A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.

Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM).

This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy (SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes (NEs) and nuclear pore complexes (NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at -80 degrees C. Demembranated sperm chromatin should take only approximately 2-3 h to prepare and can be stored at -80 degrees C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.

Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells.

Our previous work characterizing the biogenesis and structural integrity of the nuclear envelope and nuclear pore complexes (NPCs) has been based on amphibian material but has recently progressed into the analysis of tissue-culture cells. This protocol describes methods for the high resolution visualization, by field-emission scanning electron microscopy (FESEM), of the nucleus and associated structures in tissue culture cells. Imaging by fluorescence light microscopy shows general nuclear and NPC information at a resolution of approximately 200 nm, in contrast to the 3-5 nm resolution provided by FESEM or transmission electron microscopy (TEM), which generates detail at the macromolecular level. The protocols described here are applicable to all tissue culture cell lines tested to date (HeLa, A6, DLD, XTC and NIH 3T3). The processed cells can be stored long term under vacuum. The protocol can be completed in 5 d, including 3 d for cell growth, 1 d for processing and 1 d for imaging.

Electrochemical monitoring of anticancer compounds on the human ovarian carcinoma cell line A2780 and its adriamycin- and Cisplatin-resistant variants.

A novel electrochemical technique which detects and monitors real-time changes in cell behavior in vitro has been used to examine the effects of recognized anticancer drugs on the human ovarian carcinoma cell line A2780 and its adriamycin (A2780adr)- and cisplatin (A2780cispt)-resistant variants. These cells, adherent to gold electrodes or sensors, modify the extracellular microenvironment at the cell:sensor interface, producing an electrochemical potential that is different from that of the bulk culture medium. Confluent, adherent A2780 cells produced an electrochemical signal, measured as an open circuit potential (OCP), of approximately -100 mV compared to a cell-free value of approximately -15 mV. Exposure of A2780 cells to cisplatin (range 10(-4) to 10(-6) M), adriamycin (range 10(-5) to 10(-7) M), and vinblastine (10(-6) M) all produced positive shifts in the OCP signal relative to untreated control cells during 24 h of culture, but Taxotere (range 10(-5) to 10(-7) M) had no effect. These positive shifts in OCP signal were evident well before observations of reduced cellular adhesion and viability after 24 h, as judged in parallel cultures with a plastic substratum and by scanning electron microscopy. By contrast, the same treatments applied to the A2780adr and A2780cispt variants showed that each demonstrated different sensitivities to the same drugs applied to the parental A2780 cells. The effects of the same four anticancer drugs on ovarian carcinoma (A2780) and breast carcinoma (8701-BC) cell lines showed that the former was far more responsive to adriamycin and cisplatin. Such differences in drug sensitivities between the two cell lines were subsequently confirmed using the conventional MTT assay over 5 days. Although this electrochemical technology readily detects changes in cell adhesion and viability, the modified OCP signals recorded within a few hours of anticancer drug treatments are evident well before microscopic morphological changes become apparent. It is proposed that these early changes in OCP signals, relative to control untreated cells, reflect modifications of physiological/behavioral processes manifested at the cell surface.

Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos.

The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional 'spindle envelope'. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.

The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins.

The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo- cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin alpha/beta-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

Ran alters nuclear pore complex conformation.

Transport across the nuclear membranes occurs through the nuclear pore complex (NPC), and is mediated by soluble transport factors including Ran, a small GTPase that is generally GDP-bound during import and GTP-bound for export. The dynamic nature of the NPC structure suggests a possible active role for it in driving translocation. Here we show that RanGTP but not RanGDP causes alterations of NPC structure when injected into the cytoplasm of Xenopus oocytes, including compaction of the NPC and extension of the cytoplasmic filaments. RanGTP caused accumulation of nucleoplasmin-gold along the length of extended cytoplasmic filaments, whereas RanGDP caused accumulation around the cytoplasmic rim of the NPC. This suggests a possible role for Ran in altering the conformation of the cytoplasmic filaments during transport.

The nuclear pore complex: structure, function, and dynamics.

A full understanding of nucleocytoplasmic transport depends on knowledge of nuclear pore complex (NPC) structure, the functional roles of NPC components, their interactions during transport and dynamics during the cell cycle. NPC structure is conserved, flexible, and is not simply a tunnel between the nucleus and cytoplasm but appears to be actively involved in the transport process by a series of structural modifications. Transport through the NPC begins in either of its asymmetrical peripheral compartments that are both structurally reorganized during transport in different ways. The central compartment is composed of two symmetrical halves, and functions as a system of transiently open, discrete gates that is not believed to play a role in determining direction. Each NPC subunit has a specific morphology that corresponds to the functional role it plays. A complicated system of vertical and horizontal connections may allow one part of the NPC to transmit a signal to other parts, leading to an ordered series of conformational changes that drive translocation. High-resolution scanning electron microscopy has identified sequential stages of NPC assembly in vitro and revealed how the individual NPC components are assembled into a mature NPC. This review focuses on structural events during transport and on possible mechanisms of NPC assembly.

The nuclear pore complex: mediator of translocation between nucleus and cytoplasm.

The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100-200 in yeast, a few thousand in mammalian cells and approximately 50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50-100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.

Consequences associated with work-to-family conflict: a review and agenda for future research.

A comprehensive review of the outcomes associated with work-to-family conflict was conducted and effect sizes were estimated. Atypology was presented that grouped outcomes into 3 categories: work related, nonwork related, and stress related. Issues concerning the measurement of work-family conflict were also discussed. The results demonstrate the widespread and serious consequences associated with work-to-family conflict. On the basis of the results of the review, an agenda for future research was provided.

Mosaic characteristics of human endometrial epithelium in vitro: analysis of secretory markers and cell surface ultrastructure.

Specific terminal carbohydrate structures and mucin-associated glycans increase in expression within the human endometrial epithelium during the secretory phase of the menstrual cycle but exhibit wide intercellular variation. We postulated that variation in glycosylation between cells would produce differences in the glycocalyx and result in complex mixtures of cells bearing different combinations of glycans. MUC-1 mucin, keratan sulphate and fucosylated lactosaminoglycans were examined in epithelial gland fragment cultures with antibodies (HMFG1, 5D4) and a lectin (Dolichos biflorus agglutinin). The glycocalyx was examined by transmission and high resolution scanning electron microscopy. The data were related to patterns of expression seen in vivo. The MUC-1 mucin was expressed relatively uniformly in culture, but heterogeneity was evident in mucin sialylation within the epithelial cell population. Double labelling of gland explant cultures for combinations of fucosylated lactosaminoglycans, keratan sulphate and MUC-1 demonstrated cells expressing all combinations of these markers. Ultrastructural examination confirmed remarkable intercellular variation in the glycocalyx. Though the human endometrial epithelium is relatively morphologically homogeneous, these observations reveal complex variations of cell surface glycosylation between neighbouring cells and suggest that secretory function might vary in a similar fashion.

TALE homeoproteins as HOX11-interacting partners in T-cell leukemia.

The mammalian PBX and Meis proteins belong to the TALE (three-amino acid-loop-extension) superfamily of homeodomain-containing transcription factors. Members of both the PBX and Meis groups have been implicated in tumorigenesis and are known to cooperatively bind DNA with Class I (clustered) HOX homeoproteins. Here we show that PBX and Meis homeoproteins cooperatively bind the PBX-responsive sequence in vitro with the oncoprotein encoded by the non-clustered homeobox gene HOX11 activated by the t(10;14)(q24;q11) chromosomal translocation in T-cell acute lymphoblastic leukemia (T-ALL). An FPWME motif N-terminal to the homeodomain is required for interaction with PBX proteins, which appears to confer DNA-binding specificity to HOX11. PBX proteins are highly expressed in HOX11 immortalized/transformed hematopoietic cells; in particular, the 10q24 translocation-carrying T-ALL Sil and K3P lines were found to selectively express PBX2. Ectopic retroviral-directed overexpression of PBX2 in concert with HOX11 in NIH3T3 cells resulted in decreased contact inhibition of growth as evidenced by focus formation in confluent cell monolayers. The accumulated data are thus consistent with a role of TALE homeoproteins in HOX11-mediated leukemogenesis.

Nucleolar segregation during apoptosis of haemopoietic stem cell line FDCP-Mix.

The programmed elimination of cells during apoptosis is distinct from necrosis both morphologically and biochemically. Currently, the morphological description of apoptosis discriminates between the segregation of the nucleolus and the so called 'chromatin condensation'. The latter originates from observations of electron dense material adjacent to the nuclear envelope of apoptotic nuclei. Although there is ample evidence for an involvement of DNA in electron dense marginations, their true nature is still unknown. By studying apoptosis in FDCP-Mix, a pluripotent murine haemopoietic stem cell line, we found morphological and histochemical evidence that electron dense material at the nuclear envelope (NE) has emerged as a result of the segregation of nucleoli in association with the nuclear membrane. The remaining electron dense and homogenous bulk of the nucleolus labels for RNAse-gold, but even more intensely for DNAse-gold, and therefore could possibly be mistaken as 'condensed chromatin' in the light microscope. The labelling of the electron dense material for DNase-gold in FDCP-Mix could be explained by a migration of DNA into the bulk of the nucleoli at an early stage of cell death.

Nuclear envelope and nuclear pore assembly: analysis of assembly intermediates by electron microscopy.

At mitosis, the nucleus of higher eukaryotic cells disassemblies into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.

The effects of organizational citizenship behavior on performance judgments: a field study and a laboratory experiment.

The process linking organizational citizenship behavior (OCB) with performance judgments was investigated in a field and a laboratory study. In the field study, managers rated the task performance and OCB of 148 subordinates. In the laboratory research, 136 students viewed and rated videotaped segments of teaching performance that demonstrated either high or low task performance and high or low OCB. In both studies, liking and perceived affective commitment mediated the relationship between OCB and overall evaluation. Liking also mediated the relationship between OCB and reward recommendations. Further, the field study indicated that the causal motive attributed by the manager for the employee's OCB mediated the relationship between OCB and overall evaluation.