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.     

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.     

Improved visualization of vertebrate nuclear pore complexes by field emission scanning electron microscopy

Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.     

Evaluation of the effects of cryopreservation of isolated erythrocytes and leukocytes of largemouth bass by flow cytometry

We examined the effects of separation and freezing on fish leukocyte and erythrocyte morphology by light microscopy and on DNA content as measured by flow cytometry (FCM). Leukocytes and erythrocytes of largemouth bass Micropterus salmoides were isolated by density gradient centrifugation of whole blood, and frozen in liquid nitrogen in a buffer containing DMSO as a cryopreservative. The coefficient of variation (CV) of the G₀/G₁ peak of the cells was used to assess variation in nuclear DNA content within cell populations before and after separation and freezing treatments. In erythrocytes, the CV did not change significantly (P>0.05) when nuclei were isolated and stained without freezing or when erythrocytes were frozen prior to nuclear isolation and staining. In leukocytes, freezing and thawing prior to isolation and staining of nuclei significantly increased the CV (P<0.05), and produced hyperdiploid shoulders of the G₀/G₁ peak. However, the CV of leukocyte nuclei that were isolated and stained prior to freezing and the CV of non-frozen leukocyte nuclei did not differ (P>0.05). Microscopy showed that the freezing protocol had little effect on erythrocyte morphology, but caused irregular swelling in leukocytes. Freezing intact leukocytes also significantly (p<0.05) altered the apparent distribution of cells among the phases of the cell cycle as measured by FCM. The distributions of leukocyte nuclei that were isolated and stained prior to freezing were not different to non-frozen leukocytes. DNA measurements of nucleated blood cells are widely used in physiological, genetic and toxicological studies. Our results suggest that whole blood and erythrocytes for use in such studies can be frozen whole using a simple protocol, but leukocyte nuclei must be isolated and stained before freezing to avoid serious artifacts.     

Isolation and characterization ofEuglena nuclei

Summary A novel method for isolatingEuglena gracilis Z. nuclei, based on pretreatment of cells in concentrated glycerol buffer before homogenization, is described. Such a treatment weakens the tough cell pellicle facilitating cell disruption, and avoids nuclear damage induced by detergents and by freezing and thawing the cells in aqueous media. Nuclei, purified by centrifugation in dense sucrose, are obtained with a 30% yield, and only small amounts of cell wall fragments contaminate the nuclear pellets. The purified nuclei retain their ultrastructural characteristics. High molecular weight DNA, as well as undegraded RNA species and histones, can be extracted from these nuclei. Nuclease digestions and spread preparations show an unaltered nucleosomal structure of chromatin. This method has been applied to cell samples at any stage of the cell cycle, including mitosis, since inEuglena the nuclear envelope persists during cell division.     

High-resolution microscopy of cell wall formation in regenerating Mougeotia (Chlorophyceae) protoplasts

Field emission scanning electron microscopy (FESEM) preparation techniques have been successfully adapted for visualization of the internal and external ultrastructure of Mougeotia filaments and protoplasts. FESEM of the innermost layer of cell wall in Mougeotia filaments revealed that microfibrils are deposited parallel to each other in an interconnected mesh and are oriented perpendicular to the direction of elongation. For the first time, the surface of protoplasts at different stages of regeneration has been observed using FESEM. Nascent microfibril deposition occurs between 1 and 2 h after isolation and arrangement of these microfibrils is random for at least 8 h. Observation of the inner surface of the plasma membrane in burst protoplasts showed that microtubules are not strongly attached for at least 3 h after protoplast isolation.     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

Isolation and some characteristics of cell nuclei from brain cortex of adult cat

A rapid detergent method for the isolation of nuclei from cat brain cortex is described. It involves the homogenization of the tissue in buffered 0.34 M sucrose with the addition of the non-ionic detergent Cemulsol NPT 12 and the subsequent low speed centrifugal sieving of the nuclei through two layers of sucrose (0.68 M and 1.0 M). The final purification is achieved by high speed centrifugation (40,000 g) of the nuclear suspension layered over 1.8 M sucrose. Observations by light microscopy indicate that highly purified and well preserved nuclei are obtained from neurons and glial cells. Electron microscopy reveals some microsomal contaminants adhering to the nuclear membrane. According to an analysis of the nuclear size distribution, a considerable loss of smaller nuclei (10 to 20µ²), mainly from glial cells, occurs during the purification procedure. The action of different detergents is compared, the best results being obtained with Cemulsol NPT 12 or Triton X-100. Chemical analyses of the purified nuclear fraction give the following content expressed in picograms per nucleus: DNA, 6.54; RNA, 2.94; cholesterol, 1.50; and protein, 97.5. The sucrose density gradient centrifugation of nuclei isolated from cat brain cortex shows that their density is equal to or higher than that of 2.2 M sucrose and is thus similar to the density of nuclei from other tissues. The observation of a varying influence of different suspending media on the density of brain cell nuclei resolves the conflicting data in the literature on the density of these nuclei.     

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

DNA isolation protocol for seaweeds

We report a DNA isolation protocol for red seaweeds. Recovering DNA of high quality and quantity is a prerequisite for ensuring suitable applications, such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and sequencing. Isolation of DNA from seaweeds has proven difficult because of coprecipitation of polysaccharides. Our process minimizes this contamination, which is mostly due to the highly hydrocolloidal content of algal cell walls. This protocol, using 2 steps, is based on a preliminary enzymatic digestion of cell wall with specific enzymes (Novozymes) followed by centrifugation, allowing isolation of DNA on the pellet. This provides a higher yield of DNA, in the range of 40 μg (Palmaria palmata) and 18 μg (Gracilaria verrucosa) from 50 mg of fresh frozen pellet.     

Intermediate structures in nuclear morphogenesis following metaphase from HeLaS3 cells can be isolated and temporally grouped

Previously nuclear reformation following metaphase in HeLaS₃ cells was conceptualized in terms of a stepwise process which was continuous throughout anaphase and telophase. This concept was based on a three-dimensional visualization by scanning electron microscopy (SEM) of individual, organically prepared chromatid structures (prenuclei) which could be sequentially arranged. Morphologic analysis revealed unique topographies and morphometric properties which suggested that it should be possible to isolate populations of prenuclei aqueously. Such an isolation using detergents and density centrifugation is presented which yields metaphase plates and two populations of prenuclei with distinctive morphology. Essentially, prenuclei are freed from late mitotic cells in suspension cultures of synchronized HeLaS₃ cells by treatment with 0.1% Nonidet-P40 followed by treatment with a mixture of Tween 40-desoxycholate (0.5%). Critical for the isolation is the presence of a divalent cation (5 mM Mg^{+ +}) and an acid pH (~ 5.8). After density centrifugation, 2N decondensing structures (late intermediates) are recovered from 42% Percoll, and a mixture of 2N predecondensing (early intermediates) and 4N metaphase plates are recovered from 52% Percoll. The latter intermediates can be further separated into highly enriched populations (>94% pure) by fluorescence-activated sorting. Predecondensing structures are of the same overall morphology as prenuclei isolated previously by organic means, can also be ordered sequentially to demonstrate nuclear morphogenesis, and retain centromere/kinetochore loci. These chromosomal loci based on immunostaining of individual structures appear to be positioned centrally during chromatid reassociation and then appear to be dispersed prior to structural rearrangements leading to formation of a disc-like prenucleus. The significance of grouping intermediates temporally and of two protocols of isolation yielding the same structures is discussed with regard to a study of the requirements for nuclear morphogenesis in late mitosis.     

p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.     

Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.     

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c⁺), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45^hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.     

Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens

Here, we describe a rapid, convenient, and quantitative beta-galactosidase assay in liquid culture of recombinant yeast that expresses the estrogen receptor. This assay allows large-scale screening of chemicals (more than 600 samples/day) for the evaluation of their direct estrogenic potency and accurate determination of their EC50 with minimal manipulations. The assay, which is based on digestion of the yeast cell wall by lyticase (zymolase), a beta-glucanase isolated from Arthrobacter luteus, followed by a hypoosmotic shock lysis, is performed completely in 96-well plates. This protocol for using recombinant yeast with the two-hybrid technology significantly advances recombinant yeast manipulation.