Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.     

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.     

Co-localisation of membranes and actin in growing infected cells of Glycine max root nodules

The root nodule of Glycine max (L.) Merr. is almost spherical at maturity, and its central tissue consists of infected cells filled with numerous symbiosomes containing bacteroids, interspersed with uninfected cells. During the growth of the nodule, the volume of each infected cell and the number of bacteroids per cell increases, and thus abundant membranes are required for the proliferation of symbiosomes. In expanding infected cells, there are areas adjacent to the nucleus that are devoid of bacteroids, but these areas are filled with numerous membranes and actin filaments, surrounded by endoplasmic reticulum membranes, indicating a perinuclear reservoir of newly formed membranes and a role for actin in delivering membranes to proliferating symbiosomes.     

Actin organization during the cell cycle in meristematic plant cells. Actin is present in the cytokinetic phragmoplast

The distribution and organisation of F-actin during the cell cycle of meristematic root-tip cells of Allium was investigated using a rhodamine-labelled phalloidin to stain F-actin in isolated cell preparations. Such preparations could, in addition, be stained for tubulin by immunofluorescence, enabling a comparison between F-actin and microtubule distributions in the same cell. In interphase, an extensive array of actin-filament bundles was present in the cytoplasm of elongating cells, the bundles generally following the long axis of the cell and passing in close proximity to the nucleus. In contrast, the interphase microtubule array occupied the cortex of the cell and was oriented at right angles to the actin bundles. In smaller, isodiametric cells, microfilament arrays were present but less well developed. During cell division, phalloidin-specific staining was seen in the cytokinetic phragmoplast, and co-distributed with microtubules at all stages of cell plate formation; however, neither the pre-prophase band nor the mitotic spindle were stained with phalloidin. Co-distribution of F-actin and microtubules only occurs, therefore, at cytokinesis. The relationship between microfilaments and microtubules is discussed, together with the possible role of actin in the phragmoplast.     

Actin versus tubulin configuration in arbuscule-containing cells from mycorrhizal tobacco roots

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells.
Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus.
These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant–fungus interactions during the mycorrhizal symbiosis.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

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.     

骆驼刺根瘤菌的超微结构研究

用透射电子显微镜研究骆驼刺根瘤中的根瘤菌。结果表明。在成熟的骆驼刺根瘤中,根瘤菌的大小、数量、形态、分布位置及精细结构随寄主细胞的发育程度不同而异。早期侵染细胞中,根瘤菌小,数量少,一般呈球形或椭球形,位于细胞壁附近及靠近核区的地方,没有聚磷酸盐颗粒和聚羟基丁酸。成熟侵染细胞中,根瘤菌个体较大,数量较多．多呈棒状,少数为球形或椭球形。有很多根瘤菌还呈现明显的“T”形、“Y”形或“V”形,菌体占满了整个细胞,这时的根瘤菌大多数含有聚羟基丁酸和聚磷酸盐颗粒。而在衰老的侵染细胞中,根瘤菌细胞质收缩,电子密度增高．形状变得很不规则,有的根瘤菌解体,呈现膜泡状结构,菌体中含有数量不等的聚羟基丁酸和聚磷酸盐颗粒。球状根瘤菌从侵染初期到侵染细胞裂解的整个阶段中都仔在。并且观察到的处于分裂状态的根瘤菌都是球状菌,因此可以推测骆驼刺根瘤中是以球状根瘤菌来进行增殖的。     

A novel fix- symbiotic mutant of Lotus japonicus, Ljsym105, shows impaired development and premature deterioration of nodule infected cells and symbiosomes

Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix- mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.     

Membrane-associated actin filaments in the cortical cytoplasm of the rat mast cell

Cytoplasmic microfilaments are regular constituents of the cortical cytoplasm of rat mast cells. Heavy meromyosin binding to the microfilaments in glycerinated mast cells indicates that they represent actin filaments. Many of the actin filaments were found to be attached to spots of increased density of the plasma membrane. The actin filaments, possibly as part of an actomyosin system, may be involved in exocytosis of mast cell granules.     

Epidermal cells of a symbiosis-defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus

The influence of the mycorrhizal fungus Gigaspora margarita on cytoskeleton organisation in epidermal cells of Lotus japonicus roots was compared between plants of the wild type Gifu and the mutant Ljsym4-2, in which the fungus is confined to the epidermal cells. Immunofluorescence labelling of plant microtubules and microfilaments showed only limited alterations in the peripheral cytoskeleton of epidermal cells during early stages of fungal interaction with the wild type. Later, microtubules and microfilaments enveloped the growing hypha, while the host cell nucleus moved close to the fungus. In contrast, epidermal cells of the mutant responded with disorganisation and disassembly of microtubules and microfilaments before and during fungal penetration attempts. The fungus penetrated only as far as to epidermal cells, whose cytoplasm became devoid of tubulin and actin, suggesting cell death. The close relationship between host cytoskeleton organisation and compatibility with the fungus suggests that a functional Ljsym4 gene is necessary for correct reorganisation of the epidermal cell cytoskeleton in the presence of the fungus and for avoiding hypersensitivity-like reactions.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

A novel ankyrin-repeat membrane protein, IGN1, is required for persistence of nitrogen-fixing symbiosis in root nodules of Lotus japonicus

Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix(-) mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8-9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis.     

Array and distribution of actin filaments in guard cells contribute to the determination of stomatal aperture

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.     

Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study.

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 microM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.     

Embryogenesis in <Emphasis Type="Italic">Sedum acre</Emphasis> L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.     

Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in beta-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can "hijack" cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.     

Organization of the cytoskeleton in early Drosophila embryos

The cytoskeleton of early, non-cellularized Drosophila embryos has been examined by indirect immunofluorescence techniques, using whole mounts to visualize the cortical cytoplasm and sections to visualize the interior. Before the completion of outward nuclear migration at nuclear cycle 10, both actin filaments and microtubules are concentrated in a uniform surface layer a few micrometers deep, while a network of microtubules surrounds each of the nuclei in the embryo interior. These two filament-rich regions in the early embryo correspond to special regions of cytoplasm that tend to exclude cytoplasmic particles in light micrographs of histological sections. After the nuclei in the interior migrate to the cell surface and form the syncytial blastoderm, each nucleus is seen to be surrounded by its own domain of filament-rich cytoplasm, into which the cytoskeletal proteins of the original surface layer have presumably been incorporated. At interphase, the microtubules seem to be organized from the centrosome directly above each nucleus, extending to a depth of at least 40 microns throughout the cortical region of cytoplasm (the periplasm). During this stage of the cell cycle, there is also an actin "cap" underlying the plasma membrane immediately above each nucleus. As each nucleus enters mitosis, the centrosome splits and the microtubules are rearranged to form a mitotic spindle. The actin underlying the plasma membrane spreads out, and closely spaced adjacent spindles become separated by transient membrane furrows that are associated with a continuous actin filament-rich layer. Thus, each nucleus in the syncytial blastoderm is surrounded by its own individualized region of the cytoplasm, despite the fact that it shares a single cytoplasmic compartment with thousands of other nuclei.