Possible involvement of energy metabolism in the change of cytoplasm organization induced by a protein phosphatase inhibitor,calyculin A,in root hair cells of Limnobium stoloniferum

Summary.  In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN₃, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN₃. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells. Received June 26, 2002; accepted October 18, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-1297, Japan.     

Actin cytoskeleton is responsible for the change of cytoplasmic organization in root hair cells induced by a protein phosphatase inhibitor, calyculin A

Summary In root hair cells ofLimnobium stoloniferum, a protein phosphatase inhibitor, calyculin A (CA), at concentrations higher than 50 nM inhibits cytoplasmic streaming and induces remarkable morphological changes in the cytoplasm: the transvacuolar strands disperse and spherical cytoplasmic bodies emerge. The mechanism of the morphological changes of the cytoplasm induced by CA was studied by pharmacological analyses. The formation of spherical bodies in cells treated with CA was suppressed by the actin-depolymerizing and -fragmenting drugs latrunculin B and cytochalasin D at concentrations higher than 100 nM and 5 M, respectively. In contrast, 100 M propyzamide, a microtubule-depolymerizing drug, did not affect the formation of spherical bodies by CA. Interestingly, 60 mM 2,3-butanedione monoxime, an inhibitor of myosin, also suppressed the CA-induced formation of cytoplasmic spherical bodies. These results indicate that the actin cytoskeleton is intimately involved in the morphological changes of the cytoplasm induced by CA.Abbreviations APW artificial pond water - BDM 2,3-butanedione monoxime - CD cytochalasin D - DMSO dimethylsulfoxide - LB latrunculin B - Pro propyzamide     

Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells ofHydrocharis

Summary Effects of cytochalasin B and mycalolide-B on cytoplasmic streaming, organizations of actin filaments and the transvacuolar strand were studied in root hair cells ofHydrocharis, which shows reverse fountain streaming. Both toxins inhibited cytoplasmic streaming and destroyed the organizations of actin filaments and transvacuolar strands. However, we found a great difference between these toxins with respect to reversibility. The effects of cytochalasin B were reversible but not those of mycalolide B. The present results suggest that actin filaments work as a track of cytoplasmic streaming and as a cytoskeleton to maintain the transvacuolar strand. The usefulness of root hair cells ofHydrocharis in studying the dynamic organization of actin filaments of plant is discussed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ML-B mycalolide B     

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells. Received: 16 September 1999 / Accepted: 3 December 1999     

Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

The role of actin in root hair morphogenesis: studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug

Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament configuration during bulge formation, root hair initiation, sustained tip growth, growth termination, and in full-grown hairs. Lipochito-oligosaccharides (LCOs) were used to interfere with growth ( De Ruijter et al . 1998 , Plant J. 13, 341–350), and cytochalasin D (CD) was used to interfere with actin function. Actin filament bundles lie net-axially in cytoplasmic strands in the root hair tube. In the subapex of growing hairs, these bundles flare out into fine bundles. The apex is devoid of actin filament bundles. This subapical actin filament configuration is not present in full-grown hairs; instead, actin filament bundles loop through the tip. After LCO application, the tips of hairs that are terminating growth swell, and a new outgrowth appears from a site in the swelling. At the start of this outgrowth, net-axial fine bundles of actin filaments reappear, and the tip region of the outgrowth is devoid of actin filament bundles. CD at 1.0 μ m , which does not affect cytoplasmic streaming, does not inhibit bulge formation and LCO-induced swelling, but inhibits initiation of polar growth from bulges, elongation of root hairs and LCO-induced outgrowth from swellings. We conclude that elongating net-axial fine bundles of actin filaments, which we call FB-actin, function in polar growth by targeting and releasing Golgi vesicles to the vesicle-rich region, while actin filament bundles looping through the tip impede vesicle retention.     

Actins from plant and animal sources tend not to form heteropolymers in vitro and function differently in plant cells

Summary. Pollen and skeletal muscle actins were purified and labeled with fluorescent dyes that have different emission wavelengths. Observation by electron microscopy shows that the fluorescent actins are capable to polymerize into filamentous actin in vitro, bind to myosin S-1 fragments, and have a critical concentration similar to unlabeled actin, indicating that they are functionally active. The globular actins from two sources were mixed and polymerized by the addition of ATP and salts. The copolymerization experiment shows that when excited by light of the appropriate wavelength, both red actin filaments (pollen actin) and green actin filaments (muscle actin) can be visualized under the microscope, but no filaments exhibiting both green and red colors are detected. Furthermore, coprecipitations of labeled pollen actin with unlabeled pollen and skeletal muscle actin were performed. Measurements of fluorescent intensity show that the amount of labeled pollen actin precipitating with pollen actin was much higher than that with skeletal muscle actin, indicating that pollen and muscle actin tend not to form heteropolymers. Injection of labeled pollen actin into living stamen hair cells results in the formation of normal actin filaments in transvacuolar strands and the cortical cytoplasm. In contrast, labeled skeletal muscle actin has detrimental effects on the cellular architecture. The results from coinjection of the actin-disrupting reagent cytochalasin D with pollen actin show that overexpression of pollen actin prolongs the displacement of the nucleus and facilitates the recovery of the nuclear position, actin filament architecture, and transvacuolar strands. However, muscle actin perturbs actin filaments when injected into stamen hair cells. Moreover, nuclear displacement occurs more rapidly when cytochalasin D and muscle actin are coinjected into the cell. It is concluded that actins from plant and animal sources behave differently in vitro and in vivo and that they are functionally not interchangeable.     

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.     

Mechanism of inhibition of cytoplasmic streaming by a myosin inhibitor, 2,3-butanedione monoxime

Summary On the basis of the inhibition of myosin by 2,3-butanedione monoxime (BDM), the protein's involvement in various cell activities is discussed. However, it has not been established whether BDM inhibits plant myosin. In the present study, the effect of BDM on isolated plant myosin was analyzed in vitro. The sliding between myosin from lily (Lilium longiflorum) pollen tubes and actin filaments from skeletal muscle was inhibited to 25% at a concentration of 60 mM, indicating that BDM can be used as a myosin inhibitor for plant materials. Cytoplasmic streaming was completely inhibited by BDM at 30 mM in lily pollen tubes and at 70 mM in short root hair cells, and at 100 mM in long root hair cells ofHydrocharis dubia. However, BDM at high concentrations induced the disorganization of actin filament bundles in lily pollen tubes and short root hair cells. In addition, cortical microtubules were also fragmented in short root hair cells treated with BDM, suggesting a possible side effect of BDM.Abbreviations AF actin filament - BDM 2,3-butanedione monoxime - MT microtubule     

Microinjection of pollen‐specific actin‐depolymerizing factor, ZmADF1, reorientates F‐actin strands in Tradescantia stamen hair cells

Maize actin-depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein-conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re-modelling the actin after ADF1 injection may be provided by the microtubular system.     

Actin in living and fixed characean internodal cells: identification of a cortical array of fine actin strands and chloroplast actin rings

Summary We report on the novel features of the actin cytoskeleton and its development in characean internodal cells. Images obtained by confocal laser scanning microscopy after microinjection of living cells with fluorescent derivatives of F-actin-specific phallotoxins, and by modified immunofluorescence methods using fixed cells, were mutually confirmatory at all stages of internodal cell growth. The microinjection method allowed capture of 3-dimensional images of high quality even though photobleaching and apparent loss of the probes through degradation and uptake into the vacuole made it difficult to record phallotoxin-labelled actin over long periods of time. When injected at appropriate concentrations, phallotoxins affected neither the rate of cytoplasmic streaming nor the long-term viability of cells. Recently formed internodal cells have relatively disorganized actin bundles that become oriented in the subcortical cytoplasm approximately parallel to the newly established long axis and traverse the cell through transvacuolar strands. In older cells with central vacuoles not traversed by cytoplasmic strands, subcortical bundles are organized in parallel groups that associate closely with stationary chloroplasts, now in files. The parallel arrangement and continuity of actin bundles is maintained where they pass round nodal regions of the cell, even in the absence of chloroplast files. This study reports on two novel structural features of the characean internodal actin cytoskeleton: a distinct array of actin strands near the plasma membrane that is oriented transversely during cell growth and rings of actin around the chloroplasts bordering the neutral line, the zone that separates opposing flows of endoplasm.     

Confocal Microscopic Observations on Actin Filament Distribution in Lily Pollen Protoplasts

Actin filaments (F-actin) were localized in the isolated pollen protoplasts of lily using TRITC-phalloidin probe and confocal microscopy. Two kinds of pollen protoplasts were examined: one from pollen grains of non-dehiscent anthers(referred to as ‘nearly mature’ pollen); and the other from pollen grains of just dehiscent anthers(referred to as ‘just mature’ pollen). In the cytoplasm of the pollen protoplasts of the ‘nearly mature’ pollen there was a very well organized actin network made up of thick actin bundles. Two types of bundle connections were seen in the network; namely ‘branch’ connections and 'junction' connections. The ‘branch’ connection (or branching points) was formed due to branching or merging of bundies. The ‘junction’ connection (or 'junction' point) had two or more bundles associated with it. Some of the ‘junction’ points might be actin filament organization: centres. The generative cell in iht pollen protoplasts of the ‘nearly mature’ pollen also contained an actin network. But this network was structurally quite loose and the pundles made up the network were short and thick. In the cytoplasm of the pollen protoplasts of the ‘just mature’ pollen the actin net work was more densely packed. The bundles made up the network were also thinner. The actin network in the generative cell was, however, less densely packed. If the pollen protoplasts from both the ‘nearly mature’ and the 'just mature' pollen grains were transferred from a B5 medium into a Brewbaker and Kwack medium supplemented with sucrose, protoplasts rapidly (i.e. within 2 to 3 hours) developed vacuoles and transvacuolar strand. In these va cuolated protoplasts the vegetative nucleus andthe generative cell became tightly surrounded by a new actin network. In the transvacuolar strands there were numerous actin bundles. The “ends” of some of these bundles appeared to be tightly attached to the protoplast membrane indicating that some kind of structures might be present in the protoplast membrane for actin filament attachment.     

Fluorescence Microscopic Observations on the Distribution Patterns of Actin Filaments in the Cells of Developing Endosperm in Triticum aestivum

The presence and distribution patterns of actin filaments (AFs) in the cells of developing wheat (Triticum aestivum L. ) endosperm exhibiting intercellular protoplasmic movement were studied with fluorescence microscopy and video microscopy. By using TRITC-PhaIloidin as fluorescence probe and cytochalasin B (CB) treatment it was uncovered that there were a lot of AFs scattered throughout the cytoplasm and the patterns of AFs varied greatly with the actin localization. Four configurations of AFs could be recognized: an actin meshwork surrouding the nucleus; bundles of AFs radiating from nuclear “basket” and extending to the periplasm; numerous finer AFs densely and randomly distributed in the cortical cytoplasm and fusiform bodies composed of AFs appearing in the endosperm cells lying at the “cheek” of the caryopsis. Judging from the dynamic characters of intercellular movement of the cytoplasmic constituents and the reaction of cytoplasmic strands related to CB treatment, the authors have discussed and proposed that the exhibition of the two kinds of intercellular movement (extrusion of cytoplasmic strands and mass flow of ground substance) might also be in close relation to the different configurations of AF organization in the cytoplasm.     

Arabidopsis VILLIN4 is involved in root hair growth through regulating actin organization in a Ca2+-dependent manner

? Villin is one of the major actin filament bundling proteins in plants. The function of Arabidopsis VILLINs (AtVLNs) is still poorly understood in living cells. In this report, the biochemical activity and cellular function of AtVLN4 were examined. ? The biochemical property of AtVLN4 was characterized by co-sedimentation assays, fluorescence microscopy and spectroscopy of pyrene fluorescence. The in vivo function of AtVLN4 was analysed by ectopically expressing it in tobacco pollen and examining the phenotypes of its T-DNA insertional plants. ? Recombinant AtVLN4 protein exhibited multiple activities on actin, including actin filament bundling, calcium (Ca(2+))-dependent filament severing and barbed end capping. Expression of AtVLN4 in tobacco pollen induced the formation of supernumerary actin cables and reduced pollen tube growth. Loss of function of AtVLN4 resulted in slowing of root hair growth, alteration in cytoplasmic streaming routes and rate, and reduction of both axial and apical actin bundles. ? Our results demonstrated that AtVLN4 is involved in root hair growth through regulating actin organization in a Ca(2+)-dependent manner.     

Arabidopsis thaliana myosin XIK is involved in root hair as well as trichome morphogenesis on stems and leaves

Myosins form a large superfamily of molecular motors that move along actin filaments. The functions of myosins in plant cells are thought to be related to various processes: cell division, movement of mitochondria and chloroplasts, cytoplasmic streaming, rearrangement of transvacuolar strands, and statolith positioning. Class VIII and XI myosins are represented in the Arabidopsis thaliana genome by 4 and 13 potential genes, respectively. The roles of individual class XI myosins and their cellular targets in A. thaliana are still unclear. In this work we implemented a reverse genetic approach to analyse the loss-of-function mutants of XIK, a representative of class XI myosins in A. thaliana. Three different T-DNA insertion mutants in the myosin XIK gene showed similar phenotypes: impaired growth of root hair cells, twisted shape of stem trichomes, and irregular size, branch positioning, and branch expansion of leaf trichomes. Morphometric analysis of mutant seedlings showed that the average length of root hairs was reduced up to 50% in comparison with wild-type root hairs, suggesting an involvement of the class XI myosin XIK in tip growth. On leaves, the proportion of trichomes with short branches was doubleed in mutant plants, and the mutant trichomes possessed a mildly twisted shape. Therefore, we concluded that myosin XIK is involved also in the elongation of stalks and branches of trichomes.     

Actin-Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co-align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co-align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin-binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly-merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross-linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.     

Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells

The cytoskeleton that supports microvilli in intestinal epithelial cells was visualized by the quick-freeze, deep-etch, rotary-replication technique (Heuser and Salpeter. 1979. J. Cell Biol. 82: 150). Before quick freezing, cells were exposed to detergents or broken open physically to clear away the granular material in their cytoplasm that would otherwise obscure the view. After such extraction, cells still displayed a characteristic organization of cytoskeletal filaments in their interiors. Platinum replicas of these cytoskeletons had sufficient resolution to allow us to identify the filament types present, and to determine their characteristic patterns of interaction. The most important new finding was that the apical "terminal web" in these cells, which supports the microvilli via their core bundles of actin filaments, does not itself contain very much actin but instead is comprised largely of narrow strands that interconnect adjacent actin bundles with one another and with the underlying base of intermediate filaments. These strands are slightly thinner than actin, do not display actin's 53A periodicity, and do not decorate with myosin subfragment S1. On the contrary, two lines of evidence suggested that these strands, could include myosin molecules. First, other investigators have shown that myosin is present in the terminal web (Mooseker et al. 1978. J. Cell Biol. 79: 444-453), yet we could find no thick filaments in this area. Second, we found that the strands were removed completely in the process of decorating the core filament bundles with the myosin subfragment S1, suggesting that they had been competitively displaced by exogenous myosin. We conclude that myosin may play a structural role in these cells, via its cross-linking distribution, in addition to whatever role it plays in microvillar motility.     

Actin-filament-dependent remodeling of the vacuole in cultured mesophyll protoplasts

Summary. The ability of plant cells to dedifferentiate represents an important survival strategy invoked in a range of situations from repair mechanisms following wounding to apomixis. Dedifferentiation requires that somatic cells reprogram and enter the cell division cycle. This in turn necessitates the accurate partitioning of nuclear content and organelles, such as chloroplasts, to daughter cells, thereby ensuring continuity of cellular information systems. The distribution of cytoplasm and its organelle content in mature plant cells is governed by a large, central vacuole, with connections between distant cortical and perinuclear cytoplasmic domains mediated by transvacuolar strands. Here we examined the changes to vacuolar architecture in Arabidopsis thaliana protoplasts expressing a green-fluorescent protein fusion to a δ-tonoplast-intrinsic protein (δTIP). We found that vacuolar architecture became increasingly intricate during protoplast culture with the development of numerous transvacuolar strands. The development of an intricate vacuolar architecture was an actin filament- and not microtubule-dependent process, as is the case in interphase plant cells. Furthermore, we show that myosin is required for this increased complexity of vacuolar architecture and the formation of subcortical actin filament arrays. Despite the likelihood that increased vacuolar invagination would allow better redistribution of cytoplasmic organelles, we found that repositioning of chloroplasts from cortical to perinuclear cytoplasm was not dependent on transvacuolar strands. Our findings indicate that the vacuole is a dynamic entity that develops a complex architecture before dedifferentiating plant cells enter cell division. Supplementary material to this paper is available in electronic form at Correspondence and reprints: School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.     

A cytochalasin-sensitive actin filament meshwork is a prerequisite for local wound wall deposition inNitella internodal cells

Summary Reorganization of the actin cytoskeleton following cell wall puncturing of characean internodal cells was studied by immunofluorescence and confocal laser scanning microscopy. Injury locally destroyed the parallel subcortical actin filament bundles and cortical actin strands that are characteristic of unwounded regions. At wounds, a delicate three-dimensional interlaced structure of actin strands, with meshes up to 5 m wide, formed by de novo assembly of isolated filaments and by the elongation of residual subcortical actin bundles and cortical actin strands. The actin meshwork persisted for up to 2 h, corresponding to the duration of intense wound wall secretion. Actin filament bundles continuous with the subcortical bundles outside the wound then regenerated, their parallel alignment probably assisted by endoplasmic flow. Cytochalasin D concentrations that arrested cytoplasmic streaming completely inhibited the formation of the actin meshwork, wound wall deposition and recovery of actin bundles. Concentrations that only reduced streaming velocity delayed meshwork formation and wound walls were thinner than in controls. The actual amount of F-actin within the meshwork, however, was clearly greater in the presence of low cytochalasin concentrations. In late stages of recovery, the actin bundles became very thick and intervening spaces became wider thereby forming a conspicuous, three-dimensional lattice that was continuous with interwebbing subcortical bundles and cortical actin around the periphery of the wound. Our experiments suggest that actin meshwork formation is a prerequisite for plasma membrane-directed transport of vesicles involved in wounding-induced exocytosis in characean internodes. Stabilization of the meshwork by subinhibitory concentrations of cytochalasin D is probably caused by actinbinding properties of the drug that either induce bundling or impede function of associated proteins.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - CLSM confocal laser scanning microscope (microscopy) - DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - MBS m-maleimidobenzoyl N-hydroxy-succinimide ester - PBS phosphate-buffered saline - SCAB subcortical actin bundle