细胞功能测定

It is now generally agreed that the nuclei of higher eukaryotes, and particularly of mammalian cells, are highly structured and that different aspects of this structure contribute to the regulation of function (1, 2). Despite the general consensus, the key mechanisms that link nuclear struc ...

We present an experimental approach by the help of which structure and passive permeability of the nuclear envelope (NE) can be investigated thoroughly, by combining imaging, fluorescent, and electrophysiological techniques. A mature Xenopus laevis oocyte features a large nucle ...

The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyoph ...

Glycosylation is one of the most common and complex forms of posttranslational modifications of proteins in eukaryotes. Seven different protein-carbohydrate linkages have been characterized on nuclear and cytoplasmic glycoproteins, the most widespread of which is the modif ...

The formation of the nuclear envelope (NE) typically occurs once during every mitotic cycle in somatic cells, and also around the sperm nucleus following fertilization. Much of our understanding of NE assembly has been derived from systems modeling the latter event in vitro. In these systems, ...

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins in higher eukaryotes mediated by poly(ADP-ribose) polymerases (PARPs) that is involved in many physiological processes such as DNA repair, transcription, cell division, and cell death. Biochemical stu ...

Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) systems employing combinations of acetic acid/urea (AU), acetic acid/urea/Triton X-100 (AUT) and sodium dodecyl sulfate (SDS) gel formulations are uniquely effective for resolution of histone variants and the ...

Recent developments in cell biology and microscopy techniques enable us to observe macromolecular assemblies in their natural setting: the living cell. These emerging technologies have revealed novel concepts in nuclear cell biology. In order to further elucidate the biochemis ...

Many nuclear proteins contain thiols, which undergo reversible oxidation and are critical for normal function. These proteins include enzymes, transport machinery, structural proteins, and transcription factors with conserved cysteine in zinc fingers and DNA-binding doma ...

Microscopic imaging of single fluorescent molecules within cells provides a molecular, real-time view of physiological processes in vivo. Single fluorescent molecules produce diffraction-limited light spots in the image plane, which can be localised with a very high precision. In ...

Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fl ...

In this chapter we present the method of spatially modulated illumination (SMI) microscopy, a (far-field) fluorescence microscopy technique featuring structured illumination obtained via a standing wave field laser excitation pattern. While this method does not provide high ...

Visualisation of RNA at an ultrastructural level represents a major approach to study organisation and function of the cell nucleus. In addition to methods allowing one to visualise a general distribution of RNA-containing structural constituents, in situ hybridisation (ISH) is a pow ...

Our understanding of sub-nuclear organisation is largely based on fluorescence and electron microscopy methods. Conventional electron microscopy, which depends on heavy atom contrast agents, provides excellent contrast of condensed chromatin and some sub-nuclear struc ...

Nuclear architecture has been investigated intensively by various electron microscopy (EM) methods. Most of these require chemical fixation of the sample, although cryofixation has also been used in combination with cryosubstitution and resin embedding. This approach allowed ...

Protein-fragment Complementation Assays (PCAs) are a family of assays for detecting protein–protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism, or in vitro. PCAs can be used to detect PPI between pr ...

This protocol describes a method to image individual mRNA molecules in situ. About 50 oligonucleotides complementary to different regions of a target mRNA species are used simultaneously. Each probe is labeled with a single fluorescent moiety. When these probes bind to their target, each m ...

Examination of spatial and temporal gene expression pattern is a key step towards understanding gene function. Therefore, in situ hybridization of mRNA is one of the most powerful and widely used �techniques in biology. Recent advances allow the reliable and simultaneous detection of mR ...

Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. Th ...

In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and ...