Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.     

Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.     

Characterization of the cytoskeleton in human normal and otosclerotic osteoblast-like cells.

The localization and distribution of some cytoskeletal protein components were studied by immunostaining methods in normal and ostosclerotic osteoblast-like cells. The protein components investigated were microtubules (beta-tubulin), intermediate filaments (vimentin), microfilaments (actin and myosin) and structural proteins (alpha-actinin and fibronectin). Although the mechanism is not yet clear, the alterations observed in the pathological cells could well play a role in the expression of otosclerosis.     

Development and Maintenance of the Neuronal Cytoskeleton in Aggregated Cell Cultures of Fetal Rat Telencephalon and Influence of Elevated K+ Concentrations

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development-dependent expression of several cytoskeletal proteins, including the heavy- and medium-sized neurofilament subunits (H-NF and M-NF, respectively); brain spectrin; synapsin I; beta-tubulin; and the microtubule-associated proteins (MAPs) 1, 2, and 5 and tau protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular beta-tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M-NF preceded that of H-NF, with the latter being detectable only after approximately 3 weeks in culture. Furthermore, MAP 2 and tau protein showed a development-dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron-enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M-NF, H-NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145-kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron-enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton.     

Changes in the distribution of mitochondria in mouse embryos blocked at the two-cell stage

Changes in the distribution of mitochondria in the two-cell mouse embryos preceding the developmental arrest in vitro, caused by a genetically determined "two-cell block in vitro" or genisteine treatment, were examined vitally using the mitochondrial-specific probe rhodamine 123 and conventional fluorescence microscopy. In the former case, serious disturbances in the localization of mitochondria appeared already from the middle of two-cell stage, long before the time corresponding to the 2nd cleavage division. Comparison of the behavior of mitochondria in the embryos successfully developing between the one- and two-cell stages and that in the embryos that ceased to cleave suggests that the developmental arrest was accompanied by aggregation of the mitochondria into clusters. There are many such clusters unlike in the cytoplasm of normally developing embryos. Intracellular localization of clusters observed in the genisteine-treated embryos differed radically from that observed in the embryos blocked in vitro at the two-cell stage.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.     

A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo

The antigenic site recognized by a rat monoclonal antibody (clone YL 1/2) reacting with alpha-tubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) has been determined and partially characterized. YL 1/2 reacts specifically with the tyrosylated form of brain alpha-tubulin from different mammalian species. YL 1/2 reacts with the synthetic peptide Gly-(Glu)3-Gly-(Glu)2- Tyr, corresponding to the carboxyterminal amino acid sequence of tyrosylated alpha-tubulin, but does not react with Gly-(Glu)3-Gly- (Glu)2, the constituent peptide of detyrosylated alpha-tubulin. Electron microscopy as well as direct and indirect immunofluorescence microscopy shows that YL 1/2 binds to the surface of microtubules polymerized in vitro and in vivo. Further in vitro studies show that the antibody has no effect on the rate and extent of microtubule polymerization, the stability of microtubules, and the incorporation of the microtubule-associated proteins (MAP2) and tau into microtubules. In vivo studies using Swiss 3T3 fibroblasts injected with YL 1/2 show that; when injected at low concentration (2 mg IgG/ml in the injection solution), the antibody binds to microtubules without changing their distribution in the cytoplasm. Injection of larger concentration of YL 1/2 (6 mg IgG/ml) induces the formation of microtubule bundles, and still higher concentrations cause the aggregation of microtubule bundles around the nucleus (greater than 12 mg IgG/ml).     

Restoration of Resistance to Osmotic Swelling of Vitrified Mouse Embryos by Short-Term Culture

In cryopreservation of mammalian embryos, embryos can be injured by osmotic swelling during removal of the cryoprotectant after warming. We have shown that vitrified embryos are more sensitive to osmotic swelling than fresh cells but that sensitivity is reduced or abolished if vitrified cells are cultured for a short period before subjecting them to hypotonic stress. In the present study, we examined the mechanism by which vitrified two-cell mouse embryos regain their resistance to osmotic swelling by culturing the embryos in the presence of various inhibitors before hypotonic treatment. New synthesis of RNA and proteins during culture was not required for regaining resistance to osmotic swelling because actinomycin D and cycloheximide failed to inhibit restoration. Inhibitors of polymerization of microfilaments and microtubules (cytochalasin B and demecolcine, respectively) also did not affect restoration of resistance to osmotic swelling, suggesting that rearrangement or repolymerization of cytoskeletal components is not involved in this process. On the other hand, brefeldin A and concanamycin A, which inhibit intracellular vesicular transport, strongly suppressed restoration of resistance. These results suggest that the intracellular vesicular transport system plays a crucial role in restoration of resistance of vitrified embryos to osmotic swelling during short-term culture.     

Dynamics of morphofunctional organization and developmental potential of mouse embryos in the state of "two-cell block in vitro"

Vital observation in combination with electron microscopy and immunocytochemistry was used for studying structural organization and developmental potential of BALB/c mouse embryos after cleavage cessation at a two cell stage caused by the "two-cell block in vitro" phenomenon. Modification of structure and viability of embryos was followed for 2 days from the time of cleavage arrest. Several hours before cleavage arrest, changes in mitochondrian distribution were noticed in embryos, no other disturbances in structural organization of blastomeres being obvious. Embryos, whose development was arrested for 24 h, remained viable and demonstrated some morphological changes similar to those seen in normally developing embryos of the same age. Towards the end of a 48 h block period some embryos died, the surviving embryos remained morphologically intact and metabolically active. At the same time, the nuclei of the latter frequently displayed chromatin condensation near the nuclear membrane, which is similar to the pattern of chromatin reorganization in the nuclei of early apoptotic cells. Our results support a concept on the "two-cell block in vitro" phenomenon as a specific functional state of embryos, and well compare with data on a partial realization by blocked embryos of the developmental program.     

Microfilament organization and distribution in freeze substituted tobacco plant tissues

Summary The organization and distribution of microfilaments in freze substituted leaf tissues and root tips of tobacco plants (Nicotiana tabacum L. var. Maryland Mammoth) were investigated in detail. Three categories of microfilaments were recognized in interphase cells of all tissues including those in the root cap: (1) microfilament bundles; (2) single microfilaments; (3) cortical microtubuleassociated microfilaments. While the microfilament bundles appeared to be distributed throughout the cytoplasm, the single microfilaments were mainly confined to the cell periphery. All three categories of microfilaments were associated with various organelles. Our study indicates that the three categories of microfilaments are normal cytoskeletal components in higher plant cells. The implications of these findings are discussed.Abbreviations MFB microfilament bundle - SMF single microfilament - MAMF microtubule-associated microfilament - AAP actin-associated protein - MAP microtubule-associated protein - MES 2(N-morpholino)ethanesulfonic acid     

Studies on the interaction between mitochondria and the cytoskeleton

Mitochondrial movements and morphology are regulated through interactions with the cytoskeletal system, in particular the microtubules. An interaction between the microtubule-associated proteins (MAPs) and the outer surface of rat brain mitochondria has been demonstratedin vitro andin situ. One of the MAPs, MAP2, binds to specific high-affinity sites on the outer membrane. Upon binding, MAP2 is released from microtubules, and it induces a physical alteration in the outer membrane which is characterized by a tighter association of porin with the membrane. It is concluded that MAP2 either binds to porin or to a domain of the outer membrane which alters the membrane environment of porin. The possibility is raised that this domain participates in mitochondrial mobilityin situ.     

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.     

Teloplasm formation in a leech, Helobdella triserialis, is a microtubule-dependent process

Fertilized eggs of the leech Helobdella triserialis undergo a cytoplasmic reorganization which generates domains of nonyolky cytoplasm, called teloplasm, at the animal and vegetal poles. The segregation of teloplasm to one cell of the eight-cell embryo is responsible for a unique developmental fate of that cell, i.e., to give rise to segmental ectoderm and mesoderm. We have studied the cytoplasmic movements that generate teloplasm using time-lapse video microscopy; the formation and migration of rings of nonyolky cytoplasm were visualized using transmitted light, while the movements of mitochondria into these rings were monitored with epifluorescence after labeling embryos with rhodamine 123, a fluorescent mitochondrial dye. To examine the likelihood that cytoskeletal elements play a role in the mechanism of teloplasm formation in Helobdella, we examined the distribution of microtubules and microfilaments during the first cell cycle by indirect immunofluorescence and rhodamine-phalloidin labeling, respectively. The cortex of the early embryo contained a network of microtubules many of which were oriented parallel to the cell surface. As teloplasm formation ensued, microtubule networks became concentrated in the animal and the vegetal cortex relative to the equatorial cortex. More extensive microtubule arrays were found within the rings of teloplasm. Actin filaments appeared in the form of narrow rings in the cortex, but these varied apparently randomly from embryo to embryo in terms of number, size, and position. The role of microtubules and microfilaments in teloplasm formation was tested using depolymerizing agents. Teloplasm formation was blocked by microtubule inhibitors, but not by microfilament inhibitors. These results differ significantly from those obtained in embryos of the oligochaete Tubifex hattai, suggesting that the presumably homologous cytoplasmic reorganizations seen in these two annelids have different cytoskeletal dependencies.     

Plant microtubule-associated proteins: the HEAT is off in temperature-sensitive mor1.

Microtubules perform essential functions in plant cells and govern, with other cytoskeletal elements, cell division, formation of cell walls and morphogenesis. For microtubules to perform their roles in the cell their organization and dynamics must be regulated and microtubule-associated proteins bear the main responsibility for these activities. We are just beginning to identify these plant microtubule-regulating proteins. Biochemical, molecular and genetic procedures have identified plant homologues of known microtubule-associated proteins, such as kinesins, katanin and XMAP215, and novel classes of plant microtubule-associated proteins, such as MAP65 and MAP190. Showing how these proteins coordinate the microtubule cytoskeleton in vivo is now the challenge. The recent identification and characterization of the Arabidopsis thaliana microtubule organization mutant, mor1, begins to address this challenge and here we highlight the significance of this work.     

A genetic screen for suppressors and enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that regulate microtubule and microfilament stability

Coordination between cell-cycle progression and cytoskeletal dynamics is important for faithful transmission of genetic information. In early Drosophila embryos, increasing maternal cyclin B leads to higher Cdk1-CycB activity, shorter microtubules, and slower nuclear movement during cycles 5-7 and delays in nuclear migration to the cortex at cycle 10. Later during cycle 14 interphase of six cycB embryos, we observed patches of mitotic nuclei, chromosome bridges, abnormal nuclear distribution, and small and large nuclei. These phenotypes indicate disrupted coordination between the cell-cycle machinery and cytoskeletal function. Using these sensitized phenotypes, we performed a dosage-sensitive genetic screen to identify maternal proteins involved in this process. We identified 10 suppressors classified into three groups: (1) gene products regulating Cdk1 activities, cdk1 and cyclin A; (2) gene products interacting with both microtubules and microfilaments, Actin-related protein 87C; and (3) gene products interacting with microfilaments, chickadee, diaphanous, Cdc42, quail, spaghetti-squash, zipper, and scrambled. Interestingly, most of the suppressors that rescue the astral microtubule phenotype also reduce Cdk1-CycB activities and are microfilament-related genes. This suggests that the major mechanism of suppression relies on the interactions among Cdk1-CycB, microtubule, and microfilament networks. Our results indicate that the balance among these different components is vital for normal early cell cycles and for embryonic development. Our observations also indicate that microtubules and cortical microfilaments antagonize each other during the preblastoderm stage.