveal a surprising cell-type specificity of Sltr-mediated actin polymerization in myoblast fusion, and demonstrate that targeted exocytosis of prefusion vesicles is a critical step prior to plasma membrane fusion.     

Identification of a Distal GLUT4 Trafficking Event Controlled by Actin Polymerization

The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking, and fusion of GLUT4 vesicles with the plasma membrane (PM) represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the PM. In this study we have identified a previously uncharacterized role for cortical actin in the distal trafficking of GLUT4. Using high-frequency total internal reflection fluorescence microscopy (TIRFM) imaging, we show that insulin increases actin polymerization near the PM and that disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion, we found that defective actin remodeling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM, but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process.     

Effect of polyamine synthesis inhibitors separately and in combination with epidermal growth factor on fusion of lysosomes with phagosomes and F-actin level in mouse peritoneal macrophages

Effects of polyamine (PA) synthesis inhibitors--alpha-difluoromethylornithinchloride (DFMO) and alpha-methylornithinchloride (MO)--separately or in combination with the epidermal growth factor (EGF)--on lysosome-phagosome fusion (P-LF) and F-actin content in murine peritoneal macrophages were studied using fluorescent dye Acridine orange for lysosome labelling, FITC-phalloidin for F-actin, and yeast cells as a target. DFMO and MO significantly inhibited P-LF and decreased F-actin content in murine peritoneal macrophages. A combination of DFMO and MO with EGF failed to inhibit P-LF or to decrease F-actin content in these cells. The results obtained with DFMO and MO suggested new cellular targets of their effects. These results may be extended to cancer research to provide a rationale for clinical trials using combinations of EGF with DFMO or MO.     

Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.     

Polymerization of Actin from Maize Pollen

Here we describe the in vitro polymerization of actin from maize (Zea mays) pollen. The purified actin from maize pollen reported in our previous paper (X. Liu, L.F. Yen [1992] Plant Physiol 99: 1151-1155) is biologically active. In the presence of ATP, KCl, and MgCl2 the purified pollen actin polymerized into filaments. During polymerization the spectra of absorbance at 232 nm increased gradually. Polymerization of pollen actin was evidently accompanied by an increase in viscosity of the pollen actin solution. Also, the specific viscosity of pollen F-actin increased in a concentration-dependent manner. The ultraviolet difference spectrum of pollen actin is very similar to that of rabbit muscle actin. The activity of myosin ATPase from rabbit muscle was activated 7-fold by the polymerized pollen actin (F-actin). The actin filaments were visualized under the electron microscope as doubly wound strands of 7 nm diameter. If cytochalasin B was added before staining, no actin filaments were observed. When actin filaments were treated with rabbit heavy meromyosin, the actin filaments were decorated with an arrowhead structure. These results imply that there is much similarity between pollen and muscle actin.     

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.     

The actin-driven movement and formation of acetylcholine receptor clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.     

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.     

Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO-transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.     

Interaction between membrane properties and protein synthesis: In vitro synthesis of membrane proteins during erythropoiesis

Membrane protein synthesis was investigated by incubating rabbit reticulocytes, in vitro, with radioactive amino acids. The kinetics of membrane protein synthesis showed linear incorporation for approx. 15 min, after which there was only a slight increase in incorporation. On the other hand, intracellular protein synthesis was linear for an incubation period of 60 min. Membranes isolated from such rabbit reticulocytes were analysed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Two major radioactive bands were found in the 50–60 000 D region, whilst another labelled band had a molecular weight of 43 000 D. This latter band had an electrophoretic mobility identical with rabbit muscle actin (and chick brain actin), when run on one-dimensional SDS polyacrylamide gels. Absolute identity between rabbit brain actin and a newly synthesized reticulocyte membrane protein was shown by comigration on a two-dimensional (first dimension isoelectric focusing and second dimension SDS gel) electrophoresis system. Another band that was radioactively labelled was found to have a molecular weight of approx. 32 000 D. Separation of reticulocytes into different age groups showed that young reticulocytes synthesized a membrane protein species that was not radioactively labelled in the old reticulocyte population.     

Actin remodeling to facilitate membrane fusion

Actin and its associated proteins participate in several intracellular trafficking mechanisms. This review assesses recent work that shows how actin participates in the terminal trafficking event of membrane bilayer fusion. A recent flurry of reports defines a role for Rho proteins in membrane fusion and also demonstrates that this role is distinct from any vesicle transport mechanism. Rho proteins are well known to govern actin remodeling, which implicates this process as a condition of membrane fusion. A small but significant body of work examines actin-regulated events of intracellular membrane fusion, exocytosis and endocytosis. In general, actin has been shown to act as a negative regulator of exocytosis. Cortical actin filaments act as a barrier that requires transient removal to allow vesicles to undergo docking at the plasma membrane. However, once docked, F-actin synthesis may act as a positive regulator to give the final stimulus to drive membrane fusion. F-actin synthesis is clearly needed for endocytosis and intracellular membrane fusion events. What may seem like dissimilar results are perhaps snapshots of a single mechanism of membranous actin remodeling (i.e. dynamic disassembly and reassembly) that is universally needed for all membrane fusion events.