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When you look at an interphase nucleus in a living cell through a light microscope, you will see a round, static organelle separated from the cytoplasm. If you continue the live cell observation, you will easily learn that the cell nucleus does not undergo any significant morphological changes until it reaches the mitosis, where the nuclear envelope and the chromosomes show dynamic structural changes. Because of these morphological properties, the cell nucleus had previously been considered a “container” of genome that provides an enclosed space for genomic events to be carried out. However, recent progress in molecular and cellular biological approaches has led to the revelation that the cell nucleus is composed of various kinds of different “compartments,” each of which is supposed to have a distinct “structure” and “function.” These include promeyelocytic leukemia (PML) bodies, Cajal bodies, nucleolus, nuclear speckles, and nuclear foci (see figure in the Preface). Recent developments in various fluo-rescence observation techniques have revealed that these compartments are moving within a nucleus and there is a constant flow of proteins between nucleoplasm and these compartments (Fig. 1).In this chapter, therecent progress in various “visualization techniques” will be reviewed and how these techniques have been utilized to visualize the structures and the dynamics of the inner nuclear compartments and chromosomes will be described.

In 1994 a paper was published that contained a rather unusual observation made with a rather unusual technique ( Oberleithner et al. 1994 ). The unusual observation was the increase in number of nuclear pore complexes (NPCs) in nuclear envelopes of kidney cells in response to aldosterone and the unusual technique applied in this study was atomic force microscopy (AFM). At that time, aldosterone had been considered as a hormone that controlled fluid and electrolyte balance in kidney through regulation of plasma membrane ion channels and transporters but virtually nothing was known about its interaction with the nuclear barrier. Possibly, those AFM experiments were born of the desperate desire of a few renal physiologists who wanted to apply a new nanotechnique, originally developed by physicists working in the material sciences ( Binnig and Quate 1986 ), on a biological membrane with some relevance for kidney function. In the meantime 10 years have passed. Aldosterone underwent a dazzling metamorphosis in terms of site and mode of action ( Oberleithner 2004 ). Atomic force microscopy developed into a useful tool in the biological sciences ( Roco 2003 ). Finally, the nuclear envelope advanced to an extensively explored membrane system that selectively passes signals from outside into the nucleus ( Gerasimenko et al. 2003 ). In this short review chapter I will focus on recent developments in this field

The somatic division, called mitosis, is characterized by equal distribution of parental genome into two daughter cells. Mitosis involves a dramatic reorganization of both nucleus and cytoplasm driven by protein kinase cascades including master controller Cdkl-cyclin B. Mitosis is an ancient eukaryotic event, and some divergence emerged during evolution. Many single cell eukaryotes, including yeast and slime molds, undergo a closed mitosis, in which mitotic spindle formation and chromosome segregation occur within an intact nuclear envelope. However, higher eukaryotes such as animal and plant cells use open mitosis, in which nuclear envelope disassembles before the chromosomes segregate. This review primarily focuses on mitotic chromosome segregation in animal cells and refers to other organisms when regulation is mechanistically conserved. For convenience of discussion, mitotic chromosome dynamics are subdivided into six phases: prophase, prometaphase, metaphase, anaphase, telophase and cytokinesis.

In eukaryotes, the nuclear envelope encapsulates chromosomes and provides a physical framework for their organization; it also acts as a nucleo-cytoplasmic boundary for intracellular components providing a regulated chemical environment within the nucleus. The genetic activities of chromosomes are modulated within this distinct physicochemical domain. The nuclear envelope is an apparently stable structure during interphase in the cell cycle, but is dynamic during mitosis, proceeding through disassembly and reassembly in a short period of time. These processes must be precisely regulated to ensure proper progression of the cell cycle, and defects in such processes often cause cell death or disease. Owing to the advancement of imaging technologies, the dynamic behavior of the nuclear envelope during the cell cycle is now being studied in detail in the living cells of many organisms. In this chapter we describe the dynamic processes of disassembly and reassembly of the nuclear envelope as revealed by fluorescence microscopy.

The nucleolus is the most prominent structure in the nucleus, and is clearly distinguished from other nuclear substructures ( Scheer and Hock 1999 ; Olson et al. 2000 ; Spector 2003 ). Since the 1960s, it has been generally thought to be the site of ribosome biogenesis. Inner nucleolar DNA sequences encode 18S, 5.8S and 28S ribosomal RNAs and in the nucleolus, many ribosomal proteins and nascent ribosomes are detected ( Fatica and Tollervey 2002 ; Raska 2003 ). In metabolically active animal and plant somatic cells and in yeast, the nucleolus harbors tens to hundreds of active ribosomal genes, which account for roughly one half of the total cellular RNA production, while no other types of active genes have been identified within it.

The nuclear envelope is the boundary between the nucleus and cytoplasm. The nuclear envelope consists of two lipid bilayers, the nuclear lamina and nuclear pore complexes (NPCs) (Fig. 1). The transfer of materials between the nucleoplasm and cytoplasm is regulated by NPCs. The nuclear envelope is also the basis of the nuclear architecture and functions. Inner nuclear membrane proteins connect the nuclear lamina and the nuclear membrane (Fig. 1). The nuclear envelope provides a platform for chromatin. The participation of inner nuclear membrane proteins in gene replication and expression has been demonstrated. The nuclear envelope also dynamically changes in structure during the cell cycle. In vertebrates, it is disassembled in the prometaphase, and reassembled at the transition from the anaphase to the telophase. Some control systems for these dynamic changes, i.e., microtubule-dynein, Ran-importinß and phosphorylation-dephosphorylation systems, were partially revealed recently. On the other hand, it has become clear that when some proteins supporting such nuclear envelope functions are mutated, unexpected diseases, i.e., muscular dystrophies, familial partial lipodystrophy, cardiomyopathy, progeria, and others, are caused.

Gene expression is the process whereby DNA sequence information is converted into a functional transmitter or player, namely, mRNA, and then a major functional player, namely, protein. Transcription is the first step in gene expression. Since the temporal and spatial regulation of gene expression define cellular identity, transcription is the most critical and fundamental step in the cellular functions of a gene. We have classified transcriptional regulation into three functional stages on the basis of the complexity of the DNA structures involved. The first level concerns the activation/inactivation of promoters on naked DNA, the second level entails activation/inactivation of nucleosomes, while the third level involves the activation/inactivation of chromosomal regions (Fig. 1). We denote the components that determine which genes are activated or repressed at each of these levels as “gene selectors.” We have categorized the gene selectors into three main groups, namely, DNA-binding proteins, histones (non-specific DNA-binding proteins), and histone-binding proteins. These three types of gene selectors work in cooperation to select the genes that are to be expressed (Fig. 2).

Although we have a draft sequence of the human genome, little is known about how the chromatin fiber is packed in three-dimensional (3D) space, or how packing affects function ( Jackson 2003 ). We know packing plays a major role; the rate of transcription of a typical gene can vary over eight orders of magnitude ( Ivarie et al. 1983 ), but deleting local elements like promoters and enhancers reduces expression by less than 5000-fold in transient transfection assays where the 3D “context” is missing. Common sense suggests the fiber cannot be packed randomly, but elucidating what any underlying order might be has proved difficult. First, the foldings of the chromatin fiber have dimensions below the resolution (≈200 nm) of the light microscope (LM) and so can only be seen by electron microscopy (EM), but then the fixation required can distort structure. Second, DNA is so long and packed so tightly it breaks and/or aggregates easily on isolation. Third, chromatin is poised in a metastable state so small charge alterations trigger changes in structure and function, and replacing the natural environment with our buffers often promotes aggregation.

The driving force behind studies on nuclear architecture is based on the assumption that nuclear architecture is an integrated part of the complex epigenetic regulatory mechanisms which control cell type specific gene expression patterns. Epigenetic mechanisms comprise the chromatin level, including DNA methylation, histone modifications and chromatin remodeling factors, and the nuclear level, which includes the dynamics and three-dimensional (3D) spatial higher-order organization of the genome inside the cell nucleus. There is increasing evidence that such a higher-order organization of chromatin arrangement contributes essentially to the regulation of gene expression and other nuclear functions (for review see Spector 2003 ; van Driel et al. 2003 ).

A single somatic cell of humans contains DNA fibers of a total length of approximately 2 m, which are compacted, without entanglement, into the nucleus of approximately 1×10^−5 m in diameter. To greater or lesser degrees, all organisms compact their DNA. Biologically important DNA regions, such as the origins of DNA replication, regulatory regions of transcription, and recombination loci, must all be compacted. The tightly constrained DNA, however, presents the appropriate environment for replication, transcription, and recombination to take place.