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.     

Selective extraction of cotton fiber cytoplasts to identify cytoskeletal-associated proteins

Cytoplasts from cotton (Gossypium hirsutum L.) fiber cells retain microtubule and microfilament cytoskeletons through extraction with non-ionic detergent and ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid. Tubulin and actin are the most abundant proteins in extracted cytoplasts; however, many other less abundant proteins are also present. To determine if minor proteins were associated with the cytoskeleton, microtubules and microfilaments were selectively removed from extracted cytoplasts by detergent extraction in an alkaline Ca²⁺ solution. Under these extraction conditions, microtubules and microfilaments were fragmented and depolymerized unless previously stabilized by taxol and phalloidin. Associated proteins were identified by their loss in conjunction with either microtubules or microfilaments. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one protein, of roughly 115 kDa, appeared to be associated with microfilaments since it was present in Ca²⁺-extracted preparations only when microfilaments were stabilized with phalloidin. The failure of most minor proteins to associate with microtubules and microfilaments suggests that caution must be used when interpreting co-isolation as evidence for an association of low abundance proteins with cytoskeletons.     

Analysis of cytoskeleton structural changes in the granular cells of the frog bladder during the stimulation of water transport

Structural changes of the cytoskeleton of the frog urinary bladder granular cells were examined during low and high water permeability of the epithelium. A tight connection of the microfilaments and microtubules with vacuolar membranes and a great increase in the number of microtubules during a stimulated water flow was shown using different electron microscopic methods. Two populations of microtubules were discovered, respectively, with different diameter and different rate of stability. It is suggested that the thicker microtubules while interacting with actin microfilaments through associated electron dense globules may fulfil the transport function in the cell.     

Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. Received: 9 December 1996 / Accepted: 29 April 1997     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Tensegrity architecture explains linear stiffening and predicts softening of living cells

The problem of theoretical explanation of the experimentally observed linear stiffening of living cells is addressed. This explanation is based on Ingber's assumption that the cell cytoskeleton, which enjoys tensegrity architecture with compressed microtubules that provide tension to the microfilaments, affects the mechanical behavior of the living cell. Moreover, it is shown that the consideration of the extreme flexibility of microtubules and the unilateral response of microfilaments is crucial for the understanding of the living cell overall behavior. Formal nonlinear structural analysis of the cell cytoskeleton under external mechanical loads is performed. For this purpose, a general computer model for tensegrity assemblies with unilateral microfilaments and buckled microtubules is developed and applied to the theoretical analysis of the mechanical response of 2D and 3D examples of tensegrity cells mimicking the behavior of real living cells. Results of the computer simulations explain the experimentally observed cell stiffening. Moreover, the theoretical results predict the possible existence of a transient softening behavior of cells, a phenomenon, which has not been observed in experiments yet.     

Organisation of the cytoskeleton during in vitro maturation of horse oocytes

Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal anti-alpha-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TO-PRO(3)). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphase-I and telophase-I, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrel-shaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation.     

Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.     

Role of microfilaments and microtubules in the invasion of INT-407 cells by Campylobacter jejuni

The internalization mechanisms triggered by Campylobacter jejuni were studied by invasion assays conducted with different inhibitors that act on the cytoskeleton structure of eukaryotic cells. The depolymerization of microfilaments by cytochalasin-D and that of microtubules by colchicines and nocodazole inhibited the uptake of C. jejuni into INT-407 cells in a dose-dependent manner. The inhibitory effect of microfilament depolymerization on C. jejuni internalization was more pronounced than that of microtubule depolymerization. By immunofluorescence microscopic observations, it was demonstrated that both microfilaments and microtubules were localized in INT-407 cells after C. jejuni infection. These data suggest that the internalization mechanism triggered by C. jejuni is associated with the combined effect of microfilaments and microtubules of host cells.     

Organization and functions of cytoskeleton in metazoan ciliated cells

Ciliated cells are characterized by a highly organized cytoskeleton which is connected with the ciliary apparatus. The organization of microtubules, microfilaments, and cytokeratin filaments is described and the relationships of each network with the ciliary apparatus are emphasized. Possible functions of such a complex cytoskeleton are discussed.     

Rhinanthus serotinus (Schönheit) Oborny (Scrophulariaceae): immunohistochemical and ultrastructural studies of endosperm chalazal haustorium development

Chalazal endosperm haustorium in Rhinanthus serotinus consists of a single large binucleate cell. It originates from the primary endosperm cell dividing transversely into two unequal cells: a smaller micropylar cell and a larger chalazal cell. The chalazal cell undergoes a single mitotic division, then lengthens significantly during development and functions as a chalazal endosperm haustorium. In this paper, immunofluorescent techniques, rhodamine phalloidin assay, and electron microscopy were used to examine the actin and tubulin cytoskeleton during the development of the chalazal haustorium. During the differentiation stage, numerous longitudinally oriented bundles of microfilaments ran along the axis of transvacuolar strands in haustorium. Microtubules formed intensely fluorescent areas near the nuclear envelope and also formed radial perinuclear microtubule arrays. In the fully differentiated haustorium cell, the actin cytoskeleton formed dense clusters of microfilaments on the chalazal and micropylar poles of the haustorium. Numerous microfilament bundles occurred near wall ingrowths on the chalazal wall. There were numerous clusters of microfilaments and microtubules around the huge lobed polytenic haustorial nuclei. The microfilaments were oriented longitudinally to the long axis of the haustorium cell and surrounded both nuclei. The microtubules formed radial perinuclear systems which were appeared to radiate from the surface of the nuclear envelope. The early stage of degeneration of the chalazal haustorium was accompanied by the degradation of microtubules and disruption of the parallel orientation of microtubules in the chalazal area of the cell. The degree of vacuolization increased, autophagous vacuoles appeared and the number of vesicles decreased.     

The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.     

Evidence for a role of capillary pericytes in vascular growth of the developing ovine corpus luteum

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.     

Rearrangement of the keratin cytoskeleton after combined treatment with microtubule and microfilament inhibitors

In addition to containing microtubule and microfilament systems, vertebrate epithelial cells contain an elaborate keratin intermediate-filament cytoskeleton. Little is known about its structural organization or function. Using indirect immunofluorescence microscopy with an antikeratin antiserum probe, we found that destabilization of microtubules and microfilaments with cytostatic drugs induces significant alterations in the cytoskeletal organization of keratin filaments in HeLa and fetal mouse epidermal cells. Keratin filament organization was observed to undergo a rapid (1-2 h) transition from a uniform distribution to an open lattice of keratin fibers stabilized by membrane-associated focal centers. Since addition of any one drug alone did not elicit significant organizational change in the keratin cytoskeleton, we suggest that microfilaments and microtubules have a combined role in maintaining the arrangement of keratin in these cells. Vimentin filaments, the only other intermediate-sized filaments found in HeLa cells, did not co-distribute with keratin in untreated or drug-treated cells. These findings offer a new way to approach the study of the dynamics and functional roles of the keratin cytoskeleton in epithelial cells.     

Cell cytoskeleton and tensegrity

The role of tensegrity architecture of the cytoskeleton in the mechanical behavior of living cells is examined by computational studies. Plane and spatial tensegrity models of the cytoskeleton are considered as well as their non-tensegrity counterparts. Local buckling including deep postbuckling response of the compressed microtubules of the cytoskeleton is considered. The tensioned microfilaments cannot sustain compression. Large deformation of the whole model is accounted and fully nonlinear analysis is performed. It is shown that in the case of local buckling of the microtubules non-tensegrity models exhibit qualitatively the same linear stiffening as their tensegrity counterparts. This result raises the question of experimental validation of the local buckling of microtubules. If the microtubules of real cells are not straight, then tensegrity (in a narrow sense) is not a necessary attribute of the cytoskeleton architecture. If the microtubules are straight then tensegrity is more likely to be the cytoskeletal architecture.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

Cytoskeleton Structure and Composition in Choanoflagellates

The structure and composition of the cytoskeleton has been studied in Monosiga ovata (Protozoa: Order Choanofiagellida Kent 1880) using a combination of methods in association with light and electron microscopy. Supplementary observations are included for Desmarella moniliformis. The basal body of the single anterior flagellum is subtended proximally and at right angles by a second, non-flagellar basal body. The edges of the two basal bodies are connected by a fibrillar bridge. A long, narrow, striated, fibrillar rootlet extends posteriorly from the lower edge of the non-flagellar basal body towards the Golgi apparatus. It is associated throughout most of its length with the surface of a flattened sac. Rootlet microtubules pass radially from a ring of electron dense material which encircles the distal end of the flagellar basal body. These microtubules extend outwards for about one-third of the length of the cell. Within each collar tentacle is a longitudinal bundle of microfilaments composed of actin as illustrated by rhodamine-phalloidin staining for fluorescence microscopy. The base of each microfilament bundle is associated with one or more rootlet microtubules by fine fibrillar bridges. The attachment between microtubules and tentacle microfilaments is further demonstrated by their coordinated displacement when the cytoskeleton becomes dislodged. The role of the cytoskeleton in maintaining the position of the collar tentacles during interphase and cell division is discussed.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

Alterations in the Cytoskeleton Accompany Aluminum-Induced Growth Inhibition and Morphological Changes in Primary Roots of Maize

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea mays L.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.     

Assessing the Cytoskeletal System and its Elements in C6 Glioma Cells and Astrocytes by Atomic Force Microscopy

Object To investigate how the characteristic structure of the cytoskeleton in glioma cells is associated with invasiveness. Methods The whole cytoskeletal system was characterized by atomic force microscopy (AFM), while single cytoskeletal elements were exhibited by AFM and using cytoskeletal protein inhibitors to inhibit microfilaments or/and microtubules and displayed by immunofluorescence microscopy. The fluorescence intensity of F-actin was measured by flow cytometry and the structural difference between C6 glioma cells and astrocytes was studied. Results Cytoskeletons in both cells presented network structures, however, the C6 glioma cells showed an irregular edge root and their microfilaments were creber and dense. Intermediate filaments were extensive network structure with non-polarized multipoint connections. The microtubules were relatively big and long and formed tight bundles with close connections between bundles. Astrocytes had a regular and smooth edge, with sparse microfilaments, while the intermediate filaments were dense and interwoven and the microtubules were dense bundled, but only loosely connected each other. Besides, the fluorescence intensity of F-actin was significantly higher in C6 glioma cells (202.54 ± 11.06) than in the astrocytes (62.64 ± 10.23), P < 0.01. Conclusion Whole cytoskeleton and its elements of C6 cells were disclosed of characteristic structures associated with invasiveness. Meanwhile, the content of F-actin could be used as a parameter for measuring cell invasiveness.