Relations between cell volume control, microfilaments and microtubules networks in T2 and PC12 cultured cells

The possible relations between cell volume, microfilaments and microtubules networks have been studied in cultured mice fibrosarcoma cells of line T2 and rat pheochromocytoma cells of line PC12. The obtained results show that: 1. Changes in volume induced by application of hypo-osmotic medium are concomitant with a modification in the organization of the microfilaments network as visualized by immunocytochemistry. The microtubules lattice is not affected in these conditions. 2. Disruption of the microfilaments network by cytochalasin B causes a significant decrease in cell volume in isosmotic conditions. It also deeply affects the volume regulation response of cells swollen in hypo-osmotic media. 3. Disruption of the microtubules lattice by colchicine has no effect on volume in isosmotic conditions nor on the volume regulation that follows application of hypo-osmotic shock. The possible role of microfilaments in cell volume control is discussed.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.     

The nitrate reductase inhibitor,tungsten, disrupts actin microfilaments in Zea mays L.

Tungsten is a widely used inhibitor of nitrate reductase, applied to diminish the nitric oxide levels in plants. It was recently shown that tungsten also has heavy metal attributes. Since information about the toxic effects of tungsten on actin is limited, and considering that actin microfilaments are involved in the entry of tungsten inside plant cells, the effects of tungsten on them were studied in Zea mays seedlings. Treatments with sodium tungstate for 3, 6, 12 or 24 h were performed on intact seedlings and seedlings with truncated roots. Afterwards, actin microfilaments in meristematic root and leaf tissues were stained with fluorescent phalloidin, and the specimens were examined by confocal laser scanning microscopy. While the actin microfilament network was well organized in untreated seedlings, in tungstate-treated ones it was disrupted in a time-dependent manner. In protodermal root cells, the effects of tungsten were stronger as cortical microfilaments were almost completely depolymerized and the intracellular ones appeared highly bundled. Fluorescence intensity measurements confirmed the above results. In the meristematic leaf tissue of intact seedlings, no depolymerization of actin microfilaments was noticed. However, when root tips were severed prior to tungstate application, both cortical and endoplasmic actin networks of leaf cells were disrupted and bundled after 24 h of treatment. The differential response of root and leaf tissues to tungsten toxicity may be due to differential penetration and absorption, while the effects on actin microfilaments could not be attributed to the nitric oxide depletion by tungsten.     

Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells

Respreading gerbil fibroma cells (CCL146) have been found to display cytoplasmic actin-based polygonal fiber networks 10 h after replating (stage III of respreading according to Vasiliev & Gelfand, [1]). The networks have been analyzed by immunofluorescence and electron microscopy. The foci, sites of actin, α-actinin and filamin distribution, are condensed meshworks of microfilaments attached to the inner surface of the plasma membrane. The interconnecting fibers, sites of uniform actin distribution and complementary periodicities of α-actinin and myosin, are bundles of parallel microfilaments with periodic dense bodies. Heavy meromyosin (HMM) labelling of the microfilaments in the foci and interconnecting bundles confirm that they contain actin. In addition, approx. 70% of the microfilaments associated with an individual focus have a uniform polarity relative to it (arrowheads pointing away) suggesting that they have their origin there. Our results support earlier conclusions [2] that polygonal networks are structural intermediates responsible for organizing contractile proteins of the cortical microfilament layer into stress fibers.     

Nuclear-cytoskeletal interactions: evidence for physical connections between the nucleus and cell periphery and their alteration by transformation.

The overall coordination of cell structure and function that results in gene expression requires a spatial and temporal precision that would be unobtainable in the absence of structural order within the cell. Cells contain extensive and elaborate three-dimensional skeletal networks that form integral structural components of the plasma membrane, cytoplasm, and nucleus. These skeletal networks form a dynamic tissue matrix are composed of the nuclear matrix, cytoskeleton, and extracellular matrix. The tissue matrix is an interactive network which undergoes dynamic changes as cells move and change shape. Pathologists have long recognized cancer in pathologic specimens based on the altered morphology of tumor cells compared to their normal counterparts. The structural order of cells appears to be altered in transformed cells. This structural order is reflected in the altered morphology and motility observed in transformed cells compared to their normal counterparts, however, it is unclear whether the structural changes observed in cancer cells have any functional significance. We report here on the nature of the physical connections between the nucleus and cell periphery in nontransformed cells and demonstrate that the nucleus is dynamically coupled to the cell periphery via actin microfilaments. We also demonstrate that the dynamic coupling of the nucleus to the cell periphery via actin microfilaments is altered in Kirsten-ras transformed rat kidney epithelial cells. This loss of structure-function relationship may be an important factor in the process of cell transformation.     

Mechanics of Individual Keratin Bundles in Living Cells

Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻活体生殖细胞及精细胞的微丝骨架

利用绿色荧光蛋白(GFP)基因结合鼠Talin基因表达技术及水稻(Oryza sativa L.)转基因技术,筛选出表达稳定和具等位基因型的第三代转基因水稻.在其活体花粉的4个发育阶段(Ⅰ.小孢子晚期;Ⅱ.二细胞早期;Ⅲ.二细胞晚期;Ⅳ.三细胞阶段),观察了细胞内微丝骨架的分布和结构形态的变化.发现在这4个花粉发育阶段,花粉内的营养核、生殖核、生殖细胞和精细胞都在不同的发育阶段出现位移.而这些位移与微丝骨架的结构变化和运动有密切关系.在胞质中央的微丝网络以及细胞周质的网络不断变化和互动,导致营养核、生殖核或生殖细胞和精细胞的定向位移.在活体生殖细胞和精细胞内,存有一股与细胞纵轴平行排列的微丝骨架.这些微丝骨架对生殖细胞及精细胞可以提供移动的动力,这对生殖细胞或精细胞在花管内以及胚囊内的运动(包括独自游动)提供了依据.     

Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells

Dynamic reorganization of the actin microfilament networks is dependent on the reversible phosphorylation of myosin light chain. To assess the potential role of protein phosphatases in this process in living nonmuscle cells, we have microinjected the purified type-1 and type-2A phosphatases into the cytoplasm of mammalian fibroblasts. Our studies reveal that elevating type-1 phosphatase levels led to the rapid (within 30 min) and fully reversible disassembly of the actin microfilament network as determined by immunofluorescence analysis. In contrast, microinjection of equivalent amounts of the purified type-2A phosphatase had no effect on actin microfilament organization. Metabolic labeling of cells after injection of purified phosphatases was used to analyze changes in protein phosphorylation. Concomitant with the disassembly of the actin microfilaments induced by type-1 phosphatase, there was an extensive dephosphorylation of myosin light chain. No such change was observed when cells were injected with type-2A phosphatase. In addition, after extraction of fibroblasts with Triton X-100, the type-1 phosphatase could be specifically localized by immunofluorescence to a fibrillar network of microfilaments. Furthermore, neutralizing type-1 phosphatase activity in vivo by microinjection of an affinity-purified antibody, prevented the reorganization of actin microfilaments that we had previously described following injection of cAMP-dependent protein kinase. These data support the notion that type 1 and type-2 phosphatases have distinct substrate specificity in living cells, and that type-1 phosphatase plays a predominant role in the dephosphorylation of myosin light chain and thus in the modulation of actin microfilament organization in vivo in intact nonmuscle cells.     

Patterns of actin filaments during cell shaping in developing mesophyll of wheat (Triticum aestivum L.).

Young leaves of wheat exhibit a smooth developmental gradient with meristematic cells at the base and highly differentiated cells at the tip. During differentiation, mesophyll cells attain a lobed outline resembling tube-shaped balloons with almost regularly spaced isthmi. Microfilament patterns in developing wheat mesophyll cells were investigated using fluorescent-labeled phalloidin. Various patterns were found, including delicate arrays of transversely oriented microfilaments in the cortex of the cytoplasm. A close correlation between changes in the patterns of cortical microfilaments, microtubules, cell wall microfibrils, and cell shape was observed. The fine arrays of transversely oriented microfilaments coaligned with bands of microtubules occurring during cell elongation. These bands were found beneath sites of intense wall deposition. It has recently been proposed that the resulting hoops of wall reinforcement prevent cell expansion in the corresponding regions and thus give rise to the peculiar cell shape. When cell expansion ceased, and the typical lobed cell shape was attained, a dense network of microfilaments was retained in the cytoplasm, which was in contrast to what has been described for the microtubular arrays.     

Low temperature effects on growth and actin cytoskeleton organisation in suspension cells of winter oilseed rape

Rhodamine-phalloidin staining of winter oilseed rape suspension cells revealed that the structure of actin cytoskeleton changes with the phase of cell growth. In small, 4-day-old cells, entering the exponential phase of growth, a dense and uniformly distributed cortical microfilament networks was seen. In six-day-old vacuolated cells, which reached the stationary phase of growth, the actin cytoskeleton was composed of thicker microfilament cables in irregular arrangements. In cells acclimated in cold for 7 days a dense, uniformly distributed and cortical microfilament network was still seen. The fine microfilament network was sensitive to extracellular freezing since the structures underwent depolymerization at −3 °C (in the presence of extracellular ice), both in non-acclimated and cold-acclimated cells. The thicker transvacuolar cables in cells of the stationary growth phase resisted freezing to −7 °C. Acclimation of suspensions at 2 °C resulted in slowing down growth of cells and in the increased freezing tolerance of cells as indicated by a decrease of LT₅₀ from −11 °C to −17.5^o or to −25 °C when determined 7 or 20 days after the beginning of the cold treatment, respectively. Freezing tolerance of non-acclimated cells decreased from −11 °C to −8 °C during subculture, showing a transient increase to −17 °C on the day 6. Results indicate that the arrangement of actin microfilaments and their sensitivity to freezing-induced depolymerization depends on the phase of cell growth rather than on cell acclimation status. Possible mechanisms involved in the freezing-induced depolymerization of actin microfilaments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role for actin in the polarized release of rotavirus

Rotaviruses are characterized by polarized release from the apical side of infected enterocytes, and the rotavirus VP4 spike protein specifically binds to the actin network at the apical pole of differentiated enterocytic cells. To determine the functional consequences of this VP4-actin interaction, fluorescence recovery after photobleaching experiments were carried out to measure the diffusional mobility of VP4 associated with the microfilaments. Results show that VP4 binds to barbed ends of microfilaments by using actin treadmilling. Actin treadmilling inhibition results in the loss of rotavirus apical preferential release, suggesting a major role for actin in polarized rotavirus release.     

Cytoskeletal organization and tissue patterns of epithelia in the sponge Ephydatia mülleri

Comparative morphological studies on cytoskeletal patterns of sponge basal epithelium at the tissue level have been performed by diverse methods, including immunofluorescence microscopy, and scanning and transmission electron microscopy of stained whole mounts, thin sections or replicas. These methods give results consistent with each other and show the importance of actin assemblies, which function in addition to the microtubular system and in the absence of intermediate filaments. Actin microfilaments indeed are involved in the formation of cables and networks closely associated with the plasma membrane. Both the cables and the networks result from arrangements of microfilaments into bundles of variable size, and the two types of assembly are probably interconvertible. Microfilaments appear to be implicated in the establishment of spot desmosomes and as devices for cell-to-substratum attachment. Due to the desmosomal articulations from cell to cell, the actin cytoskeleton is framed throughout the complete epithelium. It supports the unitary nature of the entire tissue, which is constructed and functions as a whole. It therefore establishes the “histoskeleton” of basic epithelial tissues. The histoskeleton is involved in all epithelial activities but is not uniformly organized into identical cell patterns at the tissue level because activities are sequential and not synchronized in all cells. Similar cytoskeletal patterns exist only in groups of cells, and this suggests that, at a given time, the multiple functions of the epithelium may be mediated by the occurrence of several multicellular functional units within a single epithelial tissue.     

Tissue slices from living root caps as a model system in which to study cytodifferentiation of polar cells

Tissue slices of living root caps of cress (Lepidium sativum L.), two to three cell layers in thickness, were prepared by a microsurgical procedure. The viability, cellular structures and cytoplasmic movement of the cells were examined in the light microscope. Nuclei, amyloplasts, vacuoles and endoplasmic reticulum were identified and their positions confirmed after fixation and observation of the same cells in the electron microscope. The distribution of microtubules was shown by immunocytochemistry. During germination, microtubules appear first at the distal edges of the statocytes, while in mature statocytes a distal domain of criss-crossed microtubules could be distinguished from a proximal domain with transversally oriented microtubules. Microfilaments in young statocytes form a nuclear enclosure; in mature statocytes bundles of microfilaments fan out into the cell cortex. The transition from statocytes to secretion cells is accompanied by a more pronounced cortical network of microfilaments, while the nucleus-associated microfilaments remain visible. It is suggested that these microfilaments play a role in the positioning of the nucleus and the translocation of endoplasmic reticulum.Abbreviations ER endoplasmic reticulum - MF microfilament - MT microtubule     

Confocal Microscopic Observations of Microfilaments in germinating Pollen of Hedychium coronarium

Changes in the microfilament (actin)organization in the germinating pollen of Hedychium coronarium Koenig were followed after TRITC-phalloidin staining without fixation. Changes in the pattern of organization of the microfilaments were visualized using eonfocal microscopy. In the hydrated pollen a reticulate network of microfilament can be observed. Before the pollen tube protrudes out from the germination pore numerous microfilaments begin to converge towards the aperture. After 10–30 mins of germination,pollen tube appears. In the pollen tube a new network of microfilament forms near the tip region. Between the pollen and the pollen tube tip region there are numerous linearly arranged microfilaments. About 1 hour after germination,the pollen tube has reached a length of about 300μm Inside the pollen, tube there are numerous longitudinally oriented microfilaments. The microfilament network in the pollen tube tip region does not change much. About 2 hours after germination,the pollen tube reaches about 1000μm in length. At this stage,the pattern of distribution of microfilament in the pollen tube is very similar to that seen at the earlier stages of development ,whereas the pattern is somewhat different in the pollen. Microfilaments in the central region of the pollen grain disappear but still a parietal network in the peripheral region. About 5 hours after germination,the microfilaments in the pollen tube become abnormally variable and produce branches. Some even change into spicules, sheets and thick bundles.     

Fate of microfilaments in vero cells infected with measles virus and herpes simplex virus type 1.

In herpes simplex virus type 1-infected Vero cells, reorganization of microfilaments was observed approximately 4 h postinfection. Conversion of F (filamentous) actin to G (globular) actin, as assessed by a DNase I inhibition assay, was continuous over the next 12 to 16 h, at which time a level of G actin of about twice that observed in uninfected cells was measured. Fluorescent localization of F actin, using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin, demonstrated that microfilament fibers began to diminish at about 16 to 18 h postinfection, roughly corresponding to the time that G actin levels peaked and virus-induced cytopathology was first observable. In measles virus-infected cells, no such disassembly of microfilaments occurred. Rather, there was a modest decrease in G actin levels. Fluorescent localization of F actin showed that measles virus-infected Vero cells maintained a complex microfilament network characterized by fibers which spanned the entire length of the newly formed giant cells. Disruption of microfilaments with cytochalasin B, which inhibits measles virus-specific cytopathology, was not inhibitory to measles virus production at high multiplicities of infection (MOI) but was progressively inhibitory as the MOI was lowered. The carbobenzoxy tripeptide SV-4814, which inhibits the ability of Vero cells to fuse after measles virus infection, like cytochalasin B, inhibited measles virus production at low MOI but not at high MOI. Thus, it appears that agents which affect the ability of Vero cells to fuse after measles virus infection may be inhibitory to virus production and that the actin network is essential to this process.     

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7–14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.     

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.