Cytochalasin Rearranges Cortical Actin of the Alga Nitella into Short, Stable Rods

The cortical cytoplasm of the alga Nitella contains reticulateactin that does not survive perfusion fixation with glutaraldehydeunless prestabilized with the cross-linker 3-maleimidobenzoyl-N-hydroxysuccinimidester (MBS). Cytochalasin D remodels thiscortical actin into short rods which are more stable, survivingaldehyde fixation without MBS pre-treatment. The overall alignmentof these actin rods correlates with that of cortical microtubules(transverse in young cells, random in old cells) but probablydoes not involve one-to-one correspondence. The time course,dose dependence and reversibility of these structural changesbroadly resemble those for streaming inhibition by cytochalasinbut the cortical actin responds to concentrations that do notslow streaming. Because the structural changes concern the corticaland not the subcortical actin, they seem unlikely to directlyinhibit streaming. Formation of cortical rods is not a responseto streaming inhibition per se since it does not occur whentwo other inhibitors of streaming (2,4-dinitrophenol (DNP) andNethyl maleimide (NEM)) are used. NEM, however, resembles MBSin stabilizing the reticulate form of cortical actin even thoughit cannot cross link. ¹Address from July 1995; Department of Biology, Faculty of Science,Osaka University, Machikaneyama 1-1, Tayonaka, Osaka, 560 Japan.     

Actin Microfilaments are Required for the Expression of Nonhost Resistance in Higher Plants

We investigated the role of actin microfilaments in nonhostresistance of higher plants. Here we present several lines ofevidence to indicate that microfilaments are indeed involvedin blocking fungal penetration of nonhost plants. Erysiphe pisi,a pathogen of pea, normally fails to penetrate into nonhostplants such as barley, wheat, cucumber and tobacco. When tissuesof these nonhost plants were treated with cytochalasins, specificinhibitors of actin polymerization, this fungus became ableto penetrate and formed haustoria in epidermal cells of theseplants. Moreover, treatment of these plants with various kindsand concentrations of cytochalasins allowed several other non-pathogenicfungi, E.graminis hordei, E.graminis tritici, Sphaerotheca fuliginea,Colletotrichum graminicola, My-cosphaella pinodes, C. lagenarium,Altemaria kikuchiana and Corynespora melonis, to also penetratethe cells of these plants. The degree of microfilament depolymeriza-tionvaried depending on the kinds and concentrations of cytochalasinsapplied and we show that this is significantly correlated withthe penetration efficiency of C. graminicola. This indicatesthat the polymerized, filamentous state of actin is necessaryfor plants to block fungal penetration. These results stronglysuggest that actin microfilaments may play important roles inthe expression of nonhost resistance of higher plants. ¹Contribution no. 129 from the Laboratory of Plant Pathology,Mie University     

Toxoplasma gondii Actin Depolymerizing Factor Acts Primarily to Sequester G-actin

Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Parasites in this phylum utilize a unique process of motility termed gliding, which is dependent on parasite actin filaments. Surprisingly, 98% of parasite actin is maintained as G-actin, suggesting that filaments are rapidly assembled and turned over. Little is known about the regulated disassembly of filaments in the Apicomplexa. In higher eukaryotes, the related actin depolymerizing factor (ADF) and cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin-binding proteins conserved in apicomplexan parasites. In this study we examined the mechanism by which T. gondii ADF (TgADF) regulates actin filament turnover. Unlike other members of the ADF/cofilin (AC) family, apicomplexan ADFs lack key F-actin binding sites. Surprisingly, this promotes their enhanced disassembly of actin filaments. Restoration of the C-terminal F-actin binding site to TgADF stabilized its interaction with filaments but reduced its net filament disassembly activity. Analysis of severing activity revealed that TgADF is a weak severing protein, requiring much higher concentrations than typical AC proteins. Investigation of TgADF interaction with T. gondii actin (TgACT) revealed that TgADF disassembled short TgACT oligomers. Kinetic and steady-state polymerization assays demonstrated that TgADF has strong monomer-sequestering activity, inhibiting TgACT polymerization at very low concentrations. Collectively these data indicate that TgADF promoted the efficient turnover of actin filaments via weak severing of filaments and strong sequestering of monomers. This suggests a dual role for TgADF in maintaining high G-actin concentrations and effecting rapid filament turnover.     

Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells

To clarifyinteractions between the cytoskeleton and activity of L-typeCa²⁺ (Ca_L) channels in vascular smooth muscle(VSM) cells, we investigated the effect of disruption of actinfilaments and microtubules on the L-type Ca²⁺ current[I_Ba(L)] of cultured VSM cells (A7r5 cellline) using whole cell voltage clamp. The cells were exposed to eachdisrupter for 1 h and then examined electrophysiologically andmorphologically. Results of immunostaining using anti--actin andanti--tubulin antibodies showed that colchicine disrupted both actinfilaments and microtubules, cytochalasin D disrupted only actinfilaments, and nocodazole disrupted only microtubules.I_Ba(L) was greatly reduced in cells that wereexposed to colchicine or cytochalasin D but not to nocodazole.Colchicine even inhibited I_Ba(L) by about 40%when the actin filaments were stabilized by phalloidin or when thecells were treated with phalloidin plus taxol to stabilize bothcytoskeletal components. These results suggest that colchicine mustalso cause some inhibition of I_Ba(L) due toanother unknown mechanism, e.g., a direct block of Ca_Lchannels. In summary, actin filament disruption of VSM cells inhibitsCa_L channel activity, whereas disrupting the microtubulesdoes not.

     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.     

Actin Filaments Purified from Tobacco Cultured BY-2 Cells Can Be Translocated by Plant Myosin

Actin filaments (AFs) and microtubules (MTs) are essential constituentsof the cytoskeleton in plant cells. Sliding of motor proteinsalong these cytoskeletons is believed to be necessary in variouscellular functions. In our previous study [Yokota et al. (1995b)Plant Cell Physiol. 36: 1563], we succeeded in isolating tubulinfrom cultured tobacco BY-2 cells, which in its polymerized formcan be translocated by the MT-based motor protein, dynein, invitro. In the present study, the method was modified to purifyboth tubulin and actin. Purified actin could be polymerizedand decorated by subfragment-1 (S-1) of skeletal muscle myosin.In the motility assay in vitro, AFs, thus prepared, could betranslocated by plant myosin isolated from lily pollen tubes.The sliding velocity of those AFs was similar to that of animalAFs prepared from chicken breast muscle, and comparable withthe velocity of cytoplasmic streaming in living pollen tubesof lily. Using S-1, motility assay was carried out. The slidingvelocity of plant AFs and that of muscle AFs were also similar.As far as we know, this is the first report of the sliding ofisolated plant AFs with myosin. (Received April 30, 1999; Accepted September 7, 1999)     

Vinculin Nucleates Actin Polymerization and Modifies Actin Filament Structure

Vinculin links integrins to the actin cytoskeleton by binding F-actin. Little is known with respect to how this interaction occurs or affects actin dynamics. Here we assess the consequence of the vinculin tail (VT) on actin dynamics by examining its binding to monomeric and filamentous yeast actins. VT causes pyrene-labeled G-actin to polymerize in low ionic strength buffer (G-buffer), conditions that normally do not promote actin polymerization. Analysis by electron microscopy shows that, under these conditions, the filaments form small bundles at low VT concentrations, which gradually increase in size until saturation occurs at a ratio of 2 VT:1 actin. Addition of VT to pyrene-labeled mutant yeast G-actin (S265C) produced a fluorescence excimer band, which requires a relatively normal filament geometry. In higher ionic strength polymerization-promoting F-buffer, substoichiometric amounts of VT accelerate the polymerization of pyrene-labeled WT actin. However, the amplitude of the pyrene fluorescence caused by actin polymerization is quenched as the VT concentration increases without an effect on net actin polymerization as determined by centrifugation assays. Finally, addition of VT to preformed pyrene-labeled S265C F-actin causes a concentration-dependent decrease in the maximum amplitude of the pyrene fluorescence band demonstrating the ability of VT to remodel the conformation of the actin filament. These observations support the idea that vinculin can link adhesion plaques to the cytoskeleton by initiating the formation of bundled actin filaments or by remodeling existing filaments.Cell migration is critical for embryonic development, adult homeostasis, inflammatory responses, and wound healing. To migrate, a cell must coordinate a number of different inputs into appropriate cellular responses. The cell must polarize in the direction of migration and extend lamellipodial and/or filopodial protrusions. Nascent adhesions that assemble within the branched actin network of the lamellipodium must link to the underlying actin cytoskeleton. This process allows for the maturation of adhesions to structures that anchor the protrusion. These adhesions also provide the traction forces necessary to pull the cell body forward and break older adhesions at the cell rear. Perturbation of any of these events affects a cell''s migratory ability. For example, nascent adhesions that do not form linkages to the actin cytoskeleton cannot effectively anchor the protrusion to the substratum. The result is an extension that folds back upon itself, forming a membrane ruffle that cannot provide the traction forces necessary for migration.How adhesions establish links to the underlying actin cytoskeleton has been an area of intense investigation. Integrin-containing structures are active areas of actin polymerization suggesting that adhesion plaques can initiate actin filament formation (reviewed in Refs. 1–3). Focal complexes are small integrin clusters that are found exclusively at the tips of lamellipodia and filopodia. Formation of these structures is closely coupled with actin assembly in protruding regions of cells. Accumulating evidence indicates that adhesion complex components recruit the Arp2/3 complex, a potent nucleator of actin polymerization. Our work (4) and that of others (5–7) demonstrates that the Arp2/3 complex is recruited to focal complexes or transient adhesion structures reminiscent of focal complexes by binding vinculin. FAK has also been implicated in linking focal complexes to the actin cytoskeleton by virtue of its ability to recruit and activate the Arp2/3 complex (8). Furthermore, efficient focal complex assembly requires the actin-binding protein, cortactin, which could affect adhesion assembly by interacting with the Arp2/3 complex (9). Hence, many of the known mechanisms for initiating filament formation involve recruitment of the Arp2/3 complex, which initiates the formation of branched actin filaments (55). It is surprising then that the earliest detectable forms of actin-associated adhesions are interconnected by short actin bundles, not branched filaments (10). These observations suggest that our current understanding for how nascent adhesions initiate filament formation is incomplete.The earliest detectable actin-associated adhesions are “dots or doublets of dots” and are highly enriched in integrins, paxillin, and vinculin (10), suggesting that one of these molecules has the capability to initiate actin filament formation from such a plaque. Vinculin has long been implicated in linking adhesion plaques to the actin cytoskeleton by virtue of the ability of its tail to bind (11) and bundle F-actin (12). The interaction of vinculin with actin has been extensively studied from the perspective of vinculin (11, 13–23). Studies of recombinant proteins identified two regions of the vinculin tail (VT)² that bind F-actin independently (21, 17), but mapping these sites onto the VT crystal structure reveals that these peptides do not correspond to distinct sites (25). Upon binding actin, vinculin undergoes a conformational change that promotes dimerization suggesting that vinculin self-association may be important for its bundling activities (15).Less is known with respect to the effect of vinculin on actin filament formation and structure. This lack of knowledge stems from the fact that many of the early studies showed vinculin to have no effect on actin dynamics (26–28). However, these experiments were performed using chicken gizzard vinculin, which exists almost exclusively in a conformation where the actin binding sites are inaccessible, or from preparations that contain contaminants that produce false negatives (29). More recently, recombinant VT proteins were shown to cross-link and bundle actin (23). However, the interaction of vinculin with G-actin and the effect of vinculin on actin filament dynamics have not been explored. In this study, we have assessed the interaction of vinculin with pyrene-labeled wild-type and mutant yeast actins. We show that the VT can promote the formation of an actin nucleus from which filaments arise and alter the assembly and structure of actin filaments. These findings provide novel insights into how adhesion plaques may be linked to the actin cytoskeleton.     

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.     

Binding of WIP to Actin Is Essential for T Cell Actin Cytoskeleton Integrity and Tissue Homing

The Wiskott-Aldrich syndrome protein (WASp) is important for actin polymerization in T cells and for their migration. WASp-interacting protein (WIP) binds to and stabilizes WASp and also interacts with actin. Cytoskeletal and functional defects are more severe in WIP^−/− T cells, which lack WASp, than in WASp^−/− T cells, suggesting that WIP interaction with actin may be important for T cell cytoskeletal integrity and function. We constructed mice that lack the actin-binding domain of WIP (WIPΔABD mice). WIPΔABD associated normally with WASp but not F-actin. T cells from WIPΔABD mice had normal WASp levels but decreased cellular F-actin content, a disorganized actin cytoskeleton, impaired chemotaxis, and defective homing to lymph nodes. WIPΔABD mice exhibited a T cell intrinsic defect in contact hypersensitivity and impaired responses to cutaneous challenge with protein antigen. Adoptively transferred antigen-specific CD4⁺ T cells from WIPΔABD mice had decreased homing to antigen-challenged skin of wild-type recipients. These findings show that WIP binding to actin, independently of its binding to WASp, is critical for the integrity of the actin cytoskeleton in T cells and for their migration into tissues. Disruption of WIP binding to actin could be of therapeutic value in T cell-driven inflammatory diseases.     

MARCKS-Related Protein Binds to Actin without Significantly Affecting Actin Polymerization or Network Structure

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.     

Germination of Monocolpate Angiosperm Pollen: Evolution of the Actin Cytoskeleton and Wall during Hydration, Activation and Tube Emergence

The monocolpate pollen grain of Narcissus pseudonarcissus L.has two preferred sites for tube emergence, one at each endof the colpus. While the cellulosic microfibnls of the innerlayer of the intine are disposed circumferentially in the centreof the grain, the microfibrils in these terminal sites are shorterand randomly oriented Soon after the beginning of hydration,inclusions of the vegetative cell begin movement, firstly ina rotatory manner, and then in a pattern focused on one or bothgermination sites, where the intine bulges as hydration progresses.These changes are associated with the evolution of the actincytoskeleton. Actin is present in the unactivated grain in theform of fusiform bodies. During hydration these dissociate toform finer fibrils, initially randomly disposed. Then, correlatedwith the change of the pattern of movement in the vegetativecell, the actin fibril system becomes polarized towards thegermination sites, where shorter fibrils accumulate. Callose,absent from the ungerminated grain, is deposited within thecellulosic wall in these locations, forming a shallow dome whicheventually develops into an annulus subtending the inner calloselining of the emerging tube. The transition to cylindrical growthis associated firstly with the development of zonation in thecytoplasm of the vegetative cell, with the tip occupied by apopulation of wall precursor bodies (P-particles) and a denseaggregate of short actin fibrils; and then with the establishmentof the ‘inverse fountain’ pattern of movement characteristicof the apical part of the extending tube. Narcissus pseudonarcissus L, pollen activation, pollen germination, actin cytoskeleton, tip-growth system, pollen-tube wall development     

LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl₂ or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.     

The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.     

A Highlights from MBoC Selection: Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We used the dendritic nucleation hypothesis to formulate a mathematical model of the assembly and disassembly of actin filaments at sites of clathrin-mediated endocytosis in fission yeast. We used the wave of active WASp recruitment at the site of the patch formation to drive assembly reactions after activation of Arp2/3 complex. Capping terminated actin filament elongation. Aging of the filaments by ATP hydrolysis and γ-phosphate dissociation allowed actin filament severing by cofilin. The model could simulate the assembly and disassembly of actin and other actin patch proteins using measured cytoplasmic concentrations of the proteins. However, to account quantitatively for the numbers of proteins measured over time in the accompanying article (Sirotkin et al., 2010 , MBoC 21: 2792–2802), two reactions must be faster in cells than in vitro. Conditions inside the cell allow capping protein to bind to the barbed ends of actin filaments and Arp2/3 complex to bind to the sides of filaments faster than the purified proteins in vitro. Simulations also show that depolymerization from pointed ends cannot account for rapid loss of actin filaments from patches in 10 s. An alternative mechanism consistent with the data is that severing produces short fragments that diffuse away from the patch.     

HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.     

Tailor-Made Ezrin Actin Binding Domain to Probe Its Interaction with Actin In-Vitro

Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.     

The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism

All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action.     

Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars

Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars.     

The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.