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)     

Actin Filament Severing by Cofilin Dismantles Actin Patches and Produces Mother Filaments for New Patches

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Drebrin-induced Stabilization of Actin Filaments

Drebrin is a mammalian neuronal protein that binds to and organizes filamentous actin (F-actin) in dendritic spines, the receptive regions of most excitatory synapses that play a crucial role in higher brain functions. Here, the structural effects of drebrin on F-actin were examined in solution. Depolymerization and differential scanning calorimetry assays show that F-actin is stabilized by the binding of drebrin. Drebrin inhibits depolymerization mainly at the barbed end of F-actin. Full-length drebrin and its C-terminal truncated constructs were used to clarify the domain requirements for these effects. The actin binding domain of drebrin decreases the intrastrand disulfide cross-linking of Cys-41 (in the DNase I binding loop) to Cys-374 (C-terminal) but increases the interstrand disulfide cross-linking of Cys-265 (hydrophobic loop) to Cys-374 in the yeast mutants Q41C and S265C, respectively. We also demonstrate, using solution biochemistry methods and EM, the rescue of filament formation by drebrin in different cases of longitudinal interprotomer contact perturbation: the T203C/C374S yeast actin mutant and grimelysin-cleaved skeletal actin (between Gly-42 and Val-43). Additionally, we show that drebrin rescues the polymerization of V266G/L267G, a hydrophobic loop yeast actin mutant with an impaired lateral interface formation between the two filament strands. Overall, our data suggest that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.     

Preparation of Actin and Fluorescent Actin Analogs from Plant Cells

The plant actin cytoskeleton provides a dynamic cytoplasmic framework for many fundamental cellular processes like cytoplasmic streaming,cytokinesis and morphogenesis.Understanding the actin organization and structure in plants requires the generation of new probes for measuring actin dynamics in living cells. Fluorescent analog cytochemistry presents an unrivaled opportunity to probe the actin cytoskeleton in living cells. Such method using in the study of plant actin cytoskeleton has not been reported. By using this method, based on the affinity chromatography of profilin with PLP-Sepharose (PLP: poly-L-proline) for actin purification, the author obtained 6 mg of > 98% in purity, polymerizable actin from 10 g of maize (Zea mays L. ) pollen, and this actin was successfully labeled with Oregon Green 488 carboxylic acid. From 10 g of maize pollen, 1.2 mg with 60 % dye/protein ratio, polymerizable, fluorescent actin analog was obtained. The study yields an effective method for purifying plant actin and preparing fluorescent analog, which may provide facilities for the study of actin dynamics in plant ceils.     

Actin in Tetrahymena paravorax: Ultrastructural Localization of HMM-Binding Filaments in Glycerinated Cells1,2

Actin has been identified in the ciliated protozoon Tetrahymena paravorax on the basis of the ultrastructural detection of filaments typically decorated with heavy meromyosin (HMM) in glycerinated microstome cells. These filaments are widely distributed in endoplasmic and cortical regions and can form bundles. They are particularly numerous in elongating cells; HMM-binding filaments run approximately parallel to rib microtubules in the ectoplasm of the right wall of the buccal cavity and seem to extend to the cytopharyngeal region, suggesting some role of actin in maintenance of the crest-trough pattern of ribbed wall and/or in formation of food vacuoles. Extensive actin bundles are observed below some membranellar areas and are thought to follow the course of the microtubular “deep fiber bundle.” The “fine filamentous reticulum” underlying the oral ribs and the “apical ring” extending beneath kinetosomes of ciliary couplets display filaments that do not bind HMM and are ? 14 nm in diameter. No evidence for actin in these structures was obtained in the present study. The “specialized cytoplasm” of the cytostome-cytopharyngeal region appears as an undecorated reticulum with 20 nm-spaced nodes. Occasionally HMM-binding filaments were found inside the macronucleus, just beneath its envelope. Actin is suggested to be involved in cell shaping and in control of the transport of food vacuoles.     

Origin of Twist-Bend Coupling in Actin Filaments

Actin filaments are semiflexible polymers that display large-scale conformational twisting and bending motions. Modulation of filament bending and twisting dynamics has been linked to regulatory actin-binding protein function, filament assembly and fragmentation, and overall cell motility. The relationship between actin filament bending and twisting dynamics has not been evaluated. The numerical and analytical experiments presented here reveal that actin filaments have a strong intrinsic twist-bend coupling that obligates the reciprocal interconversion of bending energy and twisting stress. We developed a mesoscopic model of actin filaments that captures key documented features, including the subunit dimensions, interaction energies, helicity, and geometrical constraints coming from the double-stranded structure. The filament bending and torsional rigidities predicted by the model are comparable to experimental values, demonstrating the capacity of the model to assess the mechanical properties of actin filaments, including the coupling between twisting and bending motions. The predicted actin filament twist-bend coupling is strong, with a persistence length of 0.15-0.4 μm depending on the actin-bound nucleotide. Twist-bend coupling is an emergent property that introduces local asymmetry to actin filaments and contributes to their overall elasticity. Up to 60% of the filament subunit elastic free energy originates from twist-bend coupling, with the largest contributions resulting under relatively small deformations. A comparison of filaments with different architectures indicates that twist-bend coupling in actin filaments originates from their double protofilament and helical structure.     

Actin Filaments in Mature Guard Cells Are Radially Distributed and Involved in Stomatal Movement

Stomatal movements, which regulate gas exchange in plants, involve pronounced changes in the shape and volume of the guard cell. To test whether the changes are regulated by actin filaments, we visualized microfilaments in mature guard cells and examined the effects of actin antagonists on stomatal movements. Immunolocalization on fixed cells and microinjection of fluorescein isothiocyanate-phalloidin into living guard cells of Commelina communis L. showed that cortical microfilaments were radially distributed, fanning out from the stomatal pore site, resembling the known pattern of microtubules. Treatment of epidermal peels with phalloidin prior to stabilizing microfilaments with m-maleimidobenzoyl N-hydroxysuccimimide caused dense packing of radial microfilaments and an accumulation of actin around many organelles. Both stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abolished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partial opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation.     

Freeze Preservation of Cultured Plant Cells

A basic technique for successful freeze preservation and storage at -196°C of cultured plant cells and an assay of percentage survival following the freezing-storage-thawing procedure are described. These techniques have been applied to suspension cultures of carrot (3 cell lines), belladonna and sycamore. Dimethyl sulfoxide (DMSO) and glycerol, when appropriately applied, were the most effective cryoprotectants tested. Although these cryoprotectants were of low toxicity and did not cause alterations in the cytology and growth potential of the recovered cells, the cell lines differed in their sensitivity to the toxicity of these cryoprotectants. Small meristematic cells survived the freezing-thawing procedure better than larger more highly vacuolated cells. Specific differences in survival are in part explained in terms of differences in cell morphology.     

Cover Caption: Organization of Actin Filaments

Plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and plays a key role in the organization of the actin cytoskeleton. In this issue, Dong et al. (pp. 250–261) demonstrate that charged residues Arg98 and Lys100 of ADF1 are essential for both G‐ and F‐actin binding, and that basic residues on β‐strand 5 (K82/A) and α‐helix 4 (R135/A, R137/A) form another actin binding site for F‐actin.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

Fluorescence Microscopic Observations on the Distribution Patterns of Actin Filaments in Stigma Cells of Eichhornia crassipes

The distribution patterns of actin filaments in the non-fixed stigma of Eichhornia crassipes (Mart) Solms were examined with fluorescence microscopy by using FITC-phalloidin as fluorescence probe. In the finger-like papillae the distribution patterns of actin filament varied greatly with actin localization. In the basal region fusiform bodies emitting intense fluorescence were scatteredly distributed. In the middle zone(often occupied by dense cytoplasm) a network composed of numerous actin filaments appeared. These filaments of various diameters lay more or less parallelly to the cell axis, extending upwards and gradually merging into some thick dense bundles . In the apical region a few actin filaments sparsely and longitudinally distrubuted in the subcortical cytoplasm,and diffuse fluorescence often appeared in the spheroidal protrusion. Furthermore,an actin network composed of very thin filaments in the periplasm of the cell was observed ;the constituent filaments were in helical arrangement and often branched and interconnected. Considering possible relationship between the actin configurations and the physiological activities and functions of the stigma cells, it is proposed that the active cytoplasmic streaming, the translocation of solutes towards the apical region ,the active secretion of exudate from the spheroidal protrusion and maintaining of the structural integrity and stability of periplasm, all these might be considered as certain physiological events being affected or regulated by the actin filament patterns described above.     

Homocysteine Induces Hypophosphorylation of Intermediate Filaments and Reorganization of Actin Cytoskeleton in C6 Glioma Cells

In this study, we investigated the actions of high homocysteine (Hcy) levels (100 and 500 μM) on the cytoskeleton of C6 glioma cells. Results showed that the predominant cytoskeletal response was massive formation of actin-containing filopodia at the cell surface that could be related with Cdc42 activation and increased vinculin immunocontent. In cells treated with 100 μM Hcy, folic acid, trolox, and ascorbic acid, totally prevented filopodia formation, while filopodia induced by 500 μM Hcy were prevented by ascorbic acid and attenuated by folic acid and trolox. Moreover, competitive NMDA ionotropic antagonist DL-AP5 totally prevented the formation of filopodia in both 100 and 500 μM Hcy treated cells, while the metabotropic non-selective group I/II antagonist MCPG prevented the effect of 100 μM Hcy but only slightly attenuated the effect induced by of 500 μM Hcy on actin cytoskeleton. The competitive non-NMDA ionotropic antagonist CNQX was not able to prevent the effects of Hcy on the reorganization of actin cytoskeleton in the two concentrations used. Also, Hcy-induced hypophosphorylation of vimentin and glial fibrillary acidic protein (GFAP) and this effect was prevented by DL-AP5, MCPG, and CNQX. In conclusion, our results show that Hcy target the cytoskeleton of C6 cells probably by excitoxicity and/or oxidative stress mechanisms. Therefore, we could propose that the dynamic restructuring of the actin cytoskeleton of glial cells might contribute to the response to the injury provoked by elevated Hcy levels in brain.     

Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency and location along filaments. The emergent behavior of mechanically heterogeneous filaments, particularly under confinement, emphasizes that severing in cells is likely to be influenced by multiple physical and chemical factors.     

Stepwise Sliding of Single Actin and Myosin Filaments

Dynamics of sliding were explored in isolated actin and myosin filaments. Sliding occurs in steps. The steps are integer multiples of 2.7 nm, which is equal to the monomeric repeat along the actin filament. When filaments were forced to slide in the reverse direction, the size paradigm was the same. This size paradigm is parallel to that seen in the kinesin-microtubule system, where step size is an integer multiple of the tubulin repeat along the microtubule.     

Actin Purified from Maize Pollen Functions in Living Plant Cells

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal [alpha]-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.     

Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases

Changes in the organization and mechanical properties of the actin network within plant and animal cells are primary responses to cell signaling. These changes are suggested to be mediated through the regulation of G/F-actin equilibria, alterations in the amount and/or type of actin-binding proteins, the binding of myosin to F-actin, and the formation of myosin filaments associated with F-actin. In the present communication, the cell optical displacement assay was used to investigate the role of phosphatases and kinases in modifying the tension and organization within the actin network of soybean cells. The results from these biophysical measurements suggest that: (a) calcium-regulated kinases and phosphatases are involved in the regulation of tension, (b) calcium transients induce changes in the tension and organization of the actin network through the stimulation of proteins containing calmodulin-like domains or calcium/calmodulin-dependent regulatory proteins, (c) myosin and/or actin cross-linking proteins may be the principal regulator(s) of tension within the actin network, and (d) these actin cross-linking proteins may be the principal targets of calcium-regulated kinases and phosphatases.     

A New Method for Use in Plant Virology

Авторы описывают новый метод Фильм с использованием камеры и пленки для изучения формирование и развитие локальных поражений и начало и ход системных инфекции, вызванные различными вирусами растений. Метод до сих пор используется для Вирус табачной мозаики только для изучения местных поражений в Nicotiana glutinosa и системные инфекции в Nicotiana tabacum. Она также может быть использован для изучения других растений вирусов, впрочем, и его преимущество состоит в точной регистрации явлений которые не могут быть установлены на всех, или только очень неточно, по невооруженным глазом. Еще одно преимущество заключается в том, что процесс, один раз сняли, может быть прогнозируемый снова или отдельных местных поражений на различных этапах роста можно проследить на документ по копирование таблицы. Этот метод позволяет точное исследование роста, размеры и Форма местные поражения, которое не было возможности, с методами, используемыми до сих пор.