Sandra A Rutherford

HIV‐1 DNA Flap formation promotes uncoating of the pre‐integration complex at the nuclear pore

The HIV‐1 central DNA Flap acts as a cis‐acting determinant of HIV‐1 genome nuclear import. Indeed, DNA‐Flap re‐insertion within lentiviral‐derived gene transfer vectors strongly stimulates gene transfer efficiencies. In this study, we sought to understand the mechanisms by which the central DNA Flap mediates HIV‐1 nuclear import. Here, we show that reverse transcription (RT°) occurs within an intact capsid (CA) shell, independently of the routing process towards the nuclear membrane, and that uncoating is not an immediate post‐fusion event, but rather occurs at the nuclear pore upon RT° completion. We provide the first observation with ultrastructural resolution of intact intracellular HIV‐1 CA shells by scanning electron microscopy. In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore. These data show that DNA Flap formation, the very last event of HIV‐1 RT°, acts as a viral promoting element for the uncoating of HIV‐1 at the nuclear pore.

Three-dimensional surface structure analysis of the nucleus.

Publisher Summary This chapter discusses field emission sources for scanning electron microscopes (SEMs), which were approximately 1,000 times brighter than conventional sources and facilitated surface imaging at much the same effective resolution for biological material as conventional transmission electron microscopy (TEM). Specified resolution for field emission instruments is around 1 nm, and it seems highly likely that the technology of biological specimen preparation is the major limiting factor at the moment. To allow the surface imaging of subcellular components, specimens must be exposed to the electron beam at the site of interest, for example, the surface of the nucleus, and the methodologies required for this type of access. The majority of methods are based on chemical preservation, followed by dehydration, critical point drying, and thin metal coating. This approach is compatible with conventional TEM thin sectioning and has proved more accessible in terms of overall comparative observation, such as in the visualization of rare events, than the rather more limited specimen access provided by cryo approaches.

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.

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.

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, 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 °C. Demembranated sperm chromatin should take only approximately 2–3 h to prepare and can be stored at −80 °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.

Scanning electron microscopy of nuclear structure.

Accessing internal structure and retaining relative three dimensional (3D) organization within the nucleus has always proved difficult in the electron microscope. This is due to the overall size and largely fibrous nature of the contents, making large scale 3D reconstructions difficult from thin sections using transmission electron microscopy. This chapter brings together a number of methods developed for visualization of nuclear structure by scanning electron microscopy (SEM). These methods utilize the easily accessed high resolution available in field emission instruments. Surface imaging has proved particularly useful to date in studies of the nuclear envelope and pore complexes, and has also shown promise for internal nuclear organization, including the dynamic and radical reorganization of structure during cell division. Consequently, surface imaging in the SEM has the potential to make a significant contribution to our understanding of nuclear structure.

Field Emission Scanning Electron Microscopy and Visualization of the Cell Interior

Publisher Summary This chapter discusses the role of field emission scanning electron microscopy (FESEM) in helping researchers visualize intracellular surfaces. The use of low accelerating voltages in FESEM has also been shown to be of advantage, reducing charging and penetration of the electron beam, but maintaining high-resolution information content. High-pressure freezing, freeze substitution, and examination of cryohydrated specimens may all be used for FESEM. Organelles and macromolecules can be isolated by standard procedures, possibly requiring subsequent modifications in the light of HRSEM visualization, which are beyond the scope of this article. Basically, the specimens must be undamaged by osmotic shock, proteolysis, or unsuitable isolation buffers. Many cell components naturally adhere to glass coverslips, silicon chips, or carbon support film on grids. Tissue culture cells will grow in identical fashion on silicon as they do on glass or plastic, and isolated cytosol or organelles will also adhere naturally to silicon in the same way as they do to glass.

Visualization of the Dynamics of Nuclear Envelope Reformation in Mammalian Cells

In all higher eukaryotes, the nuclear envelope (NE) breaks down at the start of mitotic prophase, and is re-established in telophase as the chromatids of each daughter cell decondense to form interphase nuclei. This is in contrast to yeast and some insects, where the NE is retained throughout division, in a ‘closed’ mitosis. The series of events during NE reformation has been studied extensively in model, cell free systems, often involving amphibian egg extracts, which will reform a nuclear envelope in vitro around even naked DNA, but more usually, demembranated sperm chromatin. Our own studies (1) with this system have shown that vesicles from the extract which will form the new nuclear membrane attach to the surface of the sperm chromatin, spread out and fuse with adjacent vesicles to complete enclosure. At the same time nuclear pore complexes (NPCs) become inserted in the NE, developing through a well established series of intermediate levels of assembly, many of which can be experimentally modulated (1). Once enclosure is complete, the in vitro formed nucleus undergoes a precisely controlled DNA replication, confirming the physiological relevance of the system. In this system however, areas of novel membrane must always be present before NPCs are inserted, even if the new membrane patch is barely wide enough to accommodate the diameter of the NPC. Indeed, if NPC formation is experimentally blocked, NE formation will still be completed, allowing subsequent insertion of NPCs . In a series of investigations to compare stages of in vitro NE reformation with higher eukaryotes in vivo, we have developed approaches to visualize NE re-assembly in whole cells, ( HeLa, DLD ) This approach produces access for surface imaging by FESEM in situ in dividing cells. We have extended the protocol for accessing interphase nuclear surfaces to cells in division (2).Briefly, this involves fixation, (10 secs in 2% paraformaldehyde,0.01% glutaraldehyde), followed by extraction in 0.5% Triton (15-30mins),which may be followed by antibody incubations, then re-fixation in 3% Glutaraldehyde and 1% Osmium, dehydration and critical point drying. The cells are then subjected to a simple ‘dry-fracture’ where the Si chip on which the cells were grown is touched to double sided tape. This produces fractures which vary from exposure of the upper surface of the interphase nucleus to various depths within the nucleus itself. Both sides of the fracture can be studied in the SEM. In the case of dividing cells, the fracture removes enough of the detergent resistant cytoskeletal remains to allow direct imaging of the chromosomal surfaces. Specimens are coated with 2nms of Cr, which does not inhibit the BSE signal from either 10 nm or 5 nm Au colloid marking the secondary antigen sites. Condensed chromatin after osmium fixation itself generates a strong BSE signal, but the Au label still stands out in the BSE image. We have shown 10nm Au particles at low magnifications of 8-10,000X, which is useful for demonstrating the overall distribution across the cell. Our system of signal acquisition is to optimize the SE and BSE signals separately, and then acquire each simultaneously (on the same scan) at 2800 X 2000 pixels. This ensures that the register between SE and BSE images is maintained, for exact superimposition of images HeLa cells were accessed as above in successive stages of mitosis, and imaged with immunostaining for mAb 414 (an antibody which binds the O –linked Glyc-Nac sugar residues common to several nucleporins), and also for individual members of the 107 -160 Nup protein Copyright 2005 Microscopy Society of America DOI: 10.1017/S1431927605500576 Microsc Microanal 11(Suppl 2), 2005 1106