A diet rich in n-3 polyunsaturated fatty acids reduced prostaglandin biosynthesis, ovulation rate, and litter size in mice

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to > 2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.     

Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice

Jasplakinolide (JAS), which induces microfilament polymerization and stabilization, inhibits microfilament-mediated events in murine oocyte maturation and fertilization in a fashion unlike the effects of cytochalasin B (CCB) and latranculin A (LAT A). JAS prevents egg polar body emission at a much lower concentration than either CCB or LAT A. Microfilament bundles were detected on the entire egg cortex after JAS exposure. Conversely, microfilament patterns did not change after exposure to CCB, and few microfilaments were observed after exposure to LAT A. Eggs that were allowed to recover from JAS were unable to recover normal microfilament organization. During oocyte maturation, JAS prevented both spindle migration to the oocyte cortex and first polar body emission. During in vitro fertilization, sperm head entered the eggs and formed pronuclei, but sperm tail entry, pronuclear centration, and second polar body emission were not detected. DNA synthesis occurs in these JAS-treated zygotes. JAS inhibited not only the formation, but also the disassembly, of incorporation cones. JAS was also found to prevent cortical granule exocytosis following artificial activation, and cortical granules were still beneath the plasma membrane even after activation. Finally, incorporation of microinjected nonmuscle actin into the microfilament network of mice eggs was delayed by JAS. We conclude that JAS acts as a microfilament inhibitor during maturation and fertilization and is more powerful than other inhibitors. Its mechanism differs in that it promotes assembly and stabilization of microfilaments. JAS is a novel cell permeable tool for the investigation of microfilament-dependent events in early mammalian development.     

F-actin rings are associated with the ring canals of the Drosophila egg chamber

Staining of Drosophila egg chambers with rhodaminyl-lysine-phallotoxin (RLP), a specific stain for F-actin, has demonstrated the presence of dense F-actin rings associated with the inner surfaces of the ring canals. They were first observed in the distal part of the germarium where rings of four different size classes were found, differing in diameter by up to twofold. The ring sizes are considered to correspond to the ring canals formed at each of four successive incomplete cleavages. During the growth of the egg chamber the actin rings were found to increase in diameter from less than 1 micron to approx. 10 micron. Concomitantly a secondary outer ring of more diffuse material is built up in association with the cell membranes. A well developed array of microfilament bundles was also associated with the nurse cell plasmalemma. In stages where the transfer of the bulk of the nurse cell cytoplasm into the oocyte was occurring the rings came closer together in a central area. In late stage chambers the F-actin rings and the microfilament bundles appeared to be incorporated into large irregular masses of actin, which subsequently disappeared as the mature oocyte formed. The F-actin rings are suggested to act as mechanical strengthening elements for the canal plasmalemma, whilst cytoplasmic transport occurs through the ring canals.     

Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway

The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate filament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cytoskeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKC-deficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies involving intact cells.     

Disorders of the actin cytoskeleton in transformed epithelial cells

The actin cytoskeleton of 8 transformed epithelial cell lines was studied using electron microscopy of platinum replicas. Seven of these lines belonged to the IAR series of rat liver epithelial cells, being at different stages of neoplastic progression. One cell line (FBT) was derived from the epithelium of bovine fetal trachea. The extent of actin cytoskeleton alteration in cell lines studied has been shown to correlate with other signs of neoplastic transformation. Among various actin-containing cell structures (microfilament bundles, actin meshwork at active edges, cell-cell adherence junctions, and endoplasmic microfilament sheath) the latter was the most sensitive to transformation. The loosening of the sheath and the alteration of its fine structure were observed in all the cell lines. The degree of these changes increased in the following order: FBT; non-tumorigenic IAR lines; IAR lines transformed in vitro; IAR lines obtained from the latter by single or double selection in vivo. The alteration of sheath was the only disturbance of actin cytoskeleton in FBT cells, whereas in other groups of epithelial cell lines some other changes occurred. These involved disruption of actin-containing intercellular junctions, the cell polarization accompanied by progressive shortening of length of the cell active edge containing actin meshwork, and disappearance or reorganization of microfilament bundles.     

In vivo identification of Tetrahymena actin probed by DMSO induction of nuclear bundles

We report the first successful identification of actin, an ubiquitous contractile protein, in Tetrahymena pyriformis (strain W). We employed dimethyl sulfoxide (DMSO) as a probe to induce the formation of actin bundles in the cell nucleus [1, 2] through disruption of cytoplasmic microfilament organization [3, 4]. The cells were incubated for 30 min at 22 °C in the inorganic medium of Prescott & James [5] containing 10% DMSO, and observed under a transmission electron microscope (TEM). Microfilarment bundles were formed in interphase macronuclei, and these microfilaments, approx. 6 nm in diameter, could be decorated by rabbit skeletal muscle heavy meromyosin (HMM) in the glycerinated model. In many cases, the bundles formed closely parallel to natively existing bundles of microtubules. Interestingly, these microtubules had prominent striation with 15–16 nm periodicity. SDS-polyacrylamide gel electrophoresis was designed to show the low actin content of Tetrahymena cells in comparison with that of Dictyostelium. Actin was suggested to comprise less than 1.7% of the total protein in Tetrahymena, whereas as much as 6% was actin in Dictyostelium cells. In assessing the physiological significance of the bundle formation, we further performed HMM and myosin subfragment-1 (S1)-binding studies to clarify the organization process and the polarity of the DMSO-induced nuclear actin filaments by using the tannic acid staining technique [6]. Randomly oriented short filaments appeared in the nucleus treated with 10% DMSO for 10 min. These filaments became elongated and associated with each other to form loose bundles in the following 10 min. With 30-min treatment, the filaments were organized and large bundles with single axes developed. With these well-developed bundles, the Student's t-test was performed on 172 pairs of neighboring filaments and the probability (p) of the deviation from random polarity was 0.08, suggesting that the filaments were organized in an anti-parallel manner. The results show that the DMSO induction of nuclear actin is a powerful tool to demonstrate the existence of cellular actin in vivo and to study the mechanism of microfilament organization in relation to cell physiological activities.     

pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status

The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.     

Cytoskeleton structure and adhesion properties of human stromal precursors under conditions of simulated microgravity

During spaceflight and in simulated microgravity (SMG), cytoskeleton rearrangements were observed in lymphocytes, glial cells and osteoblasts. One potential mechanism for the cytoskeletal gravisensitivity of cells is the disruption of the extracellular matrix and integrin interactions. We investigated the effect of SMG on the structure of the actin cytoskeleton, distribution of cellular vinculin, the expression of some integrin subtypes and cellular adhesion molecules in cultured mesenchymal stem cells (hMSCs) derived from human bone marrow in vitro. Simulated microgravity was produced by desktop RPM equipment (Dutch Space, Netherlands). Cells were exposed to simulated microgravity for 30 min to 120 h. The results showed that the actin cytoskeleton was reorganized very quickly (30 min). Later (6, 24, and 48 h), the number of cells with disrupted actin cytoskeletons was increased; however, after 120 h of exposure, cells partly regained their F-actin structures. RPM exposure augmented the number of cells that express integrin-α2. We also observed a decrease in the number of VCAM-1-positive cells and changes in the expression of ICAM-1. Our findings indicate that SMG induces reversible microfilament reorganization in hMSCs and alters their adhesion properties.     

Formation of microfilament bundles in the tumor cell interphase nuclei of a rat neurinoma exposed to dimethyl sulfoxide

The action of 15% dimethyl sulfoxide (DMSO) on the ultrastructure of the rat neurinoma cells (line NGUK-I) has been studied. The agent induced the formation of microfilament bundles in interphase nuclei after 30-60 min of treatment. The microfilament bundles revealed are suggested to be actin.     

Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.     

Insulin-induced endothelial cell cortical actin filament remodeling: a requirement for trans-endothelial insulin transport

Insulin's trans-endothelial transport (TET) is critical for its metabolic action on muscle and involves trafficking of insulin bound to its receptor (or at high insulin concentrations, the IGF-I receptor) via caveolae. However, whether caveolae-mediated insulin TET involves actin cytoskeleton organization is unknown. Here we address whether insulin regulates actin filament organization in bovine aortic endothelial cells (bAEC) and whether this affects insulin uptake and TET. We found that insulin induced extensive cortical actin filament remodeling within 5 min. This remodeling was inhibited not only by disruption of actin microfilament organization but also by inhibition of phosphatidylinositol 3-kinase (PI3K) or by disruption of lipid rafts using respective specific inhibitors. Knockdown of either caveolin-1 or Akt using specific small interfering RNA also eliminated the insulin-induced cortical actin filament remodeling. Blocking either actin microfilament organization or PI3K pathway signaling inhibited both insulin uptake and TET. Disruption of actin microfilament organization also reduced the caveolin-1, insulin receptor, and IGF-I receptor located at the plasma membrane. Exposing bAEC for 6 h to either TNFα or IL-6 blocked insulin-induced cortical actin remodeling. Extended exposure (24 h) also inhibited actin expression at both mRNA and protein levels. We conclude that insulin-induced cortical actin filament remodeling in bAEC is required for insulin's TET in a PI3K/Akt and plasma membrane lipid rafts/caveolae-dependent fashion, and proinflammatory cytokines TNFα and IL-6 block this process.     

Increased actin nucleating activity in tumorigenic cells

The kinetics of actin polymerization has been used to quantitate the relative levels of actin nucleating activity in extracts from a number of related tumorigenic and non-tumorigenic cells. The level of nucleating activity was significantly elevated in the tumorigenic compared with the non-tumorigenic cell extracts whether the results were expressed on the basis of per protein (2-3 fold increase) or per total endogenous cellular actin (3-4 fold increase). It is concluded that this activity is probably due to an actin filament capping/severing regulatory protein(s) and that this protein(s) may be, at least partially, responsible for the microfilament disruption observed in transformed cells.     

Intranuclear actin bundles induced by dimethyl sulfoxide in interphase nucleus of Dictyostelium

Electron microscopic evidence demonstrated that dimethyl sulfoxide (DMSO) induces formation of giant intranuclear microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium. These giant bundles are approximately giant bundles are approximately 3 micrometer long, 0.85 micrometer wide, and composed of microfilaments 6 nm in diameter. Studies in which glycerinated cells were used showed that these microfilaments bind rabbit skeletal muscle heavy meromyosin, forming typical decorated "arrowhead" structures, and that this binding can be reverted by Mg-adenosine triphosphate. These data verify that the intranuclear microfilaments are the contractile protein actin, and that DMSO affects intranuclear actin, inducing the formation of such giant bundles. The intranuclear actin bundles appear at any developmental stage in two different species of cellular slime molds after treatment with DMSO. The native form of the intranuclear actin molecules and their possible functions are discussed, and it is proposed that the contractile protein has essential functions in the cell nucleus.     

Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism

Immunofluorescence with an antiactin antibody and electron microscopy were used to study the distribution of actin in cultured mouse fibroblasts during treatment with inhibitors of energy metabolism. The inhibitors induce gradual disorganization of actin-containing microfilament bundles. At the first stage of the process the bundles degrade into separate fragments; later only small patches of actin can be found in the inhibitor-treated cells. This transformation takes about 90 min and is fully reversible as microfilament bundles are recovered after incubation of the cells in the inhibitor-free growth medium. The inhibitors do not alter actin distribution in the presence of glucose. This shows that their action is due to a reduction of the ATP level in the cells. A 90 min incubation with the inhibitors does not markedly alter either the cell shape or the microtubule system. Inhibitors of the energy metabolism prevent cytochalasin action on cells. Cytochalasin B (CB) or cytochalasin D (CD) rapidly disorganize the microfilament bundles and cause cell arborization. However, microfilament bundle destruction in the cells incubated in the mixture of cytochalasin and any of the inhibitors requires 90 min and is not accompanied by dramatic changes in the cell morphology, so the process is indistinguishable from microfilament bundle destruction in the presence of the inhibitors alone.     

Benzyl butyl phthalate influences actin distribution and cell proliferation in rat Py1a osteoblasts

We previously reported that transient administration of phthalates induced actin cytoskeleton disruption in Py1a osteoblasts. However, the mechanism of this transient effect was not elucidated. In this study we provided evidence that the actin cytoskeletal re-established conditions are dependent on new actin expression and synthesis. To assess the role of phthalates in modulating the distribution of actin, confocal and electron microscopy studies were carried out. Results indicated a modification of actin distribution after phthalate administration. In addition, a relation with the nucleoskeletal component lamin A supports the hypothesis that phthalates may participate in regulatory cell processes involving actin in Py1a osteoblasts. The present study also supports the mitogenic effects of phthalates, which involve microfilament disruption, nuclear actin and lamin A. In particular, the increased levels of cyclin D3, which in mammalian cells plays a critical role in G1 to S transition and is a putative proto-oncogene in benzyl butyl phthalate treated cells, suggested a possible effect of the endocrine disruptor in cancer processes.     

Differences in the G/total actin ratio and microfilament stability between normal and malignant human keratinocytes

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0·30 ± 0·03 (mean ± SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0·49 ± 0·03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0·5 ± 0·07 (n = 4), indicating a 1·7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10^?6 and 10^?5 M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10^?4 M ), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted. We concluded that the G/total actin ratio was significantly increased in malignant keratinocytes. This seems to be correlated with altered microfilament morphology and resistance to cytochalasin B treatment. Our results suggest that the process of malignant transformation may be characterized by changes in the state of the polymerization of actin and in the stability of the microfilament network indicating that both features could potentially serve as markers determine the transformed state of keratinocytes.     

The effect of microtubule disruption on the distribution of the cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal and RSV-transformed fibroblasts

By double indirect immunofluorescence and interference electron microscopy, we have observed the effect of microtubule disruption by antimitotic drugs and coldness treatment on the distribution of adhesion sites and of the three cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal rat cells and in rat cells transformed by Rous sarcoma virus. This study shows that the state of organization of the microtubule--intermediate filament complex modulates the location and the arrangement of intracellular structures containing vinculin, alpha-actinin, and actin in normal as well as in transformed cells. The most important alterations are observed in transformed cells on the distribution of the rosette clusters that have been shown to characterize the transformation by Rous sarcoma virus [8]. These results suggest the microtubule-intermediate filament complex is directly or indirectly connected with the microfilament network.     

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.     

The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte

The effect of dimethylsulphoxide (DMSO) on the organization of the microtubular system of the mouse oocyte has been examined. Exposure to DMSO causes the immediate appearance of multiple, cold-resistant microtubular asters associated with the foci of pericentriolar material (PCM) normally present in the oocyte. More prolonged exposure to DMSO leads to progressive disassembly of the spindle, and as a result dispersal of the chromosomes and polar PCM foci occurs, and tubulin polymerization becomes confined to PCM-organized asters. Those astral microtubules located between the PCM foci and the cortex of the oocyte appear to be particularly stable, resulting in the development of lengthening radial bundles of microtubules between the PCM and the surface and the progressive movement of the PCM foci towards the centre of the cell. In contrast, after activation of the oocyte the microtubules generated in the presence of DMSO remain located in a cortical mesh. The effects of DMSO do not appear to be fully reversible in most oocytes. We discuss the implications of these results both for the cytoplasmic organization of the oocyte and zygote, and for the attempts at cryopreservation of human oocytes for therapeutic use in infertility programmes.     

MOESIN crosslinks actin and cell membrane in Drosophila oocytes and is required for OSKAR anchoring

In Drosophila, development of the embryonic germ cells depends on posterior transport and site-specific translation of oskar (osk) mRNA and on interdependent anchoring of the osk mRNA and protein within the posterior subcortical region of the oocyte. Transport of the osk mRNA is mediated by microtubules, while anchoring of the osk gene products at the posterior pole of the oocyte is suggested to be microfilament dependent. To date, only a single actin binding protein (TropomyosinII) has been identified with a putative role in osk mRNA and protein anchoring. This communication demonstrates that mutations in the Drosophila moesin (Dmoe) gene that encodes another actin binding protein result in delocalization of osk mRNA and protein from the posterior subcortical region and, as a consequence, in failure of embryonic germ cell development. In Dmoe mutant oocytes, the subcortical actin network is detached from the cell membrane, while the polarized microtubule cytoskeleton is unaffected. In line with the earlier observations, colocalization of ectopic actin and OSK protein in Dmoe mutants suggests that the actin cytoskeleton anchors OSK protein to the subcortical cytoplasmic area of the Drosophila oocyte.