Nuclear Dynamics: Molecular Biology and Visualization of the Nucleus

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The most striking difference between the net phosphorus image Net P , which reflects the density of nucleic acids, and the net nitrogen image Net N , which reflects the density of protein and nucleic acid, is in the extranucleolar regions of the nucleus. In the net nitrogen image, the interchromatin space does not appear open, as it does in the net phosphorus image. Instead, the interchromatin space is filled with structure, and the regions of chromatin only produce a marginally greater signal than the surrounding interchromatin structure.

These results demonstrate that there is an abundance of protein-rich, nucleic acid-depleted structure in the interchromatin space of these paraformaldehyde-fixed cells. ESI of cellular structures. The principles of ESI of chromatin, ribonucleoprotein, and protein structures are illustrated.

An energy loss series was recorded from an ultrathin section of an Indian muntjac fibroblast cell. The top series shows a reference eV energy loss image, a phosphorus-enhanced eV energy loss image, and a phosphorus distribution map Net P calculated from these two images. The bottom panels show analagous pre-edge and element-enriched images used to generate a nitrogen distribution map Net N. The images in the top two panels show structure as bright on a dark background. Bar, 2. In principle, nucleic acids and proteins can be discriminated through analysis of phosphorus and nitrogen contents in a quantitatively sensitive manner.

This principle is qualitatively validated by visual comparisons of structures such as those in Figure 1. We sought to validate this approach further through quantification of phosphorus and nitrogen contents in cellular components of well-characterized composition. The data are presented in Figure 2 and Table 1. Part of the cell nucleus has been cut along its surface, evident by the cross-sectioning of several nuclear pores 3.

A vision of 3D chromatin organization | Nature Reviews Molecular Cell Biology

Cytoskeletal elements Figure 2 , 2 and mitochondria Figure 2 , 1, arrow in eV image are also represented. These structures serve as references for structures that are predominantly protein in composition. Chromatin Figure 2 , 7 and the granular component of the nucleolus Figure 2 , 6 serve as references for structures that have a high compositional representation of nucleic acid.

The IGC Figure 2 , 5 and the interchromatin region Figure 2 , 4 were evaluated for their compositional characteristics relative to these reference structures. Relative phosphorus and nitrogen contents of compositionally defined and compositionally undefined cellular structures. The eV phosphorus-enhanced, eV nitrogen-enhanced, net nitrogen Net N , and net phosphorus Net P images are shown. The regions numbered 1—7 were analyzed quantitatively for both phosphorus and nitrogen content. The arrow in the eV image indicates the position of a mitochondrion region 1. The results of these measurements are presented in Table 1.

Table 1.

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Relative nitrogen-to-phosphorus ratios of representative cellular structures. The numbers indicate regions identified in Figure 3. Integrated density for regions containing, with the exception of nuclear pores, at least pixels were calculated in both the net nitrogen and net phosphorus images.

A quantitative analysis of the relative nitrogen and phosphorus composition is presented in Table 1. It can be seen that an interchromatin region, specifically chosen to avoid nuclear RNP granules 4 , and the IGC 5 , which contains several small ribonucleoprotein granules within a proportionately larger nonnucleoprotein mass, have a very high nitrogen-to-phosphorus ratio. This reflects the predominance of protein structures within these regions. The granular component of the nucleolus also has a much greater protein contribution to its total mass than the condensed chromatin region.

These results indicate that the combined net phosphorus and net nitrogen information can be used to determine the biochemical composition of the individual structures represented in energy filtered electron micrographs. The results of Table 1 validate the comparison of net nitrogen and net phosphorus images for the morphological identification and elemental characterization of cellular complexes. We next addressed whether a protein component could be visualized that had the basic morphological characteristics of a protein matrix.

Figure 3 shows a eV energy loss image and net phosphorus and nitrogen images of a subregion of an interphase Indian muntjac fibroblast cell nucleus. In the bottom right panel, the net phosphorus image has been false colored green and superimposed on the net nitrogen image red. As expected, the nucleic acid components of the cell nucleus contain high amounts of both nitrogen and phosphorus, and, consequently, appear yellow in the merged image.

These are likely RNP particles. Similarly, decondensed chromatin fibers are also observed and appear yellow in the merged image Figure 3 , large arrowheads in bottom right panel; also see corresponding regions of Net P image.

PASSIVE AND ACTIVE FORCES IN CHROMATIN

Despite the presence of very small structures that contain nucleic acid, most of the nitrogen-containing material outside of the compact regions of chromatin is derived from protein structures, highly depleted in nucleic acid. This appears red in the merged image. There are two nuclear pores that appear as red insertions between the blocks of condensed chromatin on the periphery of this cell nucleus Figure 3 , NP in bottom right panel.

These serve as references for structures that are protein based in composition.

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Additionally, close examination will reveal a narrow band of protein on the cytoplasmic side of the peripheral chromatin Figure 3 , NL in bottom right panel. This reflects the presence of the nuclear lamina. The intervening protein elements within the nucleoplasm have a general filamentous appearance and can be seen to connect adjacent nucleic acid-rich structures.

Protein transport into nucleus

Moreover, the smallest nucleic acid-containing complexes observed in this nuclear section Figure 3 , small arrows in bottom right panel are associated with this more massive protein architecture. Facing page. Direct visualization of protein architecture within the cell nucleus. An energy loss series of a region of an Indian muntjac fibroblast cell nucleus was collected at high magnification.

The bottom right panel shows a superimposition of the net phosphorus image false-colored green onto the net nitrogen image false-colored red. Nucleoproteins, which contain high amounts of both phosphorus and nitrogen, appear yellow in the merged image. Protein structures, which are low in phosphorus, appear red and orange. The positions of nuclear pores NP and the nuclear lamina NL are indicated.

The small arrowheads indicate the positions of small RNP granules, and the large arrowheads indicate the positions of decondensed nucleic acid-containing fibrils that are continuous with more condensed chromatin fibers. Bar, nm. The IGC represents an excellent example of compartmentalization within the cell nucleus. The best described component of the IGC is a to nm ribonucleoprotein granule Monneron and Bernhard, ; Wassef, The nature of this granule was recently revealed quantitatively by both ESI Hendzel et al. We have shown that these interchromatin granules each contain between and 10, bases of RNA.

The RNPs within the IGC domain are not so densely clustered that particle—particle associations could be responsible for their compartmentalization. In early investigations, interconnecting fibrils were observed Monneron and Bernhard, ; Puvion and Bernhard, ; Wassef, However, the specificity of the staining method for RNA is not ensured Bernhard, Thus, the method cannot be used to conclude that the fibers are composed of RNA.

We investigated the organization of IGCs using phosphorus and nitrogen Figure 4. The top two panels show low-magnification views of a region of an Indian muntjac cell nucleus containing a prominent IGC. In comparing the phosphorus-enhanced eV energy loss image with the nitrogen-enhanced eV image, it can be seen that the IGC stands out in the eV image as a nuclear region relatively depleted in phosphorus.

In contrast, the IGC is not a region of obvious signal depletion in the eV image. This reflects the presence of a large amount of protein mass within the IGC, further illustrated in the high-magnification views of the IGC and surrounding nuclear territory presented in the lower two panels. When phosphorus is imaged, the IGC appears as a number of dispersed phosphorus-rich particles Figure 4 , bottom left.

Direct Visualization of a Protein Nuclear Architecture

This contrasts with the net nitrogen image Figure 4 , bottom right , where the particle nature of the IGC is much less apparent. Instead, the IGC appears as a relatively dense network of protein fibers in which the granules are embedded. Phosphorus and Nitrogen distributions within IGCs. An energy loss series was collected for a region of an Indian muntjac fibroblast cell nucleus containing a prominent IGC. The top two panels show the eV phosphorus-enhanced and the eV nitrogen-enhanced images. Cy, cytoplasm; Chr, examples of chromatin.

Bar, and nm in the top and bottom panels, respectively. The protein components of the cell nucleus can be distinguished from nucleic acid components of the cell nucleus based on phosphorus density. Detection of low levels of phosphorus associated with most cellular components cannot always be detected reliably with only one pre-edge eV and one post-edge eV image. Instead, an energy loss spectrum derived from particular objects can be obtained to assess low levels of particular elements Vazquez-Nin et al.

Nuclear bodies, which are large, protein-based structures in the cell nucleus, and condensed chromatin were imaged at eV energy loss intervals across the phosphorus L 2,3 ionization edges of phosphorus and nitrogen. We then plotted the signal intensity at each energy loss to generate an energy loss spectrum for each structure. Figure 5 shows a net phosphorus and a net nitrogen map of a region of an SKN cell nucleus that contains a nuclear body.

The nuclear body was identified using an anti-CBP antibody and correlative immunofluorescence microscopy Hendzel et al. The nuclear body is qualitatively striking in its deficiency in phosphorus. In the energy loss spectrum obtained from the nuclear body Figure 6 , the amount of phosphorus is barely detectable, based on the continuing decline in signal beyond eV the phosphorus ionization edge.

Even less is detected in background regions of the nucleus, represented by regions that do not correspond to structural features. As expected, no phosphorus was detected over regions of the plastic embedding material. Because the phosphorus signal through this body is diffuse and near the nucleoplasmic background, it likely represents the presence of phosphorylated proteins in the nucleoplasm. The nuclear body, on the other hand, is rich in nitrogen Figure 6 , lower panel , contributing to a signal close to that of the chromatin and well above the background nuclear signal.

Phosphorus and nitrogen mapping of a nuclear body. An energy loss series was then collected on a subregion of the cell nucleus containing the nuclear body. The net phosphorus and net nitrogen maps are shown. NB, nuclear body; Chr, chromatin. Detection of protein phosphorylation. The integrated density of a nuclear body Pml Body , DNA Chr in Figure 5 , and a background region of the nucleoplasm were calculated across an energy loss series, spanning both the phosphorus ionization edge upper graph and the nitrogen ionization edge lower graph , using a constant exposure time.

Under these conditions, the phosphorus and nitrogen signals generated by the plastic decay at the expected rates. In contrast, all three nuclear structures give detectable phosphorus signals between and eV energy losses, characteristic of the presence of a phosphorus L 2,3 ionization edge.

There are many reasons to believe that the cell nucleus contains a structural component that is capable of organizing and spatially sequestering biochemicals within the cell nucleus. The increased application of confocal microscopy to the understanding of biomolecular organization within the nucleus of fixed biological specimens has provided much of the most compelling evidence.

Most biomolecules show some degree of spatial sequestration. These include transcription factors van Steensel et al. More recently, the study of biomolecular organization in living cells, using fluorescence microscopy, has further indicated the presence of a component of the cell nucleus that is capable of spatially restricting the movement of biomolecular structures within the cell nucleus.

The best characterized example is chromatin. Chromatin has been clearly shown not to undergo substantial Brownian motion Abney et al. The absence of substantial Brownian motion of chromatin within the living cell nucleus has led several investigators to suggest that chromatin is confined in situ by a structure analagous to a nuclear matrix Marshall et al. The quantitative resolution of both phosphorus and nitrogen content within the specimen using ESI is sufficient to resolve the compositional differences between many structures within the cell nucleus.

For example, the granular component of the nucleolus clearly contains a higher protein-to-nucleic acid stoichiometry than does chromatin. Similarly, the granular component of the nucleolus, which is intermediate in nucleic acid density, is also clearly resolved from protein-based components such as nuclear pores. This has not been accomplished using heavy atom staining, because it is not possible to visualize all cellular components while also discriminating between protein and nucleic acid components. In particular, decondensed chromatin and RNA fibrils cannot be differentiated from protein filaments of similar dimensions.

Moreover, heavy atom contrast agents required in conventional electron microscopy frequently fail to stain some structures or overrepresent others, based on the degree of their chemical reactivity Abholhassani-Dadras et al. Paraformaldehyde fixation is the preferred method of fixation for the preservation of chromatin structure and nuclear volume Robinett et al. It has also become a standard for indirect immunofluorescence analysis of the cell nucleus. In such preparations, transcription and splicing factors have been shown to occupy distinct compartments within the cell nucleus.

We conclude that there is a protein-based architecture that has the potential to organize factors in the nucleus. Whether the nucleus is similarly organized in a dynamic living state remains to be determined conclusively. It is noteworthy, however, that at least some factors are found in foci Htun et al. The IGC represents the single most striking example of a compartmentalization process within the extranucleolar regions of the cell nucleus. Importantly, this compartment can be visualized as a structurally dynamic domain in which splicing factors enrich in living cells Misteli et al.

Our results demonstrate that the particle density of ribonucleoprotein granules within the IGC is not sufficient to explain the compartmentalization of these particles through interparticle association. We have found that the IGC contains structures that link the individual granules. Moreover, most of the biological mass that appears to integrate the IGC is contributed by protein.

In the case of the IGC, then, there is strong reason to propose that the integration of particles through a protein architecture occurs in living cells. Since the initial characterization of a nuclease and high-salt—resistant matrix-insoluble component of the cell nucleus, the nuclear matrix has been actively studied by a small but persistent group of biochemists and cell biologists. As a means of studying high-affinity interactions between specific nuclear components, the original biochemical fractionation procedure of Berezney and Coffey and subsequent variations on this protocol Jackson and Cook, ; He et al.

As a cytological preparation for the investigation of structure—function relationships by transmission electron microscopy, however, the procedure has significant shortcomings.

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The identification and characterization of the nuclear matrix is absolutely dependent on elution of chromatin before fixation Jackson and Cook, ; He et al. Because the interaction between a nuclear matrix and chromatin is of fundamental importance for its role in organizing chromatin within the cell nucleus, this shortcoming is very serious.

The potential to reorganize components through chromatin elution is the basis for much of the skepticism surrounding the nuclear matrix field. The ideal procedure would leave chromatin components intact and would enable the study of interactions that involve not only protein and RNA but also chromatin, which is the substrate in so many biochemical processes occurring in the cell nucleus. In this study, we have presented, for the first time, compelling evidence of a nuclear protein architecture within a standard cytological preparation, based on the direct discrimination of protein-based and nucleic acid-based structures, visualized in situ.

We thank Manfred Herfort and Maryse Fillion for excellent technical assistance. We also thank Dr. Charlotte Spencer for critical reading of the manuscript. This work was supported by an operating grant provided by the Cancer Research Society. Submitted: 17 March Accepted: 31 March Molecular Biology of the Cell Vol. Document from the year in the subject Biology - Micro- and Molecular Biology, , Document from the year in the subject Biology - Micro- and Molecular Biology, , language: English, abstract: A laboratory Text book of Biochemistry, Molecular Biology and Microbiology is intended to prepare the undergraduate, postgraduate and research students to perform View Product.

This book contains basic of molecular biology i. The chemical properties of nucleic acid, types structure, functions of DNA-RNA, replication, transcription, translation, post translation modifications, etc. Concise, well-organised text with annotated Concise, well-organised text with annotated study diagrams.


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