Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide.

BACKGROUND: In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer. RESULTS: In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide. CONCLUSIONS: Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.     

Inhibition of actin filament depolymerization by the Dictyostelium 30,000-D actin-bundling protein

We have studied the effect of the Dictyostelium discoideum 30,000-D actin-bundling protein on the assembly and disassembly of pyrenyl-labeled actin in vitro. The results indicate that the protein is a potent inhibitor of the rate of actin depolymerization. The inhibition is rapid, dose dependent, and is observed at both ends of the filament. There is little effect of 30-kD protein on the initial rate of elongation from F-actin seeds or on the spontaneous nucleation of actin polymerization. We could detect little or no effect on the critical concentration. The novel feature of these results is that the filament ends are free for assembly but are significantly impaired in disassembly with little change in the critical concentration at steady state. The effects appear to be largely independent of the cross-linking of actin filaments by the 30-kD protein. Actin cross-linking proteins may not only cross-link actin filaments, but may also differentially protect filaments in cells from disassembly and promote the formation of localized filament arrays with enhanced stability.     

Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway

Although actin filaments can form by oligomer annealing in vitro, they are assumed to assemble exclusively from actin monomers in vivo. In this study, we show that a pool of actin resistant to the monomer-sequestering drug latrunculin A (lat A) contributes to filament assembly in vivo. Furthermore, we show that the cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro. Additionally, in aip1Δ mutant cells, lat A–insensitive actin assembly is significantly enhanced. We conclude that actin oligomer annealing is a physiologically relevant actin filament assembly pathway in vivo and identify Aip1 as a crucial factor for shifting the distribution of short actin oligomers toward monomers during disassembly.     

Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth

The dynamic activity of tip-localized filamentous actin (F-actin) in pollen tubes is controlled by counteracting RIC4 and RIC3 pathways downstream of the ROP1 guanosine triphosphatase promoting actin assembly and disassembly, respectively. We show here that ROP1 activation is required for both the polar accumulation and the exocytosis of vesicles at the plasma membrane apex. The apical accumulation of exocytic vesicles oscillated in phase with, but slightly behind, apical actin assembly and was enhanced by overexpression of RIC4. However, RIC4 overexpression inhibited exocytosis, and this inhibition could be suppressed by latrunculin B treatment or RIC3 overexpression. We conclude that RIC4-dependent actin assembly is required for polar vesicle accumulation, whereas RIC3-mediated actin disassembly is required for exocytosis. Thus ROP1-dependent F-actin dynamics control tip growth through spatiotemporal coordination of vesicle targeting and exocytosis.     

Quantitative analysis of actin turnover in Listeria comet tails: evidence for catastrophic filament turnover

Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over.     

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers.     

Dynamics of a protein polymer: the assembly and disassembly pathways of the MuB transposition target complex

MuB assembles into a polymer on DNA in the presence of ATP and is directly involved in the selection of an appropriate site on the Escherichia coli chromosome for the insertion of the bacteriophage Mu genome. We have developed an assay using fluorescently tagged proteins to monitor the polymeric state of MuB via fluorescence resonance energy transfer. We show that polymer assembly is initiated by the formation of an ATP-MuB complex. MuB then self-associates into a protomer before binding to DNA. Upon binding to DNA, a dramatic increase in energy transfer is observed, suggesting a conformational change within MuB. Polymer disassembly is much slower than assembly and is greatly stimulated by the MuA transposase. Additionally, MuB is readily exchanged between polymers, and ATP hydrolysis is directly coupled to polymer disassembly. Our data support a model in which a combination of rapid polymer assembly, MuA-mediated disassembly, followed by rapid reassembly of the polymer allows MuB to sample multiple DNA targets until an appropriate site is located for the insertion of the bacteriophage genome.     

Confocal and video imaging of cytoskeleton dynamics in the leech zygote

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.     

Reorganization of microfilaments in macrophages after LPS stimulation

Lipopolysaccharide (LPS), a potent activating substance of macrophages, induced the reorganization of microfilaments in macrophages obtained from C3H/HeN mice. At 1 min after LPS addition, a slight disassembly of actin was observed. At 2 to 4 min, there was a gradual assembly; then, at 5 and 6 min, a subsequent rapid disassembly occurred. We employed two methods to observe this process. One was the RITC-phalloidin staining of actin filaments and the other was the extraction of monomeric actin and unstable actin filaments with Triton X-100 solution. The results obtained by the two methods were basically in agreement. Nevertheless, there was a discrepancy between the results from the two methods, concerning the ratio of assembly and disassembly. The RITC-phalloidin staining was more sensitive in detecting actin assembly and less sensitive in detecting the disassembly than the extraction with Triton X-100 solution was. This difference suggests that some of the unstable filaments, which were extracted with Triton X-100 solution and fixed with formalin, were formed during the LPS-induced reorganization process. This reversible actin assembly could not be observed in the LPS-nonresponder, C3H/HeJ mouse macrophages. We concluded that the observed process could be attributed to LPS-signal triggering pathways subsequent to LPS binding and that a necessary component to initiate effective LPS-signaling, which is probably deficient in C3H/HeJ mice, is involved in this reorganization process of LPS-stimulated macrophages.     

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.     

A pharmacological cocktail for arresting actin dynamics in living cells

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.     

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.     

MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes

The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.     

Assembly of GMPCPP-Bound Tubulin into Helical Ribbons and Tubes and Effect of Colchicine

Microtubule assembly and disassembly is a complex structural process that doesnot proceed by simple addition and subtraction of individual subunits to and from ahelical polymer, as would be the case for actin and other helical assemblies. Thedynamic process of microtubule growth and shrinking involves short-lasting polymerforms that differ substantially from the microtubule itself and constitute crucial assemblyand disassembly intermediates. Structural characterization thus depends on thestabilization of these brief intermediates and their preservation as polymericassemblies. This paper gives experimental details on the polymerization of GMPCPPtubulininto low-temperature stable polymers that we propose to correspond to the earlystages in microtubule assembly and includes new data on the effect of colchicine onGMPCPP-tubulin polymerization. Finally, we include our thoughts on the possiblebiological meaning of tubulin polymerization versatility.     

Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers

Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315-324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.     

Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Changes in mobility of chromaffin granules in actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin.

As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin-network barrier must dissociate as a prelude. To elucidate dynamical behaviors of secretory granules in actin networks, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullas and found that the entanglement of actin filaments rapidly formed cages that confined granules in them. We also studied the effect of gelsolin, one of actin-severing proteins, on the network of actin filaments preformed in the presence of chromaffin granules. It turned out that the cages that confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. A semiquantitative analysis of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or the translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca(2+)-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in the literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.     

Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation– or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.     

Distinct pathways for the early recruitment of myosin II and actin to the cytokinetic furrow

Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new analytical approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities and argue against mechanisms that are coupled to global cortical movements.     

Under physiological conditions actin disassembles slowly from the nonpreferred end of an actin filament

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. Because of the taper of these cross-linked bundles of actin filaments, one can distinguish directly the preferred end for assembly from the nonpreferred end. Loss of growth with time from the nonpreferred end was directly assessed by electron microscopy and found to be dependent upon salt concentration. Under physiological conditions (100 mM KCl, 1 mM MgCl2) and excess ATP (0.5 mM), depolymerization of the newly assembled actin filaments at the nonpreferred end over an 8-h period was 0.024 micron/h. Thus, even after 8 h, 63% of the bundles retained significant growth on their nonpreferred ends, the average length being 0.21 micron +/- 0.04. However, in the presence of 1.2 mM CaCl2, disassembly of actin monomers from the nonpreferred end increased substantially. By 8 h, only 7% of the bundles retained any actin growth on the nonpreferred ends, and the depolymerization rate off the nonpreferred end was 0.087 micron/h. From these results we conclude that, in the absence of other cellular factors, disassembly of actin subunits from actin filaments (subunit exchange) is too slow to influence most of the motile events that occur in cells. We discuss how this relates to treadmilling.