Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

Disturbance of endomembrane trafficking by brefeldin A and calyculin A reorganizes the actin cytoskeleton of <Emphasis Type="Italic">Lilium longiflorum</Emphasis> pollen tubes

We investigated the effect of brefeldin A on membrane trafficking and the actin cytoskeleton of pollen tubes of Lilium longiflorum with fluorescent dyes, inhibitor experiments, and confocal laser scanning microscopy. The formation of a subapical brefeldin A-induced membrane aggregation (BIA) was associated with the formation of an actin basket from which filaments extended towards the tip. The orientation of these actin filaments correlated with the trajectories of membrane material stained by FM dyes, suggesting that the BIA-associated actin filaments are used as tracks for retrograde transport. Analysis of time series indicated that these tracks (actin filaments) were either stationary or glided along the plasma membrane towards the BIA together with the attached membranes or organelles. Disturbance of the actin cytoskeleton by cytochalasin D or latrunculin B caused immediate arrest of membrane trafficking, dissipation of the BIA and the BIA-associated actin basket, and reorganization into randomly oriented actin rods. Our observations suggest that brefeldin A causes ectopic activation of actin-nucleating proteins at the BIA, resulting in retrograde movement of membranes not only along but also together with actin filaments. We show further that subapical membrane aggregations and actin baskets supporting retrograde membrane flow can also be induced by calyculin A, indicating that dephosphorylation by type 2 protein phosphatases is required for proper formation of membrane coats and polar membrane trafficking.     

Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi-to-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI-coated and uncoated vesicles contain β/γ-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.     

A radiolabel and electron microscopy study of the interaction of liposomes with synaptosomal membranes

The quantitative and qualitative interaction of liposomes with synaptosomes isolated from rat brain was examined using radiolabeled phospholipids and electron microscopy. Liposomes were prepared by sonication and detergent dialysis. Binding (adsorption) of radiolabeled phospholipid to synaptosomes was saturable when liposomes were in the liquid-crystalline state, were electrically neutral (egg-phosphatidylcholine), or carried increasing fractions (10:2 and 10:4 molar ratio) of negatively charged phosphatidic acid. Analysis using the Langmuir isotherm equation indicated a biphasic adsorption behavior. Adsorption increased with increasing temperature (4 degrees C and 37 degrees C). Binding was nonsaturable when liposomes were positively charged with stearylamine or composed of dimyristoylphosphatidylcholine and phosphatidylinositol (10:2 molar ratio). Due to the latter composition's solid state at 4 degrees C, temperature dependency was inverse. Electron micrographs revealed disc-shaped areas of adsorption that were free of integral membrane particles which appeared to form a condensed layer surrounding the areas of liposome adsorption. Following interaction with stearylamine-containing liposomes the vesicular structure of synaptosomes appeared largely destroyed. It is concluded that both liposome surface charge and membrane fluidity determine the extent of interaction with biological membranes.     

Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities

Chemoattractants stimulate actin polymerization in lamellipodia of polymorphonuclear leukocytes. We find that removal of chemoattractant results in rapid (within 10 s at 37 degrees C) and selective depolymerization of the F-actin located in lamellipodia. Addition of 10 microM cytochalasin B, in the presence of chemoattractant, also resulted in rapid and selective depolymerization of lamellar F-actin. The elevated F-actin level induced by chemoattractant rapidly returns to the level present in unstimulated cells after (a) a 10-fold decrease in chemoattractant concentration; (b) the addition of 10 microM cytochalasin B; or (c) cooling to 4 degrees C. The F-actin levels of unstimulated cells are only slightly affected by these treatments. Based on the similar effects of cytochalasin addition and chemoattractant dilution, it is likely that both treatments result in actin depolymerization from the pointed ends of filaments. Based on our results we propose that chemoattractant-stimulated polymorphonuclear leukocytes contain two distinct populations of actin filaments. The actin filaments within the lamellipodia are highly labile and in the continued presence of chemoattractant these filaments are rapidly turning over, continually polymerizing at their plus (barbed) ends, and depolymerizing at their minus ends. In contrast, the cortical F-actin filaments of both stimulated and unstimulated cells are differentially stable.     

Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro

To compare the effects of cytochalasins on the cellular level with those on the molecular level, 24 cytochalasins, 20 natural compounds and 4 derivatives, were used. The following effects were tested for each of 24 cytochalasins; (a) four high dose (2-20 muM) effects on the cellular level: rounding up of fibroblastic cells, contraction of actin cables, formation of hairy filaments containing actin, and inhibition of lymphocyte capping; (b) a low dose (0.2-2 muM) effect: inhibition of membrane ruffling; and (c) two in vitro effects: an inhibition of actin filament elongation (the high affinity effect [low dose effect] in vitro) and an effect on viscosity of actin filaments(the low affinity effect [high dose effect] in vitro). These results indicated that there are almost the same hierarchic orders of relative effectiveness of different cytochalasins between low and high dose effects and between cellular and molecular effects. From the data obtained with the 24 cytochalasins, we have calculated correlation coefficients of 0.87 and 0.79 between an effect in vivo, inhibition of capping, and an effect in vitro, inhibition of actin filament elongation, as well as between inhibition of capping and another effect in vitro, effect on viscosity of actin filaments, respectively. Furthermore, a correlation coefficient between the high affinity effect and the low affinity effect determined in vitro was calculated to be 0.90 from the data obtained in this study. The strong positive correlation among low and high dose effects in vivo and those in vitro suggests that most of the effects caused by a cytochalasin, irrespective of doses or affected phenomena, might be attributed to the interaction between the drug and the common target protein, actin. In the course of the immunofluorescence microscope study on cytochalasin-treated cells using actin antibody, we have found that aspochalasin D, a 10-isopropylcytochalasin, strongly induced the formation of rodlets containing actin in the cytoplasm of the treated fibroblasts. In contrast, the other cytochalasins, including cytochalasin B, cytochalasin C, cytochalasin D, and cytochalasin H, were found to induce the formation of nuclear rodlets. Both cytoplasmic and nuclear rodlets found in the cytochalasin-treated cells were similar in ultrastructures to those induced by 5 to 10 percent (vol/vol) dimethyl sulfoxide in the same type of cells.     

Facile platelet adhesion to collagen requires metabolic energy and actin polymerization and evokes intracellular free calcium mobilization

The attachment of platelets to collagen-coated microtiter plates at 20 degrees C was inhibited strongly by depletion of metabolic energy or by addition of cytochalasins and was slightly inhibited by the intracellular Ca2+ chelator BAPTA. In keeping with their respective potencies as inhibitors of actin polymerization, cytochalasins D and H were the most potent inhibitors of adhesion, while cytochalasin B was the least potent. Energy depletion, cytochalasin D or, to a much lesser extent, BAPTA also inhibited platelet adhesion to collagen in a suspension assay system at 37 degrees C. Collagen-induced platelet cytosolic Ca2+ mobilization was inhibited up to 70% by cytochalasin D and abolished by energy depletion or BAPTA. Elevation of intracellular platelet calcium by treatment with ionomycin had little effect on platelet adhesion to collagen. We propose that rapid platelet spreading along collagen fibers is both energy- and actin-dependent and necessary to produce maximal adhesion needed to elicit Ca2+ mobilization required for subsequent responses.     

Atomic force microscopy and light scattering of small unilamellar actin-containing liposomes

Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K(+) ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.     

Differential effects of actin cytoskeleton dynamics on equine infectious anemia virus particle production

Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.     

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.     

Capping one end of an actin filament affects elongation at the other end

The rates of elongation at the free ends of actin filaments were compared to those of intact filaments, when the one end was masked with muscle beta-actinin or cytochalasin D, using fixed actoheavy meromyosin and Limulus acrosomal actin bundles as seeds. Experimental conditions were chosen so as to prevent spontaneous filament formation as far as possible. The rate of elongation at the barbed end of fixed actoheavy meromyosin was reduced to about one-fourth when the other pointed end was capped by beta-actinin, and that at the pointed end was reduced to one-third when the barbed end was blocked by cytochalasin D. Similar effects were also observed with the packed actin bundles of horseshoe crab sperm, although the decreases in elongation were less marked: 50-60% of the control both in the presence of beta-actinin and cytochalasin D. To explain the peculiar "end effect" described above, it is proposed that possible conformational changes at one end of an actin filament caused by the binding of a capping substance are transmitted successively to the other end so as to affect the elongation there.     

Actin filaments are involved in the regulation of trafficking of two closely related chemokine receptors, CXCR1 and CXCR2

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37 degrees C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.     

Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking

To study the morphogenesis of cells caused by the organization of their internal cytoskeletal network, we characterized the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, alpha-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.     

Subpopulations of tau interact with microtubules and actin filaments in various cell types

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.     

Influence of temperature on stability of multilamellar liposomes in wool dyeing

Liposomes are lipid vesicles that are composed of amphiphile molecules and can carry hydrophobic and hydrophilic materials. In this research work liposomes used as carrier for transfer of dye molecules into wool fibers. The preparation and production of multilamellar liposomes (MLV) from Soya lecithin were carried out and the behavior of liposomes at different temperature was studied. The effect of different concentration of liposomes in the dye exhaustion profile of two dyes (Namely, Irgalan Blue FBL and Lanaset Blue 2R) at two different temperatures of 85 degrees C and 95 degrees C on the wool fabric was investigated. The results showed that presence of liposomes in the dye-bath helps to increase the dye absorption on the wool fabric before 80 degrees C. Dyeing at higher temperature and longer time leads to a decrease in the final exhaustion along with increase in the liposomes concentration. Liposomes at high temperature converted to the disperse phospholipids unimers that may deposited on the fabric surface and may produce a hydrophobic barrier against absorption of dye. The presence of 1% o.w.f. (on weight of fabric) of liposomes at 85 degrees C improved the dye exhaustion of Irgalan Blue FBL on the wool fabric. The wash fastness properties of samples which dyed in the dye-bath containing liposomes also improved.     

Morphological and Topological Transformation of Membrane Vesicles

Liposomes are micro-compartments made of lipid bilayer membranes withcharacteristics quite similar to those of biological membranes. To formartificial cell-like structures, we generated liposomes that containedsubunit proteins of cytoskeletons: tubulin or actin. Spherical liposomeswere transformed into bipolar or cell-like shapes by mechanical forcesgenerated by the polymerization of encapsulated subunits of microtubules.Disk- or dumbbell-shaped liposomes were developed by the polymerizationof encapsulated actin. Dynamic processes of morphological transformationsof liposomes were visualized by high intensity dark-field lightmicroscopy.Topological changes, such as fusion and division of membrane vesicles,play an essential role in cellular activities. To investigate themechanism of these processes, we visualized in real time the liposomesundergoing topological transformation. A variety of novel topologicaltransformations were found, including the opening-up of liposomes and thedirect expulsion of inner vesicles.