The distribution and requirements of microtubules and microfilaments in bovine oocytes during in vitro maturation

Microtubules and microfilaments are major cytoskeletal components and important modulators for chromosomal movement and cellular division in mammalian oocytes. In this study we observed microtubule and microfilament organisation in bovine oocytes by laser scanning confocal microscopy, and determined requirements of their assembly during in vitro maturation. After germinal vesicle breakdown, small microtubular asters were observed near the condensed chromatin. The asters appeared to elongate and encompass condensed chromatin particles. At the metaphase stage, microtubules were observed in the second meiotic spindle at the metaphase stage. The meiotic spindle was a symmetrical, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Treatment with nocodazole did not inhibit germinal vesicle breakdown. However, progression to metaphase failed to occur in oocytes treated with nocodazole. In contrast, microfilaments were observed as a relatively thick uniform area around the cell cortex and overlying chromatin following germinal vesicle breakdown. Treatment with cytochalasin B inhibited microfilament polymerisation but did not prevent either germinal vesicle breakdown or metaphase formation. However, movement of chromatin to the proper position was inhibited in oocytes treated with cytochalasin B. These results suggest that both microtubules and microfilaments are closely associated with reconstruction and proper positioning of chromatin during meiotic maturation in bovine oocytes.     

The arrangement of microfilaments and microtubules in the periphery of spreading fibroblasts and glial cells

Studies of spreading fibroblasts and glial cells showed that the initial phase of the spreading process on a solid substratum proceeds by sequential development of different kinds of protrusions. Initially there is a high blebbing activity which is followed by development of small lamellipodia and somewhat later microspikes are formed. In the periphery of the spreading cells several types of microfilament organizations are displayed, these seem to be related to different stages in the cycles of extensions and retractions performed by the lamellipodia. The presence of microtubules and their relation to the different microfilament organizations are also shown.     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.     

Circular distribution of microfilaments in cells spreading in vitro

In the peripheral cytoplasm of spreading epithelial cells (JTC-12), circular bundles of microfilaments appear running in parallel to the cell outline at the level of the cell-substratum contact.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.     

Three-dimensional organization of microfilaments and microtubules in the cytoskeleton : Immunoperoxidase labelling and stereo-electron microscopy of detergent-extracted cells

Stereo-electron microscopy has been combined with indirect immunoperoxidase labelling to describe the three-dimensional organization of microfilaments and microtubules in spreading cells of a cultured cell line with fibroblastic morphology. Labelling was carried out after extraction of the cells with a non-ionic detergent in a buffer system allowing retention of many of the cytoskeletal elements. Preservation of the three-dimensional organization was ensured by critical point drying. The peroxidase reaction product is readily detectable in electron micrographs both at high and low magnification. Thus, visualization of the three-dimensional organization of the labelled cytoskeletal elements is possible at a magnification where entire cells or large parts of them can be examined in whole mounts. The microfilament system is shown to constitute a continuous, three-dimensional sheath enclosing the bulk of the cytoplasm and most of the microtubular system. In cytoskeletons labelled with actin antibodies, the unlabelled intermediate filaments (10 nm filaments) can be identified by their size and morphology. They constitute a network throughout the cytoplasm which is in part interwoven with the large actin cables located near the lower surface of the cell.     

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.     

The role of microtubules and microfilaments in the micronucleus ofParamecium bursaria during mitosis

Summary A unique spindle apparatus develops during mitosis in the micronucleus ofParamecium bursaria. During interphase the micronucleus contains short microtubule profiles and clumps of condensed chromatin. Throughout mitosis the nuclear envelope remains intact. During prophase, cup-shaped structures termed microlamellae develop in close association with regions of condensed chromatin. Each micromella consists of an outer sublamella, an inner sublamellae, and ring-shaped structures termed microsepta that join the two sublamellae. Microtubules elongate parallel to the division axis. During metaphase, the microlamellae appear to act as kinetochorelike structures that aid in the alignment of the chromosomes. The microlamellae appear conical and join to a meshwork of microfilaments at their apices. Further toward the polar regions the microfilaments join with microtubules that converge and terminate near the nuclear envelope. During metaphase-anaphase and anaphase the chromosomes are apparently moved by the microfilaments pulling on the kinetochorelike microlamellae. Also during metaphase-anaphase, extranuclear microtubules join the nuclear envelope of the micronucleus to microtubule elements of the cell cortex. By anaphasetelophase, microlamellae and the microfilament meshwork degenerate and microtubules represent the only spindle elements. The evidence of this report supports the hypothesis that microfilaments can participate with microtubules in the movement of chromosomes.This report is part of a Ph.D. Thesis presented by the senior author at Fordham University.     

Tropomyosin co-localizes with actin microfilaments and microtubules within supporting cells of the inner ear

Summary The distribution of tropomyosin, actin and tubulin in the supporting cells of the organ of Corti was studied by immunofluorescent localization of antibodies to these proteins. Tropomyosin colocalizes with actin and tubulin in the regions of the tunnel pillar and Deiters cells where actin microfilaments and microtubules had previously been observed ultrastructurally. Despite the implications of the presence of antiparallel actin filaments in the supporting cells, the presence of tropomyosin and the absence of myosin suggest that the role of tropomyosin may be to confer rigidity to the actin filaments. Thus the primary function of the cytoskeletal proteins in the supporting cells may be structural.     

Nuclear centering in Spirogyra: force integration by microfilaments along microtubules

The contribution of microtubules and microfilaments to the cytomechanics of transverse nuclear centering were investigated in the charophycean green alga Spirogyracrassa (Zygnematales). Cytoplasmic strands of enhanced rigidity and fasciate appearance radiate from the rim of the lenticular nucleus through the vacuole, frequently split once or twice and are attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and a web of F-actin. Bundles of microtubules, emerging from the nuclear rim, are organized into dividing fascicles within the strands and reach to the inner surface of the chloroplast envelope. Organelles are translocated in both directions along similarly arranged fascicles of microfilament bundles which extend from the nucleus to the peripheral actin cytoskeleton. Application of microtubule- and/or microfilament-depolymerizing drugs affected the position of the nucleus only slowly, but in distinct ways. The differential effects suggest that nuclear centering depends on the tensional integrity of the perinuclear scaffold, with microfilaments conveying tension along stabilized microtubules and the actin cytoskeleton integrating the translocation forces generated within the scaffold. Received: 9 December 1996 / Accepted: 29 April 1997     

Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments

Studies were conducted to compare viability of immature and mature porcine oocytes vitrified in ethylene glycol (EG) using open-pulled straws (OPS). Oocytes that had been allowed to mature for 12 h (germinal vesicle group; GV) and 40 h (metaphase II group; MII) were divided into three treatments: (1) control; (2) treated with cytochalasin B and exposed to EG; and (3) treated with cytochalasin B and vitrified by stepwise exposure to EG in OPS. After warming, a sample of oocytes was fixed and evaluated by specific fluorescent probes before visualization using confocal microscopy. The remaining oocytes were fertilized and cleavage rate was recorded. Exposure of GV oocytes to EG or vitrification had a dramatic effect on spindle and chromosome configurations and no cleavage was obtained after in vitro fertilization. When MII oocytes were exposed to EG or were vitrified, 18 and 11% of oocytes, respectively, maintained the spindle structure and either EG exposure or vitrification resulted in substantial disruption in microfilament organization. The cleavage rates of mature oocytes after being exposed to EG or after vitrification were similar (14 and 13%, respectively) but were significantly less than that of control oocytes (69%). These results indicate that porcine oocytes at different meiotic stages respond differently to cryopreservation and MII porcine oocytes had better resistance to cryopreservation than GV stage oocytes.     

Evidence for active interactions between microfilaments and microtubules in myxomycete flagellates

We have previously observed the apparent displacement of microfilaments over microtubules in the backbone structure of permeabilized flagellates of Physarum polycephalum upon addition of ATP (Uyeda, T. Q. P., and M. Furuya. 1987. Protoplasma. 140:190-192). We now report that disrupting the microtubular cytoskeleton by treatment with 0.2 mM Ca2+ for 3-30 s inhibits the movement of the microfilaments induced by subsequent treatment with 1 mM Mg-ATP and 10 mM EGTA. Stabilization of microtubules by pretreatment with 50 microM taxol retarded both the disintegrative effect of Ca2+ on the microtubules and the inhibitory effect of Ca2+ on the subsequent, ATP-induced movement of the microfilaments. These results suggest that the movement of the microfilaments depends on the integrity of the microtubular cytoskeleton. EM observation showed that the backbone structure in control permeabilized flagellates consists of two arrays of microtubules closely aligned with bundles of microfilaments of uniform polarity. The microtubular arrays after ATP treatment were no longer associated with microfilaments, yet their alignment was not affected by the ATP treatment. These results imply that the ATP treatment induces reciprocal sliding between the microfilaments and the microtubules, rather than between the microfilaments themselves or between the microtubules themselves. While sliding was best stimulated by ATP, the movement was partially induced by GTP or ATP gamma S, but not by ADP or adenylyl-imidodiphosphate (AMP-PNP). AMP-PNP added in excess to ATP, 50 microM vanadate, or 2 mM erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibited the sliding. Thus, the pharmacological characteristics of this motility were partly similar to, although not the same as, those of the known microtubule-dependent motilities.     

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

Reorganization of microfilaments and microtubules by thermal stress in two-cell bovine embryos

Two-cell bovine embryos become arrested in development when exposed to a physiologically relevant heat shock. One of the major ultrastructural modifications caused by heat shock is translocation of organelles toward the center of the blastomere. The objective of the present study was to determine if heat- shock-induced movement of organelles is a result of cytoskeletal rearrangement. Two-cell bovine embryos were cultured at 38.5 degrees C (homeothermic temperature of the cow), 41.0 degrees C (physiologically relevant heat shock), or 43.0 degrees C (severe heat shock) for 6 h in the presence of either vehicle, latrunculin B (a microfilament depolymerizer), rhizoxin (a microtubule depolymerizer), or paclitaxel (a microtubule stabilizer). Heat shock caused a rearrangement of actin-containing filaments as detected by staining with phalloidin. Moreover, latrunculin B reduced the heat-shock-induced movement of organelles at 41.0 degrees C but not at 43.0 degrees C. In contrast, movement of organelles caused by heat shock was inhibited by rhizoxin at both temperatures. Furthermore, rhizoxin, but not latrunculin B, reduced the swelling of mitochondria caused by heat shock. Paclitaxel, while causing major changes in ultrastructure, did not prevent the movement of organelles or mitochondrial swelling. It is concluded that heat shock disrupts microtubule and microfilaments in the two-cell bovine embryo and that these changes are responsible for movement of organelles away from the periphery. In addition, intact microtubules are a requirement for heat-shock-induced swelling of mitochondria. Differences in response to rhizoxin and paclitaxel are interpreted to mean that deformation of microtubules can occur through a mechanism independent of microtubule depolymerization.