Actin assembly by cadmium ions

Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. Metal ions that influence intracellular Ca²⁺ concentrations or compete with Ca²⁺ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much evidence demonstrates that cadmium interferes with the dynamics of actin filaments in various types of cells. Here we show that, at high (0.8–1.0 mM) concentrations, CdCl₂ causes actin denaturation. At such Cd²⁺ concentrations, actin precipitates (really actin, as shown by SDS-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd²⁺ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl₂ is more effective as an actin polymerizing agent than both MgCl₂ and CaCl₂. The Cd-related increase in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration measurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close (C_c ranges from 0.25 to 0.5 μM), while Ca-polymerized actin shows a polymerization extent markedly lower (C_c=4.0 μM). By both the fluorescent Ca²⁺ chelator Quin-2 assay and limited proteolysis of actin by trypsin and α-chymotrypsin, the real substitution of G-actin-bound Ca²⁺ by Cd²⁺ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl₂ indicates that, in our experimental conditions, Ca²⁺ tightly-bound to actin is partially (60–70%) replaced by Cd²⁺, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61–69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd²⁺-treated cells; however, considering the positive effect of Cd²⁺ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd²⁺ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable. © 1997 Elsevier Science B.V. All rights reserved.     

Actin assembly at membranes controlled by ARF6

The small GTPase, ADP-ribosylation factor-6 (ARF6), has been implicated in regulating membrane traffic and remodeling cortical F-actin. Using real-time video analysis of actin assembly in living cells, we investigated the function and mechanism of ARF6 in control of actin assembly. Expression of an activated form of ARF6 that mimicks the GTP-bound form of the GTPase induced actin assembly resulting in the movement of vesicle-like particles, some of which contain markers for pinosomes. Activated ARF6 also stimulated actin assembly at foci on the ventral surface of the cell and stimulated fluid phase pinocytosis. Particle motility induced by ARF6 involved Arp2/3 complex, tyrosine kinase activity, phospholipase D (PLD) and D3-phosphoinositides, but not phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We conclude that ARF6 regulates actin assembly for pinosome motility and at foci on the ventral cell surface.     

Actin and myosin: control of filament assembly

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.     

Actin assembly in electropermeabilized neutrophils: role of G-proteins

Polymerization of microfilaments, one of the responses triggered in neutrophils by stimuli such as the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), involves the conversion of actin from the monomeric to the filamentous form. The exact sequence of events responsible for this conversion remains to be defined, but its susceptibility to inhibition by pertussis toxin provides indirect evidence that GTP-binding proteins (G-proteins) are involved. In this report, electropermeabilized cells were used to obtain more direct evidence of a role for G-proteins in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin and flow cytometry were used to monitor the formation of filamentous actin. GTP-gamma-S, a nonhydrolyzable analogue of GTP and aluminum fluoride, which in combination with GDP can activate G-proteins, stimulated actin assembly in electropermeabilized cells but had only marginal effects on intact cells. fMLP-induced actin polymerization in permeabilized cells was inhibited by pretreatment with GDP-beta-S, an analogue of GDP that stabilizes the inactive form of G-proteins. In contrast, stimulation by phorbol 12-myristate 13-acetate (PMA) was largely unaffected by GDP-3-S. These observations indicate that activation of G-proteins is essential for actin assembly induced by receptor-dependent stimuli such as fMLP. Moreover, GTP-binding proteins do not seem to be required in the late stages of the signalling cascade, i.e. after stimulation of protein kinase C.     

Actin dynamics: assembly and disassembly of actin networks

Assembly of branched actin filament networks at the leading edge of migrating cells requires stimulation of the Arp2/3 complex by WASp proteins, in concert with the WASp activators Cdc42, PIP(2) and profilin. Network disassembly and debranching appears to be linked to actin-bound ATP hydrolysis as filaments age.     

Irreversible inhibition of pig kidney copper-containing amine oxidase by sodium and lithium ions.

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.     

Actin assembly in electropermeabilized neutrophils: role of intracellular calcium

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.     

Actin assembly induced by polylysine beads or purified phagosomes: quantitation by a new flow cytometry assay

BACKGROUND: Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments. METHODS: Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation. RESULTS: Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane. CONCLUSIONS: We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.     

Role of lithium ions in proline transport in Escherichia coli.

Mechanisms of Li+ stimulation of proline transport were studied in cells of Escherichia coli 7 and NR70, a mutant of strain 7 lacking adenosine triphosphatase (EC 3.6.1.3). An electrochemical potential difference of Li+ induced in an inward direction of energy-depleted cells caused a transient uptake of proline depending on the driving force provided. When proline was added to unbuffered cell suspensions under anaerobic conditions, the medium was found to be acidified only in the presence of Li+ but not in the presence of Na+ or K+. This acidification was abolished by the addition of a permeant anion, SCN-, to the medium containing Li+, but this was not demonstrated with cells of a mutant strain deficient in a carrier protein specific for proline. These results support the assumption that proline is taken up by a mechanism of Li+-proline cotransport in E. coli.     

Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis.

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO4(2-) inhibit the tubulin polymerization at an IC50 of 14-24 microM with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 microM. BIAcore measurements for tubulin-tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO4(2-). No inhibitory effect of Cu2+ or CrO4(2-) on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO4(2-) ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10-14 microM with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.     

Actin in striated muscle: recent insights into assembly and maintenance

Striated muscle cells are characterised by a para-crystalline arrangement of their contractile proteins actin and myosin in sarcomeres, the basic unit of the myofibrils. A multitude of proteins is required to build and maintain the structure of this regular arrangement as well as to ensure regulation of contraction and to respond to alterations in demand. This review focuses on the actin filaments (also called thin filaments) of the sarcomere and will discuss how they are assembled during myofibrillogenesis and in hypertrophy and how their integrity is maintained in the working myocardium.     

Actin assembly controlled by GDP-Rab27a is essential for endocytosis of the insulin secretory membrane

We have recently reported that GDP-bound Rab27a regulates endocytosis of the insulin secretory membrane via its binding to coronin 3, an actin-binding protein. The aim of this study was to examine the participation of actin assembly in the Rab27a-dependent regulation of endocytosis using a pancreatic beta cell line, MIN6. Coronin 3 promoted F-actin bundling only in the presence of GDP-Rab27a. This effect was independent of coronin-3-binding to the actin-related proteins 2 and 3 (Arp2/3). Uptake of anti-phogrin-lumen antibody into MIN6 was inhibited by anti-coronin-3-C antibody which recognizes the actin-binding site. This inhibition was also observed with coronin-3-R28D, which lacks in actin binding. These results suggest that coronin 3 is a genuine GDP-Rab27a effector, and that controls endocytosis of the secretory membrane via modulating actin assembly in pancreatic β-cells.     

Nerve cell and nematocyte production in Hydra is deregulated by lithium ions

Summary LiCl, a well-known vegetalising agent, interferes with the commitment of stem cells to nerve cells and nematocytes in Hydra attenuata. Treatment with 20 mM LiCl inhibits commitment to nerve cells, treatment with 1 mM LiCl inhibits commitment to nematocytes. However, LiCl does not prevent stem cells committed to the nematocyte pathway from dividing and differentiating into nests of nematocytes. Following LiCl treatment, determination to nerve cells and nematocytes is triggered again. Commitment to nerve cells is strongly stimulated within the first 3 h following pulse treatment with LiCl if the animals have been fed immediately prior to treatment. In Hydra exposed to LiCl for 10 days the stem cell density is reduced by at least 90% of the initial value, and nematocytes are almost completely missing, whereas the density of nerve cells is within the normal range in animals with normal morphology. Animals which developed a transverse constriction in the middle of the body axis contain a 1.7-fold higher nerve cell density in the lower part than is observed in control animals.     

Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.     

Actin filament assembly by myristoylated alanine-rich C kinase substrate-phosphatidylinositol-4,5-diphosphate signaling is critical for dendrite branching

Dendrites undergo extensive growth and branching at early stages, but relatively little is known about the molecular mechanisms underlying these processes. Here, we show that increasing the level of myristoylated, alanine-rich C kinase substrate (MARCKS), a prominent substrate of protein kinase C and a phosphatidylinositol-4,5-diphosphate [PI(4,5)P2] sequestration protein highly expressed in the brain, enhanced branching and growth of dendrites both in vitro and in vivo. Conversely, knockdown of endogenous MARCKS by RNA interference reduced dendritic arborization. Results from expression of different mutants indicated that membrane binding is essential for MARCKS-induced dendritic morphogenesis. Furthermore, MARCKS increased the number and length of filamentous actin-based filopodia along neurites, as well as the motility of filopodia, in a PI(4,5)P2-dependent manner. Time-lapse imaging showed that MARCKS increased frequency of filopodia initiation but did not affect filopodia longevity, suggesting that MARCKS may increase dendritic branching through its action on filopodia initiation. These findings demonstrate a critical role for MARCKS–PI(4,5)P2 signaling in regulating dendrite development.     

Initiation of DNA synthesis in mammary epithelium and mammary tumors by lithium ions

The growth promoting effects of lithium and insulin on cultures of mammary gland epithelium and CZF mouse mammary tumor cells were investigated. Lithium chloride exerts a 450-fold increase in the rate of DNA synthesis in mammary epithelium from mid-pregnant mice in organ culture or monolayer culture. There is an increase in both the percentage of cells initiating DNA synthesis and the net accumulation of DNA. The most effective lithium concentration is 10 mM, and the maximally effective rate of stimulation is reached 48 hours after addition. The magnitude of response to lithium varies with the physiological state of the mammary epithelial cell donor: epithelium from non-pregnant or lactating mice is less responsive than that from mid-pregnant mice. In combination, insulin and lithium produce either a synergistic or an additive effect on the growth of epithelium dependent upon the physiological state of the donor animal. Lithium also promotes the growth of mammary tumor cells in the absence of serum or other mitogens. The action of lithium on DNA synthesis appears to be a direct effect on the epithelial cells.