The effects of hydrostatic pressure-induced changes on the cytoskeleton and on the regulation of gene expression in pheochromocytoma (PC-12) cells.

The purpose of this investigation was to determine the relationship of hydrostatic pressure-induced changes in the cytoarchitecture to regulation of gene expression in PC-12 cells. Hydrostatic pressure disrupts the cytoskeleton, decreases tubulin and actin mRNA levels and causes changes in the localization of tubulin and actin mRNA. Actin mRNA levels, at 6000 and 10,000 psi for 20 min, were reduced to 78% and 64%, respectively, in undifferentiated cells and to 81% and 72%, respectively, in 4-day differentiating cells, relative to untreated controls. Tubulin mRNA levels, at 6000 and 10,000 psi for 20 min, were reduced to 75% and 67%, respectively, in undifferentiated cells and to 84% and 74%, respectively, in 4-day differentiating cells. Changes in the localization of mRNA in the soluble and cytoskeletal fractions were determined by measuring the pressure level where the mRNA level in the cytoskeletal fraction equals the mRNA level in the soluble fraction. This measurement was designated the cytoskeletal/soluble fraction index (CSFI(50)). CSFI(50)measurements indicated that following hydrostatic pressure, actin mRNA cytoskeletal association was more stable than tubulin mRNA cytoskeletal association. The addition of chemicals which stabilize or destabilize microtubules and microfilaments to pressure treatment resulted in additional changes in the CSFI(50).     

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.     

Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization

Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis.     

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.     

CANNABINOID ENANTIOMER ACTION ON THE CYTOARCHITECTURE

The negative and positive enantiomers of 7-hydroxy- Δ⁶-tetrahydrocannabinol-dimethylheptyl (designated HU-210 and HU-211 respectively) differentially affect undifferentiated and differentiating cultured pheochromocytoma cells (PC-12 cells). In general, cell viability and cell proliferation were suppressed to a much greater extent with HU-210 than with HU-211 in differentiating cells. The effects of these synthetic cannabinoids on the cytoskeleton of PC-12 cells were examined by epifluorescence and confocal microscopy. In both undifferentiated and differentiating PC-12 cells, HU-211 has little effect on the cytoarchitecture whereas HU-210 disrupts the distribution of microtubules and microfilaments. Vacuoles (2–4 μm) were evident in the cytoplasm of HU-210-treated cells but not in the cytoplasm of HU-211-treated cells or in vehicle controls. Tubulin and actin mRNA levels were reduced to 5 and 40 %, respectively (relative to untreated controls) in 10 μmHU-210-treated cells whereas the same concentration of HU-211 reduced tubulin and actin mRNA levels to 90 and 95 %, respectively. A comparison of the effects of the paired enantiomers and Δ¹-THC on the cellular parameters studied reveals that in differentiating cells the action of Δ¹-THC is intermediate between that of HU-210 and HU-211. This study demonstrates that compared to HU-210 and Δ¹-THC the positive enantiomer HU-211 has little cellular activity.     

Role of cytoskeleton in gravisensing of the root elongation zone in Arabidopsis thaliana plants

In order to reveal the involvement of tubulin microtubules and actin microfilaments in gravisensing reactions in the distal elongation zone of root, Arabidopsis thaliana plants stably transformed with MAP4-GFP construct were grown under slow clinorotation. Experiments have shown that stabilization of cell growth in the distal elongation zone of Arabidopsis seedling root is provided by common structural organization of microtubules and microfilaments, and interrelations between microtubules and microfilaments is highly dependent upon the type of cell differential growth. Less pronounced effect of microfilament disruption on microtubule organization has been observed under clinorotation and it suggests the existence of complex mechanism of cooperation between microtubules and microfilaments which is probably, masked on earth.     

A comparison of HEp-2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli

In order to further characterize cellular invasion by enteropathogenic Escherichia coli (EPEC), we compared invasion of HEp-2 cells by EPEC and enteroinvasive E. coli (EIEC). We used a gentamicin HEp-2 cell assay and measured bacterial recovery under conditions of varying incubation time and temperature, and in the presence or absence of inhibitors of cellular microfilaments and microtubules. We found that, unlike EIEC, EPEC did not rapidly multiply within HEp-2 cell but invaded well at 32 degrees C. While microfilament inhibitors reduced invasion by both EIEC and EPEC, microtubule inhibitors reduced invasion by EPEC only. These results suggest that EPEC and EIEC differ in their mechanisms of epithelial cell invasion.     

Hypersensitivity to cytoskeletal antagonists demonstrates microtubule-microfilament cross-talk in the control of root elongation in Arabidopsis thaliana

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.     

Formation and maintenance of viroplasmic centers in Tipula iridescent virus-infected mosquito cells with deranged cytoskeletons

We have examined the role of cytoskeletal elements with respect to the formation and maintenance of viroplasmic centers (VCs) in Tipula iridescent virus (TIV)-infected mosquito Aedes albopictus (C6/36) cells. Filamentous systems consisting of microtubules and microfilaments were detected by immunofluorescence microscopy. Inoculation of cells with TIV resulted in an alteration of microtubule and microfilament organization whether or not VCs developed. The formation of short arrays of microtubules induced by taxol or the depolymerization of microtubules by colchicine, as observed by immunofluorescence microscopy, had no apparent effect upon the development of VCs as detected by Hoechst staining and electron microscopy. The dissolution of the actin-containing filamentous system by cytochalasin B also had no effect upon development. We conclude from these results that microtubules and microfilaments are not involved in the formation or maintenance of VCs in TIV-infected A. albopictus (C6/36) cells.     

Motor proteins regulate force interactions between microtubules and microfilaments in the axon

It has long been known that microtubule depletion causes axons to retract in a microfilament-dependent manner, although it was not known whether these effects are the result of motor-generated forces on these cytoskeletal elements. Here we show that inhibition of the motor activity of cytoplasmic dynein causes the axon to retract in the presence of microtubules. This response is obliterated if microfilaments are depleted or if myosin motors are inhibited. We conclude that axonal retraction results from myosin-mediated forces on the microfilament array, and that these forces are counterbalanced or attenuated by dynein-mediated forces between the microfilament and microtubule arrays.     

Locomotion of epithelial cells : Factors involved in extension of the leading edge

Epithelial cells, like fibroblasts, spread by the active extension of their flattened leading edge. The role of microtubules, microfilaments, and internal hydrostatic pressure in this spreading was studied. While colchicine and colcemid have no effect, cytochalasin B, which affects the microfilament meshwork in the region of the leading edge without affecting general protein synthesis, and sorbitol, which presumably reduces the cell's internal hydrostatic pressure, reversibly prevent the extension of the flattened leading edge. These results are discussed in relation to the mechanism of this extension.     

Cytoskeleton and ion movements during volume regulation in cultured PC12 cells

Summary The present study investigates the role of cytoskeletal elements, microtubules and microfilaments, on ion transport systems activated during volume regulatory processes in PC12 pheochromocytoma cells. Disruption of microtubule network by colchicine (0.1 mm) or vinblastine sulfate (10 m) has no significant effect on PC12 cell hydration or on changes of the intracellular K⁺, Cl^– and Na⁺ content observed in hypo-osmotic conditions. Disruption of microfilament network by cytochalasin B strongly affects volume regulation in a dose-dependent manner. Cytochalasin B leads to a potentiation of the initial cell swelling and the regulatory volume decrease is suppressed. Although, the internal K⁺ and Cl^– level decreases significantly, as demonstrated by measurements of intracellular ion content and ⁸⁶Rb fluxes. Using the patch-clamp technique, we could demonstrate in PC12 cell membranes an ion channel whose gating is affected by application of a negative hydrostatic pressure (mechanical stress) to the membrane patch, by exposure of the cell to hypoosmotic medium (osmotic stress), or by disruption of the microfilament network with cytochalasin B.Water and ion content measurements, as well as ⁸⁶Rb fluxes have been carried out in the Laboratory of Animal Physiology from Professor R. Gilles, University of Liège, Belgium. M. Cornet was supported by the F.N.R.S., Belgium.     

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.     

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     

Sensitivity of Fibroblasts and Their Cytoskeletons to Substratum Topographies: Topographic Guidance and Topographic Compensation by Micromachined Grooves of Different Dimensions

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-μm-wide and 0.5-μm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.     

Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo

During development Caenorhabditis elegans changes from an embryo that is relatively spherical in shape to a long thin worm. This paper provides evidence that the elongation of the body is caused by the outermost layer of embryonic cells, the hypodermis, squeezing the embryo circumferentially. The hypodermal cells surround the embryo and are linked together by cellular junctions. Numerous circumferentially oriented bundles of microfilaments are present at the outer surfaces of the hypodermal cells as the embryo elongates. Elongation is associated with an apparent pressure on the internal cells of the embryo, and cytochalasin D reversibly inhibits both elongation and the increase in pressure. Circumferentially oriented microtubules also are associated with the outer membranes of the hypodermal cells during elongation. Experiments with the microtubule inhibitors colcemid, griseofulvin, and nocodazole suggest that the microtubules function to distribute across the membrane stresses resulting from microfilament contraction, such that the embryo decreases in circumference uniformly during elongation. While the cytoskeletal organization of the hypodermal cells appears to determine the shape of the embryo during elongation, an extracellular cuticle appears to maintain the body shape after elongation.     

‘Rings’ of F-actin form around the nucleus in cultured human MCF7 adenocarcinoma cells upon exposure to both taxol and taxotere

The anti-cancer taxoids, Taxol® (paclitaxel) and Taxotere® (docetaxel), are the most promising anti-mitotic agents developed for cancer treatment in the past decade. The effectiveness of this new class of compounds lies in their unique mechanism of action on the cytoskeleton. Both taxol and taxotere bind to microtubules and shift the normal equilibrium between monomeric and polymerized tubulin to favor the polymerized form by strongly promoting tubulin assembly and inhibiting microtubule depolymerization. Although very similar in structure, these two compounds have recently demonstrated different in vitro, in vivo, and clinical activities; however, no study to date has effectively compared specific cytoskeletal alterations induced by taxol and taxotere in cultured cells. Using specific staining techniques for both F-actin and α-tubulin, this study provides the first detailed immunohistochemical comparison of the effects of equimolar concentrations of taxol and taxotere on both the microfilament and microtubule networks in a cultured cell line. Using human MCF7 breast adenocarcinoma cells, new observations of taxotere/taxol alterations of the cytoskeleton include: an increased abundance of parallel microtubule ‘bundles’ in taxotere treated cells and a definitive reorganization of the microfilament network which results in novel ring-like formations of F-actin condensed exclusively in the perinuclear zone. Reorganization of the actin cytoskeleton induced by a taxoid disruption of the microtubule equilibrium is indicative of the interdependence between microtubules and microfilaments in this transformed cell line and suggests that the indirect role of the taxoids on the microfilament network may have been overlooked in their mechanism of action as chemotherapeutic agents.     

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.     

白芨萜类化合物对人脐静脉内皮细胞凋亡和细胞骨架的作用

利用白芨萜类化合物处理人脐静脉内皮细胞（HUVECs）并进一步研究了细胞凋亡及细胞骨架.白芨萜类化合物可拮抗血管内皮细胞生长因子（VEGF）和碱性成纤维细胞生物因子（bFGF）刺激的HUVECs增殖并诱导细胞凋亡.在处理的HUVECs中caspase-8活性明显增加.流式细胞术分析显示经处理的HUVECs的凋亡率随处理时间延长而升高.通过对微管进行免疫荧光染色和微丝进行荧光染色后,用激光共焦扫描显微观察表明,白芨萜类化合物处理的HUVECs中的微管和微丝发生改变甚至被破坏.因此,白芨萜类化合物造成HUVECs凋亡很可能是通过促使微管解体以及微丝去组装造成的.     

Effects of injected inhibitors of microfilament and microtubule function on the gastrulation movement in Xenopus laevis

It was suggested recently that gastrulation movements in amphibian embryos are caused by the active cell locomotion of individual cells. In order to elucidate the role of microfilaments and microtubules in the cell locomotion occurring during gastrulation, cytochalasin B, colchicine, and other microtubule-disrupting drugs were injected into the blastocoel of early gastrulae of Xenopus laevis. Hypertonic solutions of sorbitol were also injected to elucidate the influence of the internal hydrostatic pressure on the migrating cells. The effects were examined in 1-μm Epon sections of serially fixed embryos and by transmission electron microscopy. Cytochalasin B strongly inhibits cell migration even under conditions that do not cause dissociation into single cells. The cells become round, and have only a few thin cell processes. Electron microscopy shows an alteration in the cortical microfilament network. Colchicine and other microtubule-disrupting drugs have little effect on the rate of cell migration before they cause the accumulation of many mitotic cells and the dissociation of the embryo. The interphase cells are angular and have thin processes like those in the control embryos. The microtubules disappear, and bundles of 10-nm filaments are observed in the cytoplasm of colchicine-injected embryos. Hypertonic sorbitol solutions strongly inhibit cell migration.