The vacuolated morphology of chordoma cells is dependent on cytokeratin intermediate filaments.

Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA-mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration.

Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D.

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.

The antitumour activities induced by pegylated liposomal cytochalasin D in murine models.

Cytochalasin D targets actin and is ubiquitous in eukaryotic cells. When cytochalasin D is used as a cytotoxic agent in cancer therapy, it causes significant side effects. To prevent this, cytochalasin D can be encapsulated in polyethylene liposomes. In this study, high-performance liquid chromatography observation of the biodistribution of pegylated liposomal cytochalasin D in tumour-bearing mice showed that liposomal cytochalasin D could be conveniently dissolved in water for i.v. injection and that it specifically accumulated in tumour tissues, more than natural cytochalasin D did. The half-time of liposomal cytochalasin D in the plasma was also significantly longer than that of natural cytochalasin D (4h versus 10 min). MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that liposomal cytochalasin D treatment could cause significant inhibition of cell proliferation in vitro in a manner similar to that of natural cytochalasin D. The antitumour activities of liposomal cytochalasin D were investigated in B16 melanoma, CT26 colorectal carcinoma and H22 hepatoma models, and the results indicated that liposomal cytochalasin D could significantly inhibit tumour growth and prolong survival in a manner similar to that of cisplatin. TUNEL-based apoptosis assays showed that liposomal cytochalasin D induced significant tumour cell apoptosis. Significant inhibition of tumour angiogenesis was observed in mice treated with liposomal cytochalasin D. In addition, no significant side effects were observed in mice treated with liposomal cytochalasin D. Our results show that liposomal cytochalasin D increases solubility and bioavailability, a lower incidence of side effects and improves antitumour effects, indicating its potential as a chemical agent for cancer therapy.

Remodeling of actin cytoskeleton in mouse periosteal cells under mechanical loading induces periosteal cell proliferation during bone formation.

BACKGROUND: The adaptive nature of bone formation under mechanical loading is well known; however, the molecular and cellular mechanisms in vivo of mechanical loading in bone formation are not fully understood. To investigate both mechanisms at the early response against mechanotransduction in vivo, we employed a noninvasive 3-point bone bending method for mouse tibiae. It is important to investigate periosteal woven bone formation to elucidate the adaptive nature against mechanical stress. We hypothesize that cell morphological alteration at the early stage of mechanical loading is essential for bone formation in vivo. PRINCIPAL FINDINGS: We found the significant bone formation on the bone surface subjected to change of the stress toward compression by this method. The histological analysis revealed the proliferation of periosteal cells, and we successively observed the appearance of ALP-positive osteoblasts and increase of mature BMP-2, resulting in woven bone formation in the hypertrophic area. To investigate the mechanism underlying the response to mechanical loading at the molecular level, we established an in-situ immunofluorescence imaging method to visualize molecules in these periosteal cells, and with it examined their cytoskeletal actin and nuclei and the extracellular matrix proteins produced by them. The results demonstrated that the actin cytoskeleton of the periosteal cells was disorganized, and the shapes of their nuclei were drastically changed, under the mechanical loading. Moreover, the disorganized actin cytoskeleton was reorganized after release from the load. Further, inhibition of onset of the actin remodeling blocked the proliferation of the periosteal cells. CONCLUSIONS: These results suggest that the structural change in cell shape via disorganization and remodeling of the actin cytoskeleton played an important role in the mechanical loading-dependent proliferation of cells in the periosteum during bone formation.

Dynamic changes of acoustic load and complex impedance as reporters for the cytotoxicity of small molecule inhibitors.

Cellular motility is the major driving force of numerous biological phenomena including wound healing, immune response, embryogenesis, cancer formation, and metastasis. We studied the response of epithelial FaDu monolayers cultured on gold electrodes of an acoustic resonator (quartz crystal microbalance, QCM) and impedance sensor (electric cell-substrate impedance sensing, ECIS) to externally applied chemical stimuli interfering with cytoskeleton organization. Epithelial cell motility of confluent monolayers is characterized by subtle cell shape changes and variations in the cell-substrate as well as cell-cell distance without net directionality of individual cells. The impact of small molecules such as cytochalasin D, phalloidin, and blebbistatin as well as paclitaxel, nocodazol, and colchicin on actin and microtubules organization was quantified by conventional sensors' readouts and by comparing the noise pattern of the signals which is attributed to cellular dynamics. The responsiveness of noninvasive and label-free techniques relying on cellular dynamics is compared to classical viability assays and changes of the overall impedance of ultrasmall electrodes or acoustic loads of a thickness shear mode resonator. Depending on the agent used, a distinct sensor response was found, which can be used as a fingerprint of the cellular response. Cytoskeletal rearrangements and nuclear integrity were corroborated by fluorescence microscopy and correlated to the readouts of QCM and ECIS.

PI3K-dependent host cell actin rearrangements are required for Cronobacter sakazakii invasion of human brain microvascular endothelial cells.

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that can cause neonatal sepsis and meningitis. The mechanism involved in the pathogenesis of C. sakazakii meningitis remains largely unknown. Previous studies indicated that bacterial invasion of brain microvascular endothelial cells is required for penetration into the central nervous system. In this study, we found that C. sakazakii invasion of human brain microvascular endothelial cells (HBMEC) was significantly inhibited by cytochalasin D, a disrupting agent of actin microfilaments. Disassembly of actin stress fibers and cortical actin fibers was observed in HBMEC infected with C. sakazakii. C. sakazakii infection leads to increased Akt phosphorylation in HBMEC, which was blocked by treatment with PI3K inhibitors. Meanwhile, PI3K and Akt inhibitors significantly inhibited C. sakazakii invasion of HBMEC. Our further results illustrated that the C. sakazakii-induced Akt activation and C. sakazakii invasion were attenuated in HBMEC transfected with dominant-negative PI3K (Δp110). More importantly, the actin filaments rearrangements in HBMEC induced by C. sakazakii were effectively blocked by PI3K inhibitors treatment and transfection with Δp110. Taken together, our findings demonstrated that PI3K-mediated actin rearrangements are required for C. sakazakii invasion of HBMEC.

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata. All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 degrees C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata.

Actin dysfunction activates ERK1/2 and delays entry into mitosis in mammalian cells.

Investigations of actin function during the cell cycle have focused primarily on cytokinesis. Here, we describe the role of actin at the entry into mitosis in primary mammalian cells. Depolymerization of actin with cytochalasin D or inhibition of myosin ATPase with butanedione-2-monoxime (BDM) at G(2) blocked the mitotic spindle formation and central positioning of the nucleus in synchronized MEF and IMR90 cells. Time-lapse microscopy confirmed that these treatments inhibit both spindle formation and separation of duplicated centrosomes to the opposite poles. Concurrent with actin dysfunction, activation of Cdc2 and nuclear localization of cyclin B1 were delayed. Furthermore, cyclin A degradation that is necessary for nuclear envelope breakdown (NEBD) in early mitosis was deferred, supporting the conclusion that mitotic onset was delayed. The activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was sustained in these cells, and the use of a specific ERK inhibitor or a dominant negative form of ERK2 abrogated this delay of entry into mitosis. This delay of mitotic entry and the sustained ERK1/2 activity by actin dysfunction was observed only in primary cells and not in transformed cancer cell lines. These observations demonstrate that an intact actin cytoskeleton is necessary for entry into mitosis and that ERK1/2 is involved in monitoring actin dysfunction to control the onset of mitosis, suggesting the presence of an actin checkpoint at the G(2)/M transition in primary mammalian cells.

A role for cytoskeletal elements in the light-driven translocation of proteins in rod photoreceptors.

PURPOSE: Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. METHODS: Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. RESULTS: Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. CONCLUSIONS: The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin.

[Human cultured lens cells. II. Characterization of established lens cell lines and test of their suitability for cytotoxicity tests]. / Humane Linsenzellen in der Kultur. II. Charakterisierung von etablierten Linsenzelllinien und Testung auf Eignung für Zytotoxizitätsuntersuchungen.

BACKGROUND: Human lens cells in culture may be valuable tools to discover cataractogenic risk factors. Here we report on the characterization of established human lens cells and their use in cytotoxicity tests. MATERIAL AND METHODS: Adhesion dependence was tested by an autoradiographic method. Soft agar test was performed to secure anchorage-independent growth. Laser scan microscopy was used for measuring the nucleus-cytoplasmic relationship. The cytoskeleton was visualized by fluorescence microscopy. Investigations on cytotoxicity were done by neutral red cytotoxicity assay and 3H-thymidine incorporation. The labeling index was determined by the BrdU-method. RESULTS: Two lens cell lines were transformed into fast growing cells. They are characterized by a partial loss of adhesion dependence, cell growth in soft agar, high cloning efficiency and reduced serum requirement. However, the nucleus-cytoplasmic relationship did not change very much in comparison to non-transformed lens cells. The organization of the cytoskeleton was cell shape dependent. The intermediate filaments were from the vimentin type. The established cell lines proved to be highly sensitive to ethanol and mitomycin C. CONCLUSIONS: The established human lens cell lines are well suited to screen for cytotoxic substances in vitro which might be cataractogenic risk factors in vivo.

Role of vascular endothelial growth factor (VEGF) in endothelial cell protection against cytotoxic agents.

Autocrine expression of VEGF has been detected in endothelial cells under hypoxia or oxidative stress. However, the functional significance of this VEGF autocrine expression remains undefined. To analyze the role of autocrine VEGF in the endothelial response against injury, cultured bovine aorta endothelial cells (BAEC) were challenged with potentially cytotoxic substances with different chemical structure and pharmacologic properties, namely cytochalasin D (CyD), hydrogen peroxide (H2O2) and cyclosporine A (CsA). Our results revealed that: i. In particular conditions, exposure to potentially cytotoxic agents as CyD, H2O2 or CsA results in significant BAEC cytoprotection rather than injury. ii. The response to the 3 agents is shifted to a cell damaging pattern in the presence of a specific anti VEGF monoclonal antibody (mAb). iii. CyD and H2O2 markedly stimulate the autocrine expression of VEGF mRNA and VEGF protein. In conclusion, the present study reveals a protective mechanism of endothelial cells against injury involving autocrine VEGF production. Moreover, the occurrence of a significant increase in VEGF expression accompanying this defensive mechanism is further disclosed.

Bending of the neural plate during mouse spinal neurulation is independent of actin microfilaments.

To examine the role of actin microfilaments in mouse spinal neurulation, we stained cryosections of E8.5-10.5 CBA/Ca embryos with FITC-phalloidin. Microfilaments are present in the apical region of all cells throughout the neuroepithelium, irrespective of whether they are involved in bending of the neural plate. Disruption of the microfilaments with cytochalasin D inhibited closure of the cranial neural folds in cultured embryos, even at the lowest concentrations tested, and prevented the initiation of spinal neurulation (Closure 1) at higher concentrations. In contrast, closure of the posterior neuropore was resistant to cytochalasin D at the highest concentrations tested. Phalloidin staining and transmission electron microscopy confirmed that cytochalasin D is effective in disassembling microfilaments in spinal neuroepithelial cells. We conclude that spinal neural tube closure does not require microfilament function, in contrast to cranial neurulation which is strongly microfilament-dependent. Histological examination of cytochalasin D-treated embryos revealed that bending at hinge points, both in the midline (MHP) and dorsolaterally (DLHPs), continues in the absence of microfilaments, whereas the rigidity of non-bending regions of the neural plate is lost. This suggests that spinal neurulation can continue in the presence of cytochalasin D largely as a result of intrinsic bending of the neural plate at hinge points. Cytochalasin D treatment is a useful tool for revealing the localisation of hinge points in the neural plate. Analysis of treated embryos demonstrates a transition, along the spinal axis, from closure solely involving midline bending, at high levels of the spinal axis, to closure solely involving dorsolateral bending, low in the spinal region. These findings support the idea of mechanistic heterogeneity in mouse neurulation, along the body axis, and demonstrate that contraction of actin microfilaments is not obligatory for epithelial bending during embryonic morphogenesis. Dev Dyn 1999;215:273-283.

A novel bioassay for P-glycoprotein functionality using cytochalasin D.

The functional contribution of both P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) to multidrug resistance (MDR) in tumor cells is commonly determined by drug cytotoxicity and/or accumulation/efflux tests. We report on a bioassay developed for the specific detection of functional P-gp levels and the efficacy of related chemosensitizers (CD-P-gp-assay). The assay is based on the flow cytometric measurement of changes in the > or = G2M cell cycle compartment which are due to the induction of polykaryons after exposure of proliferating cells to three defined cytochalasin D (CD) concentrations with and without verapamil. As demonstrated in 13 well-characterized MDR cell models (20 resistant sublines), there is a significant correlation between cytokinesis-blocking CD doses, as well as responsiveness to chemosensitizers and MDR1 gene expression (mRNA and P-gp) allowing discrimination between different levels of P-gp-MDR. CD-P-gp-assay specificity was assessed by testing 23 compounds: 19 known as potent inhibitors of P-gp-MDR, some of them, though to a lesser extent, also of MRP-MDR; 1 inhibiting MRP-but not P-gp-MDR; 3 inactive in both types of MDR. A modulation of CD activity was confined exclusively to both P-gp-expressing cell lines and P-gp chemosensitizers. CD cytoskeletal activity measured by FACS is a specific and sensitive tool with which to detect functional P-gp and related chemosensitizers.

Vitamin C reduces cytochalasin D cataractogenesis.

The effect of cytochalasin D (CD), an actin monomer-stabilizer, has been studied on cataract development in rat lenses. Cataractogenesis was induced by incubating the rat lenses in medium 199 (M199) containing 10(-5) M CD; by the end of 24 h, lenses first developed a visible opacity. The increased lactate dehydrogenase (LDH) activity in the culture medium, leakage of lens cytosolic proteins into the culture medium and observable development of opacity through a dissection microscope were correlated with cell damage associated with cataract formation. Non-denaturing polyacrylamide gel electrophoresis was used to separate three lens LDH isoenzymes. The effect of 1 mM vitamin C (VC) in reducing LDH leakage was also examined. The protective effect of VC on CD-initiated cataractous lenses is significant. This suggest that a portion of the opacity and lens damage may involve oxidative damage to the membrane-cytoskeleton complex which is started by CD, but partially prevented by VC

Analysis of the tissue movements of embryonic wound healing--DiI studies in the limb bud stage mouse embryo.

The tissue movements of epithelial spreading and mesenchymal contraction play key roles in many aspects of embryonic morphogenesis. One way of studying these movements in a controlled manner is to make an excisional skin wound to an embryo and watch the wound heal. In this paper we report our studies of healing of a simple excisional lesion made to the limb bud stage mouse embryo. The wounded, living embryo is cultured in a roller bottle; under such conditions the wound heals with a highly reproducible time course and is completely closed by 24 hr. During the healing period the environment bathing the wound can be simply manipulated by adding drugs or factors to the culture medium. We have used DiI to label mesenchymal cells exposed at the margin of the initial wound and, by following their fate and measuring the area of mesenchyme remaining exposed at various time points during the healing process, we have quantified both the extent of mesenchymal contraction and the extent of reepithelialisation by movement of epidermis over mesenchyme. We show that the two types of tissue movement contribute almost equally (50:50) to the total wound closure rate. We have gone on to investigate the cell machinery underlying these processes. In adult wounds the epidermis migrates by means of lamellipodial crawling, but we show that reepithelialisation in the embryo is achieved instead by purse-string contraction of a cable of filamentous actin which assembles in the basal layer of cells at the free edge of the epidermis. Addition of cytochalasin D to the culture medium blocks formation of this actin cable and leads to failure of reepithelialisation. Contraction of adult wound connective tissue appears to be driven by conversion of dermal fibroblasts into a specialist smooth muscle-like fibroblast, the myofibroblast. However, using an antibody recognising the alpha-isoform of smooth muscle actin and specific for smooth muscle cells and myofibroblasts, we show that a similar conversion into myofibroblasts does not occur at any stage during the embryonic wound healing process. These observations indicate that both of the tissue movements of embryonic wound healing utilise cell machinery fundamentally different from that driving the analogous tissue movements of adult healing.

Inhibition of the spore polar filament extrusion of the microsporidium, Encephalitozoon hellem, isolated from an AIDS patient.

Spores of the microsporidian parasitic protozoan Encephalitozoon hellem were purified and incubated at 37 degrees C in a solution with an electrolyte composition similar to that of mammalian extracellular fluid, and in solution in which the calcium had been replaced with 0.2 mM EGTA. Polar filament extrusion (germination) was monitored by both scanning electron microscopy and light microscopy. Germination was pH-dependent, with optima at pH 7.4 and 9.5, and was significantly greater in the presence of medium calcium. Hydrogen peroxide caused a concentration-dependent increase in germination that was also reduced in a calcium-free medium. Four agents were found to inhibit spontaneous and H2O2-stimulated polar filament extrusion: the microfilament disrupter, cytochalasin D; the microtubule disrupter, demecolcine; the calcium channel blocker, nifedipine; and the antifungal agent, itraconazole. These results are consistent with the existence of a calcium-channel-mediated step, and requirements for an F-actin- and for a tubulin-containing element in the germination process of the spore of this parasite. Nifedipine, cytochalasin D and itraconazole all have different sites of action and were therefore able to potentiate one another when used in paired combination to inhibit germination.

Effects of ORG 2766, a neurotrophic ACTH4-9 analogue, in neuroblastoma cells.

Treatment of Neuro2a cells with drugs known to affect the integrity of microfilaments and microtubules, as well as with a calcium ionophore produced damage to the cellular membrane that was quantifiable by measuring the release of LDH into the culture medium. Concurrent exposure of the cells to ORG 2766 was found to modulate the release of LDH in a dose- and time-dependent fashion. ORG 2766 treatment was also able to reduce the basal release of LDH into the culture medium. [table: see text] The ORG 2766-induced reduction in LDH release was not due to down-regulation of protein synthesis. The peptide produced significant increases in protein synthesis relative to control conditions at concentrations of 10(-11) to 10(-6) M with 10(-8) M being an optimal dose. SDS-PAGE and 2-D PAGE analysis showed that de novo synthesis of most polypeptides was increased by about 40%. Additionally, a family of polypeptides tentatively identified as actins appear to undergo ORG 2766-dependent post translational charge modifications. These data are consistent with the hypothesis that regulation of transcription and/or translation are mechanisms important to the neurotrophic actions of ORG 2766.