Microfilament Inhibitor Cytochalasin Research Articles

Role of cytoskeleton in regulating fusion of nucleoli: a study using the activated mouse oocyte model.

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF.

Effects of aspect ratios of stem cells on lineage commitments with and without induction media

The present study is aimed to examine the shape effect on lineage commitment of stem cells in growth medium free of external chemical induction factors. Aspect ratios (ARs) of cells were controlled by micropatterns with cell-adhesive microislands of AR 1, 2 and 8 on the potent nonfouling background of poly(ethylene glycol) hydrogels, and the single stem cells were well shaped for 19 days. Mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured in osteogenic medium, adipogenic medium, mixed coinduction medium, and also growth medium; alkaline phosphatase (ALP) and oil droplets were employed as indicators of osteoblasts and adipocytes, respectively. Those indicators were well observed in all of three induction media as early as day 7, and also in growth medium at a longer culture time till day 13. While a significant monotonic decrease of adipogenesis was observed with the increase of AR, a non-monotonic change of osteogenesis was found with optimal AR about 2. The relative gene expressions further verified the above findings. As a result, cell shape itself is an inherent cue to regulate stem cell differentiation, let alone with or without external chemical induction factors. Such a shape effect disappeared upon addition of a microfilament inhibitor cytochalasin D or a Rho-associated protein kinase (ROCK) inhibitor Y-27632. So, formation of cytoskeleton is necessary for the shape effect, and the ROCK-pathway-related cell tension is responsible for the shape effect on the lineage commitment of stem cells even in growth medium.

Neuron Division or Enucleation

The classical Bielschowsky-Gross neurohistological method was used to reproduce all the morphological phenomena interpreted by many authors as signs of neuron division, budding, and fission. It is suggested that these signs are associated with the effects of enucleation, which occurs in many cells of other tissue types in response to a variety of chemical and physical treatments. Studies were performed using neurons isolated from the mollusk Lymnaea stagnalis and exposed in tissue culture to the actin microfilament inhibitor cytochalasin B. Phase contrast time-lapse video recording over periods of 4-8 h demonstrated nuclear displacement, ectopization, and budding, to the level of almost complete fission of the neuron body. This repeats the pattern seen in static fixed preparations in "normal" conditions and after different experimental treatments. Budding of the cytoplasm was also sometimes seen at the early stages of the experiments. Control experiments in which cultured neurons were exposed to the solvent for cytochalasin B, i.e., dimethylsulfoxide (DMSO), did not reveal any changes in neurons over a period of 8 h. We take the view that the picture previously interpreted as neuron division and fission can be explained in terms of the inhibition of actin microfilaments, sometimes developing spontaneously in cells undergoing individual metabolic changes preventing the maintenance of cytoskeleton stability.

Human Platelets AttenuateAspergillusSpecies via Granule‐Dependent Mechanisms

Using laser scanning microscopy, we investigated whether platelets are capable of internalizing Aspergillus conidia and examined Aspergillus-platelet adherence. The influence of platelets on fungal growth was evaluated by assessing galactomannan (GM) release, hyphal elongation, and colony size. A secretion assay with [(3)H]-serotonin (5-hydroxytryptamine [5-HT]) was performed. Exposure to platelets resulted in significantly decreased GM release (p<.05), hyphal elongation (p<.001), colony size, pigmentation, and 5-HT release ( p<.05). A lack of antifungal effects was observed with the microfilament inhibitor cytochalasin D. Platelets attenuate the virulence of Aspergillus species in vitro on the basis of granule-dependent effects.

Mitochondrial behavior during oogenesis in zebrafish: A confocal microscopy analysis

The behavior of mitochondria during early oogenesis remains largely unknown in zebrafish. We used three mitochondrial probes (Mito Tracker Red CMXRos, Mito Tracker Green FM, and JC-1) to stain early zebrafish oocyte mitochondria, and confocal microscopy to analyze mitochondrial aggregation and distribution. By using fluorescence recovery after photobleaching (FRAP), we traced mitochondrial movement. The microtubule assembly inhibitor nocodazole and microfilament inhibitor cytochalasin B (CB) were used to analyze the role of microtubules and microfilaments on mitochondrial movement. By using the dual emission probe, JC-1, and oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), we determined the distribution of active and inactive (low-active) mitochondria. Green/red fluorescence ratios of different sublocations in different oocyte groups stained by JC-1 were detected in merged (green and red) images. Our results showed that mitochondria exhibited a unique distribution pattern in early zebrafish oocytes. They tended to aggregate into large clusters in early stage I oocytes, but in a threadlike state in latter stage I oocytes. We detected a lower density mitochondrial area and a higher density mitochondrial area on opposite sides of the germinal vesicle. The green/red fluorescence ratios in different sublocations in normal oocytes were about 1:1. This implies that active mitochondria were distributed in all sublocations. FCCP treatment caused significant increases in the ratios. CB and nocodazole treatment caused an increase of the ratios in clusters and mitochondrial cloud, but not in dispersed areas. Mitochondria in different sublocations underwent fast dynamic movement. Inhibition or disruption of microtubules or microfilaments resulted in even faster mitochondrial free movement.

Ooplasm segregation in the squid embryo, Loligo pealeii.

Ooplasm segregation in the squid embryo, Loligo pealeii.

Bile acids induce eosinophil degranulation by two different mechanisms.

Eosinophil infiltration and activation occur in the vicinity of the bile ducts in immune cholangiopathies. When cholangiocytes are injured, bile acids may cross the damaged biliary epithelia and affect periductal immune cells. Although immunomodulatory actions of bile acids have been well explored, their effects on eosinophils have never been examined. In this study, we tested the hypothesis that bile acids directly activate eosinophils and induce their effector functions. We found that a hydrophobic bile acid, taurine-con- jugated chenodeoxycholic acid (TCDCA), induces eosinophil degranulation in vitro via 2 different mechanisms depending on its concentration. Degranulation induced by 100 to 500 micromol/L TCDCA was an active and regulated release because it was completely abolished by a tyrosine kinase inhibitor (genistein), by a microfilament inhibitor (cytochalasin B), and by incubation at 4 degrees C. Furthermore, eosinophils stimulated with 10 to 250 micromol/L TCDCA vigorously produced superoxide and interleukin-8 (IL-8). In contrast, at higher concentrations (e.g., > or = 1,000 micromol/L), TCDCA induced granule protein release without concomitant superoxide production and IL-8 production. Further genistein and cytochalasin B failed to inhibit eosinophil degranulation induced by 2,500 micromol/L TCDCA, suggesting that TCDCA at this concentration induced passive degranulation via cytolysis. The analyses of cell morphology and functional viability also supported the presence of 2 mechanisms for TCDCA-induced eosinophil degranulation. Taurine-conjugated ursodeoxycholic acid, a hydrophilic bile acid, similarly activated human eosinophils at relatively low concentrations, although the potency was always lower compared with that of TCDCA. In conclusion, we have shown that bile acids are capable of directly activating eosinophils.

Necrotizing bacteria and protein targeting

A type III-like secretion mechanism for injection of effector molecules into the cytoplasm of host cells has not yet been described for Gram-positive bacteria. These bacteria are known to secrete multiple proteins into their environments, such as those secreted by Streptococcus pyogenes, the agent responsible for strep throat and necrotizing fasciitis of soft tissues. Previous work using keratinocytes demonstrated that mutants defective in production of the hemolysin streptolysin O (SLO) lost the ability to manipulate host signaling pathways. SLO also has characteristics that would suggest it has a role in translocation. SLO is a member of a highly conserved family of pore-forming cytolysins known as cholesterol-dependent cytolysins (CDCs). Analysis of their pore size suggests that CDCs could translocate effector molecules from Gram-positive bacteria. Madden and colleagues 1xCytolysin-mediated translocation (CMT): a functional equivalent of type III secretion in Gram-positive bacteria. Madden, J.C et al. Cell. 2001; 104: 143–152Abstract | Full Text | Full Text PDF | PubMed | Scopus (180)See all References1 recently presented evidence that a molecule of streptococcal origin with eukaryotic signal transduction properties is translocated into the keratinocyte cytosol in an SLO-dependent manner.Crossreactivity with a commercially available anti-streptolysin O antiserum revealed the presence of a 52-kDa streptococcal protein in infected keratinocytes, which was not present in SLO-deficient mutants or uninfected host cells. Using cytoplasmic fractions prepared from host cells under conditions that lyse keratinocytes but not bacteria, the 52-kDa protein was found to be translocated in an SLO-dependent manner. Other host cell types were analyzed and similar results were obtained. A streptococcal NAD-glycohydrolase (SPN)-deficient mutant did not translocate the 52-kDa protein into host cells however, indicating that this particular protein was derived from the bacteria and was not of host origin. Co-infection with bacteria that were deficient in either SLO or SPN did not rescue protein transport.These observations suggest that SPN enters the cytosol through the SLO pore. The SPN protein is biochemically active in the host cell. The presence of the microfilament inhibitor cytochalasin D did not alter enzymatic activity in wild-type cells, indicating that the presence of SPN activity was not the result of endocytosis or bacterial invasion. Furthermore, SPN activity was dependent on SLO and was not attributed to an endogenous NAD-glycohydrolase. To demonstrate that SPN and SLO work in a synergistic manner, a cytotoxic response assay was used where keratinocytes were infected with either the wild-type strain, the SLO-mutant or the SPN-mutant. At various times, samples were removed and stained with the fluorescent probes calcein AM (activated by cellular esterases) and ethidium homodimer-1 (EtH-1) (which stains the nucleus). Loss of staining with calcein AM and positive staining with EtH-1 would be indicative of membrane damage. By 3h, ∼35% of the wild-type cells have lost their ability to exclude EtH-1. At all time points, infection with SLO-deficient mutants resembled SPN-deficient mutants in terms of the number of cells damaged and number of cells that remained adherent. Both mutants had reduced levels of permeability compared with the wild-type cell. These data are consistent with a model where SLO is required for the translocation of SPN into the cytosol of the host cell.With these data and previous studies of protein targeting in Gram-positive bacteria, a working model of protein translocation suggests that effector molecules are exported across the bacterial cellular membrane and cell wall via the generalized secretion pathway and translocated into the target cell using a CDC. Other than in Listeria, little is known about the function of CDCs in pathogenesis. These interesting and novel findings set the stage for analysis of function of virulence factors secreted from Gram-positive pathogens.

Osmotically induced cytosolic free Ca 2+ changes in human neutrophils

Cytosolic free Ca 2+ concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300–600 mM) or sucrose (600–1200 mM), caused a rise in cytosolic free Ca 2+ (Δ max≅600 nM). This was not due to cell lysis as the cytosolic free Ca 2+ concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca 2+ could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca 2+ concentration occurred in the absence of extracellular Ca 2+, demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca 2+ was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca 2+ may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca 2+ (Δ max≅600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca 2+ to physiologically important levels.

Involvement of the Cytoskeleton in Localization ofParacentrotus lividusMaternal BEP mRNAs and Proteins

The maternal bep1 and bep4 mRNAs and their protein products are localized at the animal pole ofParacentrotus lividuseggs. We have examined the role of the cytoskeleton in localization both of bep RNAs and BEP proteins in unfertilized and fertilized eggs and in determining the polarity ofP. lividuseggs. The use of drugs such as colchicine and cytochalasin B, which depolymerize microtubules and microfilaments respectively, revealed a perturbation of localization of bep1 and bep4 mRNAs. In contrast, the microfilament inhibitor cytochalasin B had no effect on localization of BEP1 and BEP4 antigens localization, which appears to be due only to microtubules. Moreover, the presence of bep mRNAs and BEP proteins in a microtubule preparation has been demonstrated. Maintenance of the asymmetric distribution of BEP proteins during cellular division of eggs and embryos, by association with mitotic spindle, is also shown.

Polarity and endoplasmic microtubules in food‐conducting cells of mosses: an experimental study

SUMMARYThe effects of decapitation and of drugs affecting microtubules, microfilaments and the endomembrane system, on the ultrastructure of food‐conducting cells have been studied in the sporophyte of the mosses Polytrichum juniperinum Hedw. and Mnium hornum Hedw. In both species decapitation caused a marked reduction of cellular polarity. Moreover in Mnium but not in Polytrichum decapitation induced the disappearance of endoplasmic microtubules (MTs), the loss of longitudinal alignment of organdies and the accumulation of abundant starch. Starch accumulated in the cortical parenchyma cells in Polytrichum. In both species extensive MT depolymerization was induced by 100 μM oryzalin, whereas 10 μM oryzalin was less effective and 2·5 mM colchicine had no apparent effect. Treatment with 100 μM oryzalin also caused disruption of organdie alignment, alterations in the shape and position of the nucleus and a marked reduction in, or disappearance of, endoplasmic reticulum, trans Golgi network and membrane‐bounded tubules, that were abundant in the controls. Treatment with 10 μM monensin partially mimicked the effects of oryzalin, causing a reduction of the endomembrane system and disappearance of most endoplasmic MTs. Neither oryzalin nor monensin suppressed cellular polarity. Cells treated with D2O or the actin microfilament inhibitor cytochalasin D showed no structural alterations. The data indicate that: (a) endoplasmic MTs have a major role in organelle alignment and shaping in food‐conducting cells of mosses; (b) cellular polarity depends on the presence of a source/sink gradient and is not directly associated with MTs; (c) interactions between MTS and organelles, notably the ER and other components of the endomembrane system, have mutually stabilizing effects.

Cytoskeletal inhibitors impair Ca 2+ elevations via neuropeptide Y and other G i-coupled receptors

Neuropeptide Y, α 2-adrenoceptors, thrombin and certain muscarinic acetylcholine receptors can couple to elevations of intracellular free Ca 2+ concentrations via G i-proteins. We have studied the effects of inhibitors of microtubules (colchicine, nocodazole, vinblastine) and microfilaments (cytochalasin B, cytochalasin D) on these effects in human erythroleukemia (HEL) cells. Both types of inhibitors reduced neuropeptide Y-, adrenaline- (via α 2A-adrenoceptors) and thrombin-stimulated Ca 2+ elevations while the inactive analog β-lumicolchicine was without inhibitory effects. Similarly, in SK-N-MC cells vinblastine inhibited neuropeptide Y and carbachol (via muscarinic receptors) stimulated Ca 2+ elevations. In HEL cells the inhibitory effects of the microfilament inhibitor cytochalasin D and the microtubule inhibitor colchicine were not additive. Colchicine, nocodazole or cytochalasin D did not affect the binding of the agonist neuropeptide Y. On the other hand, neuropeptide Y and thrombin significantly stimulated GTP γS binding in the absence but not in the presence of colchicine, vinblastine or cytochalasin B. This was not due to sequestration of G-protein α-subunits, since nocodazole did not affect the distribution of immunodetectable G i α1/2 or G iα3 between membrane and cytosolic fractions. We conclude that disruption of microfilaments or microtubules impairs Ca 2+ elevations by neuropeptide Y and other G i-coupled receptors by inhibiting receptor/G i-protein interaction; this does not involve impairment of agonist binding to the receptor or redistribution of G i-protein α-subunits.

Stability of nuclear segments in human neutrophils and evidence against a role for microfilaments or microtubules in their genesis during differentiation of HL60 myelocytes.

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949-954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.

Differences in the interactions of Nocardia asteroides with macrophage, endothelial, and astrocytoma cell lines

An in vitro model for studying host cell interactions with Nocardia asteroides was developed. Thus, macrophage cell lines J774A.1 and P388D1, pulmonary artery endothelium cell line CPAE, rat glial tumor cell line C6, and human astrocytoma cell lines CCF-STTG1 and U-373 MG were infected with either log- or stationary-phase cells of N. asteroides GUH-2, and the host cell-nocardia interactions were determined by light microscopy and electron microscopy. Polyclonal antinocardial antibody did not enhance uptake of nocardiae by any of these cell lines; however, log-phase cells of GUH-2 infected a higher percentage of J774A.1 and P388D1 than did stationary-phase organisms. When cells infected with stationary-phase GUH-2 were incubated for 6 h, filaments developed, which indicated that nocardial growth had occurred. In J774A.1 and P388D1, only 31 to 57% of the total stationary-phase coccobacillary cells that were phagocytized formed filaments within 6 h. This indicated that there was some inhibition of growth of the phagocytized nocardiae within these macrophage cell lines; however, the nocardiae grew within the endothelial (> 87% filaments) and astrocytoma (100% filaments) cell lines. Microfilament inhibitor cytochalasin B inhibited uptake of GUH-2 by macrophages and other cell lines, except that there was no effect on uptake of nocardial cells by astrocytoma cell line U-373 MG. Scanning and transmission electron microscopy showed phagocytosis of GUH-2 by the different cell lines. In cytochalasin B-treated cells, nocardiae were shown to penetrate through the cell surface and become internalized in a manner distinct from typical phagocytosis, suggesting that filamentous forms of this organism have a phagocytosis-independent invasion factor. The extent of this cytochalasin-resistant cellular penetration by the nocardiae differed in the different cell lines.

Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus

The directional movement of many cellular organelles in neurons is dependent on polarized microtubules and direction-specific motor molecules. Microtubules are also thought to mediate the retrograde transport of herpes simplex virus (HSV) in sensory neurons. To define the cellular machinery responsible for retrograde axonal transport of HSV, we have investigated the polarity of microtubules in the peripheral axons of trigeminal ganglion neurons. The long ciliary nerves of rabbits were prepared for a standard "hook assay" of microtubule polarity. Axons in cross-sectioned nerves contained microtubules with almost uniform orientation. The fast-growing, plus ends of these axonal microtubules are located distal to the cell body and the slow-growing, minus ends are directed centrally. To determine the role played by microtubules in the retrograde transport of HSV in these axons, we injected the retrobulbar space of mice with the microtubule-inhibiting drugs colchicine, vinblastine, or nocodazole or with the microfilament inhibitor cytochalasin D and 1 d later inoculated the cornea with HSV. We found that colchicine, vinblastine, or nocodazole reduced by 52-87% the amount of virus recovered from the ganglion 3 d postinoculation, compared to vehicle-treated animals. In contrast, cytochalasin D or beta-lumicolchicine did not significantly reduce the amount of HSV recovered from the ganglion. We conclude that the retrograde axonal transport of HSV from axon endings in the cornea to the trigeminal ganglion cell bodies requires intact microtubules and occurs in a plus-to-minus direction on the microtubules. Our data are consistent with the hypothesis that the retrograde axonal transport of HSV is mediated by a minus end-directed motor molecule, for example, cytoplasmic dynein.

Role of cytoskeleton in volume regulation of rabbit proximal tubule in dilute medium

When proximal tubules are immersed in hypotonic medium, they quickly swell to a peak volume. In a second, slower phase, termed volume regulatory decrease (VRD), they shrink as K, anion, and water leave the cells. We investigated the role of the cytoskeleton during this biphasic hypotonic volume regulatory response. Isolated, collapsed rabbit proximal convoluted tubules (PCT) were crimped tightly between two pipettes, and their volume was assessed optically. PCT volume increased to a peak 70-80% above baseline on sudden immersion in dilute medium (150 mosmol/kgH2O). After completing VRD, control tubules had regulated their volume 73 +/- 2% back toward baseline. Tubules exposed to the microtubule inhibitor vincristine (5 microM) regulated 75 +/- 2%. Tubules exposed to the microfilament inhibitor cytochalasin B (50 microM) regulated less (57 +/- 5%), and tubules exposed to both inhibitors regulated only 39 +/- 3% (P less than 0.01 vs. control). Hypotonic VRD was unimpaired in PCT loaded with NaCl by prior exposure to ouabain but was significantly reduced by cytochalasin B. We conclude that VRD is not cation specific and that intact microtubules and microfilaments play a synergistic role in the VRD of rabbit PCT in hyposmotic medium.

Cytoskeletal rearrangements accompanying Salmonella entry into epithelial cells

Salmonella bacteria can enter (invade) eukaryotic cells, and exist as intracellular parasites. Confocal, light immunofluorescence and electron microscopy were used to examine various cytoskeletal components of cultured Madin Darby canine kidney (MDCK) and HeLa epithelial cells after infection with Salmonella typhimurium. These bacteria entered and remained within membrane-bound vacuoles and were surrounded by large (5-10 microns) dense structures composed of various cytoskeletal components. These structures consisted of extensive aggregations of polymerized actin, alpha-actinin and tropomyosin above and beside the invading bacterium in both epithelial cell lines. These structures were evident soon after bacterial addition (maximal at 20 min for HeLa cells, 60 min for MDCK cells), and disappeared later in the infection as the cytoskeletal components returned to a more normal distribution after bacterial internalization. Surprisingly, tubulin also aggregated above internalized Salmonella although bacterial entry or penetration through polarized monolayers was not disrupted by the microtubule-inhibiting agent nocadazole (this treatment actually enhanced tubulin accumulation around these organisms). There were little if any rearrangements in intermediate filaments composed of keratin or vimentin. Large amounts of talin also accumulated above and around invading Salmonella, but there was only a minor accumulation of vinculin around a few organisms. Pretreatment of epithelial cells with the microfilament inhibitor cytochalasin D blocked bacterial internalization but did not prevent accumulation of polymerized actin and alpha-actinin directly beneath uninternalized bacteria, yet prevented accumulation of the other cytoskeletal components. These results suggest that Salmonella bind to the surface and trigger a signal in epithelial cells that causes marked rearrangements in various cytoskeletal components, including recruitment of actin filaments and alpha-actinin, which then generates the force necessary for bacterial uptake.

Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalasin D

The microfilament inhibitor cytochalasin D inhibits extrusion of the first polar body when present during the first meiotic division of mouse oocytes; however, it does not interfere with anaphase movement of chromosomes, and thus induces the formation of tetraploid oocytes. After the separation of chromosomes in anaphase, two spindles start to assemble. However, they merge rapidly and a single meiotic spindle forms. During the transition between metaphase I and metaphase II, in the presence of cytochalasin D, a drop in histone kinase activity takes place demonstrating a transitional decrease in the activity of the maturation promoting factor. These oocytes can be activated parthenogenetically a few hours after washing out the inhibitor. After completion of the second meiotic division and extrusion of a polar body, they contain a diploid number of chromosomes. They are genetically identical to each other and to their mother. Such eggs develop to the blastocyst stage and can implant in the uteri of foster mothers. Most of these fetuses die before the 9th day of gestation, as do diploid control fetuses treated with cytochalasin D during the second meiotic division. The heterozygous state of the experimental embryos obtained after activation of eggs recovered from heterozygous females and treated with cytochalasin D during the first meiotic division was confirmed using a glucose-phosphate isomerase assay. This technique allows the production of genetic clones of parthenogenetic embryos by simple means.

Fasciola hepatica: Disruption of spermatogenesis by the microfilament inhibitor cytochalasin B

The distribution of actin filaments in the spermatogenic cells of Fasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100 micrograms/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.

Fasciola hepatica: the effect of the microfilament inhibitor cytochalasin B on the ultrastructure of the adult fluke

The effect of the microfilament inhibitor cytochalasin B (10 and 100 micrograms/ml) on the ultrastructure of adult Fasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100 micrograms/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100 micrograms/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100 micrograms/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down "yolk" globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).