Research Design and Methods Our strategy is to develop a streamlined correlative optical and electron microscopy imaging method that will provide biochemical, along with ultrastructural, information of important chromatin-based domains involved in regulating gene expression. The correlative microscopy approach will readily permit the identification of structures of interest that are rare in the cell or are spatially restricted, after embedding and ultramicrotomy. Therefore, a major advantage of the technique is that such structures can be found very quickly, eliminating the prohibitive requirement to screen large areas and numbers of specimens in the electron microscope. The correlative microscopy approach will involve examining insulator bodies in cell culture. Kc cells were immunolabeled against the protein CP190, a consistent and reliable marker for insulator bodies, using a fluoronanogold conjugated antibody probe. The samples were embedded in plastic, and thin sections were collected on locator grids so that the same structures can be tracked and examined using the different forms of microscopy. The thin sections were imaged first by fluorescence microscopy to determine which cells are good candidates for imaging by electron microscopy and to provide a spatial map for pinpointing the location of insulator bodies within the nucleus. To help support the visual map of insulator bodies obtained by fluorescence microscopy, we used another specialized TEM imaging mode, scanning transmission electron microscopy (STEM),which is optimized for visualizing nanogold particles. Importantly, STEM also provides an image of the nucleus, which can be registered with the images collected from EFTEM. Subsequent imaging of the same cells by EFTEM generated element specific maps of the nucleus, and insulator bodies in particular. Specifically we wish to determine the dimensions and biochemical composition of the insulator bodies by imaging their protein and nucleic acid components. Recent evidence suggests that in addition to DNA and protein, chromatin insulators contain an RNA component that may be important for the organization of insulator bodies. Distinguishing DNA from RNA is an experimental limitation of EFTEM that we must consider, but this issue can be addressed in the in situ context. The fundamental and conserved nucleosomal organization of DNA in chromatin structure provides chromatin with a consistent and quantifiable phosphorus to mass ratio per unit density, which is distinct from RNA and protein. DNA, naturally double-stranded, is present as large chromosome molecules that are highly organized into chromatin structure. In comparison, RNA is primarily single stranded, represents a small transcript length of the chromosome and is bound to numerous large RNA-binding proteins to form RNP granules, which have a different phosphorus to nitrogen ratio compared to chromatin. To help determine whether the elemental image maps obtained using EFTEM are accurately representing phosphorus to nitrogen ratios of chromatin, we will use well characterized and highly abundant ribosomes as an internal control to assess the fidelity of our quantitative measurements. One suggested function of insulator bodies is to act as a scaffold for the association of many different insulator sequences and the formation of higher order chromatin domains. Our preliminary EFTEM imaging results in Kc cells indicate that insulator bodies contain low net phosphorus but high net nitrogen compared to surrounding chromatin marked by high net phosphorus suggesting that insulator bodies may actually be mostly juxtaposed to chromatin as opposed to organizing it directly.
In Aim 2, in order to better define the functional significance of the chromatin in the local vicinity of insulator bodies, we will perform double labeling with simulataneous immunodetection of CP190 and lamin followed by EFTEM imaging. These experiments will provide valuable information regarding the location of insulator bodies relative to the nuclear lamina in each of the three imaging modes. Conclusions The application of EFTEM imaging to study complex sub-nuclear regulatory structures has significant potential but is currently underutilized. This is in part due to the lack of exposure of such a specialized imaging method to biomedical researchers. Correlative microscopy provides the advantage of obtaining, with a high success rate, the ultrastructural detail in complexes that are temporally or spatially rare. By combining correlative microscopy with acquisition of energy-filtered tomographic data, high-resolution ultrastructural information in three dimensions will be obtained of the relationships between protein-based and nucleic acid-based macromolecular complexes in the cell nucleus. The group involved in this plan has the collective expertise to apply EFTEM imaging to elucidate the ultrastructural details of chromatin insulator bodies. Overall, this work will have high impact in the field of gene regulatory mechanisms as well as promote the use of a novel imaging method to study nuclear architecture.
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