Ligand-induced changes in the location of actin, myosin, 95K (alpha- actinin), and 120K protein in amebae of Dictyostelium discoideum

In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier proposals based on the in vitro properties of these two proteins (Condeelis, J., M. Vahey, J. M. Carboni, J. DeMey, S. Ogihara, 1984, J. Cell Biol., 99:119s-126s).     

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.     

F-actin capping by cap32/34 requires heterodimeric conformation and can be inhibited with PIP2.

The heterodimeric F-actin capping protein cap32/34 from Dictyostelium discoideum is a typical member of a widely distributed family of cytoskeletal proteins. To analyze its regulation and structure/function relationships we cloned and expressed the subunits separately in Escherichia coli using the ATG-expression vector pT7-7. Studies on the viscosity of F-actin solutions and the kinetics of actin polymerization in the presence of single subunits or the reconstituted protein showed that capping of F-actin absolutely requires the heterodimeric conformation. This activity can be inhibited by phosphatidyl bisphosphate (PIP2), an important component in signal transduction. The regulation of cap32/34 by PIP2 suggests an involvement of this protein in the re-organization of the actin cytoskeleton upon stimulation of D. discoideum cells with chemoattractant.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae.

Amoeboid chemotaxis involves a regulated increase in actin nucleation activity that is correlated with an increase in actin polymerization occurring seconds after chemotactic stimulation (Carson, M., Weber, A., and Zigmond, S. H. (1986) J. Cell Biol. 103, 2707-2714; Hall, A. L., Warren, V., Dharmawardhane, S., and Condeelis, J. (1989) J. Cell Biol. 109, 2207-2213). We report the isolation and characterization of an agonist-regulated capping protein, aginactin, from Dictyostelium that may regulate these changes in actin nucleation activity. Aginactin is isolated from low speed supernatants of starved amoebae by sequential anion exchange, hydrophobic interaction, fast protein liquid chromatography anion exchange, and hydroxyapatite chromatography. Aginactin migrates with an apparent molecular weight of 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and gel filtration columns, suggesting that it is a globular monomer. Aginactin is a barbed-end capping protein by several criteria. It inhibits the rate and final extent of actin polymerization and increases the apparent critical concentration at substoichiometric ratios to actin. It also inhibits depolymerization of F-actin and inhibits polymerization at the barbed end of Limulus acrosomal bundles. Aginactin is unaffected by micromolar Ca2+, and it neither severs F-actin nor nucleates actin polymerization in either the presence or absence of Ca2+. Aginactin binds to and cosediments with F-actin and has an apparent Kd for capping F-actin of 2.7 nM.     

Dynamic rearrangement of F-actin organization triggered by host-specific plant signal is linked to morphogenesis of Aphanomyces cochlioides zoospores

Cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone), a root releasing host-specific plant signal triggers chemotaxis and subsequent morphological changes in pathogenic Aphanomyces cochlioides zoospores before host penetration. The present study illustrates time-course changing patterns of cytoskeletal filamentous actin (F-actin) organization in the zoospores of A. cochlioides during rapid morphological changes (encystment and germination) after exposure to cochliophilin A. Confocal laser scanning microscopic analysis revealed that F-actin microfilaments remained concentrated at ventral groove and diffusely distributed in peripheral cytoplasm of the zoospore. These microfilaments dramatically rearranged and changed into granular F-actin plaques interconnected with fine arrays during encystment. A large patch of actin arrays accumulated at one pole of the cystospores just before germination. Then the actin plaques moved to the emerging germ tube where a distinct cap of microfilaments was seen at the tip of the emerging hypha. Zoospores treated with an inhibitor of F-actin polymerization, latrunculin B or motility halting and regeneration inducing compound nicotinamide, displayed different patterns of F-actin in both zoospores and cystospores than those obtained by the induction of cochliophilin A. Collectively, these results indicate that the host-specific plant signal cochliophilin A triggers a dynamic polymerization/depolymerization of F-actin in pathogenic A. cochlioides zoospores during early events of plant-peronosporomycete interactions.     

The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34.

During isolation of the F-actin capping protein cap32/34 from Dictyostelium discoideum, a 70 kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP-dependent interaction with the heterodimeric capping protein. To investigate the protein-protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 and 34 kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene-labeled actin showed that hsc70 increased the capping activity of cap32/34 up to 10-fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N-terminal 42 kDa ATPase region and the C-terminal 30 kDa tail of hsc70 were expressed separately in E. coli. The 32 and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C-terminal tail of hsc70 was involved in folding of the subunits of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.     

Lymphocyte mechanical response triggered by cross-linking surface receptors

Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (sIg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of sIg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when sIg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was approximately 0.15 mdyn/micron; for cells treated with antibodies against sIg or with Con A the stiffness increased to approximately 0.6 or 0.4 mdyn/micron, respectively. The stiffness decreased after completion of the capping of sIg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against sIg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface.     

High microfilament concentration results in barbed-end ADP caps.

Current theory and experiments describing actin polymerization suggest that site-specific cleavage of bound nucleotide following F-actin filament formation causes the barbed ends of microfilaments to be capped first with ATP subunits, then with ADP bound to inorganic phosphate (ADP.Pi) at steady-state. The barbed ends of depolymerizing filaments consist of ADP subunits. The decrease in stability of the barbed-end cap accompanying the transition from ADP.Pi to ADP allows nucleotide hydrolysis and subsequent loss of Pi to regulate F-actin filament dynamics. We describe a novel computational model of nucleotide capping that simulates both the spatial and temporal properties of actin polymerization. This model has been used to test the effects of high filament concentration on the behavior of the ATP hydrolysis cycle observed during polymerization. The model predicts that under conditions of high microfilament concentration an ADP cap can appear during steady-state at the barbed ends of filaments. We show that the presence of the cap can be accounted for by a kinetic model and predict the relationship between the nucleotide concentration ratio [ATP]/[ADP], the F-actin filament concentration, and the steady-state distribution of barbed-end ADP cap lengths. The possible consequences of this previously unreported phenomenon as a regulator of cytoskeletal behavior are discussed.     

Lymphocyte activation and capping of hormone receptors

In this study both a ligand-dependent treatment [concanavalin A (Con A)] and a ligand-independent treatment [high-voltage pulsed galvanic stimulation (HVPGS)] have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interleukin-2 (IL-2)] receptors within the first 5 min of stimulation. When either insulin or IL-2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intracellular Ca2+ activity; (2) aggregation of insulin or IL-2 receptors into patch/cap structures; (3) tyrosine-kinase-specific phosphorylation of a 32-kd membrane protein; and finally (4) induction of DNA synthesis. Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W-5, W-7, and W-12 drugs, implying a need for Ca2+/calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine-kinase-specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca2+/calmodulin, and myosin light-chain kinase are all closely associated with the insulin and IL-2 receptor cap structures. These findings strongly suggest that an actomyosin-mediated contractile system (regulated by Ca2+, calmodulin, and myosin light-chain kinase in an energy-dependent manner) is required not only for the collection of insulin and IL-2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for the activation of mouse splenic T-lymphocytes.     

Capping of Con A receptors and actin distribution are influenced by disruption of microtubules in Dictyostelium discoideum

Capping of Concanavalin A (Con A) receptors can be inhibited in Dictyostelium by treatment of amoebae with the microtubular drug tubulozole. In cells that were incubated with Con A or with fluorescent Con A conjugate the capping process was completed in 30 min as could be demonstrated by fluorescence microscopy and Con A peroxidase labeling. In the presence of 10(-5) M tubulozole redistribution of the receptors did not proceed beyond a stage that can be characterized as patching. The effect of the drug on microtubule integrity was checked by electron microscopy and immunofluorescence of tubulin. Treatment resulted in shortening of the peripheral parts of the microtubules, in agreement with results described by other authors. Electron microscopy confirmed that the Con A receptor complexes remained on the plasma membrane and were not internalized. The distribution of F-actin in Con A-treated cells showed a pattern closely resembling that of Con A. Cells that were also treated with tubulozole remained spherical and did not resume significant directional movement until tubulozole was removed from the medium. It is concluded that microtubules are involved in the rearrangement of the microfilament network in moving cells.     

Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

Multifunctional roles of tropomodulin-3 in regulating actin dynamics

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.     

Biochemical analysis of ligand-induced receptor patching and capping using a novel immunolactoperoxidase iodination technique

A novel approach for the analysis of membrane proteins involved in ligand-induced surface receptor patching and capping is described. The technique is based on the use of immunolactoperoxidase (immuno-LPO) conjugates which catalyze the iodination of those surface proteins with available tyrosine groups that are located in the immediate vicinity of the patch or cap of a particular antigen. We have used the patching and capping of the H-2 (histocompatibility) antigen on mouse thymocytes to illustrate this method. However, this technique should be generally applicable to any cell surface proteins which can be induced to form patches or caps by a specific ligand. Cytochemical analysis indicates that the immuno-LPO conjugates induce the same patching and capping of the H-2 antigen as does the unconjugated antibody. Biochemical analysis of the 125I-labeled proteins by SDS polyacrylamide gel electrophoresis indicates that a large membrane protein (mol wt of approximately 200,000 daltons) is closely associated with H-2 patches and caps. Since a number of other prominent membrane proteins are not labeled by this procedure, selective redistribution of certain surface proteins must be occurring during H-2 antibody-induced patching and capping.     

Correlation between movement of concanavalin A membrane receptors and cytolysis. A scanning electron microscopy study

The present study was undertaken to test whether cytolysis induced by Concanavalin A (Con A) requires lateral mobility of membranal lectin receptor sites into caps. Treatment of interphase murine mastocytoma cells with 10(-4) M colchicine promoted cap formation by Con A in about 30% of the cells, followed by cytolysis. Pretreatment of the cells with NaN3, low temperature, or glutaraldehyde decreased the degree of capping and, to the same extent, the degree of cytolysis. The addition of antibodies to cells bound with Con A increased the appearance of capping and cytolysis. A linear relationship with a high correlation coefficient exists between the degree of capping and cytolysis, suggesting that lateral mobility of membrane Con A receptors is required for cytolysis by the lectin. The process of cap formation by Con A up to the stage of cytolysis was followed by scanning electron microscopy.     

Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor patching and capping

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.     

Real-time monitoring of actin polymerization in living cells using split luciferase

Real-time monitoring of actin polymerization in living cells is beneficial for characterizing cellular activities such as migration, proliferation, and death. We developed new bioluminescence-based probe proteins that enable the monitoring of actin polymerization in living cells. Unlike other ordinary split luciferase probes, our probes were incorporated in endogenous actin filament that enabled it to measure the actin polymerization quantitatively. The probe proteins exhibited a dose-responsive decrease in photon emission intensity in response to the filamentous (F)-actin-disrupting agent latrunculin A. This technique has a high sensitivity with a high signal-to-noise ratio and is nontoxic compared with other methods of monitoring actin polymerization in living cells. Using this technique, we succeeded in monitoring the F-actin level in living cells during apoptosis progression induced by UV irradiation continuously for 12 h. F-actin was transiently upregulated after UV irradiation. Since UV-induced cell death was enhanced by treatment with latrunculin A during the period which F-actin is increased, transient upregulation of F-actin after UV is likely a protective reaction against UV-induced cell death. Our novel technique is an effective tool for investigating actin polymerization in living cells.     

Interaction of tumour promoters with epithelial cells in culture : An immunofluorescence study

12-O-tetradecanoyl phorbol-13-acetate (TPA) has a profound and rapid influence on the cytoskeleton of Madin-Darby Canine Kidney (MDCK) cells. Within 10 min, TPA induces a rapid change in morphology, from a flat, cuboidal state to a rounded or elongated morphology in which the cell membranes become convoluted. Concomitant with this morphological change is a rapid dissolution of stress fibres and a redistribution of F-actin from microfilament bundles to a membrane or sub-membranous location. The rearrangement of actin is paralleled by a rearrangement of alpha-actinin and a reduction in the number of vinculin-containing adhesion plaques. Unusual F-actin configurations are often found emanating from a perinuclear location, usually containing alpha-actinin and terminating in a vinculin-containing adhesion plaque. The cytoskeletal rearrangements occur in the presence of inhibitors of protein synthesis or oxidative phosphorylation, but do not occur if glycolysis is also inhibited. The rearrangements are partly abrogated by the presence of cytochalasin B (CB). Despite these dramatic changes in microfilaments the polymerization state of actin remained unaltered after TPA treatment. Furthermore, although changes in the movement of membrane lipids have been reported, no obvious differences in the ability of glycoproteins to redistribute in the plane of the membrane were found as judged by FITC-concanavalin A (conA) induced patching. The rapidity of the morphological response of MDCK cells to TPA indicates that the cytoskeleton is one of the primary targets of TPA, but that tumour promoters differ from RNA tumour viruses in their effect on the state of actin polymerization.     

A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex

A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.