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The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I-dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching to measure the relative binding kinetics of enhanced green fluorescent protein (EGFP)-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction." RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased binding affinity of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells., (Copyright © 2010 Wiley-Liss, Inc.)

Nuclear microenvironments are architecturally organized subnuclear sites where the regulatory machinery for gene expression, replication, and repair resides. This compartmentalization is necessary to attain required stoichiometry for organization and assembly of regulatory complexes for combinatorial control. Combined and methodical application of molecular, cellular, biochemical, and in vivo genetic approaches is required to fully understand complexities of biological control. Here we provide methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.

Regulatory machinery for gene expression, replication, and repair are architecturally organized in nuclear microenvironments. This compartmentalization provides threshold concentrations of macromolecules for the organization and assembly of regulatory complexes for combinatorial control. A mechanistic under standing of biological control requires the combined application of molecular, cellular, biochemical, and in vivo genetic approaches. This chapter provides methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies., (Copyright (c) 2005 Wiley-Liss, Inc.)

We present an overview of Runx involvement in regulatory mechanisms that are requisite for fidelity of bone cell growth and differentiation, as well as for skeletal homeostasis and the structural and functional integrity of skeletal tissue. Runx-mediated control is addressed from the perspective of support for biological parameters of skeletal gene expression. We review recent findings that are consistent with an active role for Runx proteins as scaffolds for integration, organization and combinatorial assembly of nucleic acids and regulatory factors within the three-dimensional context of nuclear architecture.

Postmitotic gene expression requires restoration of nuclear organization and assembly of regulatory complexes. The hematopoietic and osteogenic Runx (Cbfa/AML) transcription factors are punctately organized in the interphase nucleus and provide a model for understanding the subnuclear organization of tissue-specific regulatory proteins after mitosis. Here we have used quantitative in situ immunofluorescence microscopy and quantitative image analysis to show that Runx factors undergo progressive changes in cellular localization during mitosis while retaining a punctate distribution. In comparison, the acetyl transferase p300 and acetylated histone H4 remain localized with DNA throughout mitosis while the RNA processing factor SC35 is excluded from mitotic chromatin. Subnuclear organization of Runx foci is completely restored in telophase, and Runx proteins are equally partitioned into progeny nuclei. In contrast, subnuclear organization of SC35 is restored subsequent to telophase. Our results show a sequential reorganization of Runx and its coregulatory proteins that precedes restoration of RNA processing speckles. Thus, mitotic partitioning and spatiotemporal reorganization of regulatory proteins together render progeny cells equivalently competent to support phenotypic gene expression.

Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias., (Copyright 2000 Wiley-Liss, Inc.)

Mechanisms that coordinate the spatial organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture contribute to the sorting of regulatory information as well as the assembly and activity of sites within the nucleus that support gene expression. In this article we will present an overview of experimental approaches that provide insight into the trafficking of the hematopoietic and bone-specific AML/CBF family of regulatory factors to transcriptionally active subnuclear sites., (Copyright 1999 Academic Press.)

Evidence is accruing that the architectural organization of nucleic acids and regulatory proteins within the cell nucleus support functional interrelationships between nuclear structure and gene expression. The punctate distribution of several transcription factors has provided several paradigms for pursuing mechanisms that direct these regulatory proteins to subnuclear sites where gene activation or suppression occurs. Sequences that are necessary and sufficient to direct regulatory proteins to transcriptionally active nuclear domains have been identified. Mutations that disrupt intranuclear targeting signals lead to modified subnuclear distribution of transcription factors and aberrant expression in tumor cells. J. Cell. Biochem. Suppls. 32/33:24-31, 1999., (Copyright 1999 Wiley-Liss, Inc.)

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear organization of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostic and targeted therapy.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear organization of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostics and targeted therapy. J. Cell. Biochem. Suppls. 30/31:220-231, 1998. © 1998 Wiley-Liss, Inc., (Copyright © 1998 Wiley-Liss, Inc.)

The cell cycle regulating Cdc2 protein kinase helps orchestrate cell cycle dependent changes in cell structure and function. This report shows that Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying an independence of the stage in the cell cycle, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes were labeled with an antibody to the PSTAIRE domain of Cdc2. Embedment-free, immunogold electron micrographs of extracted cell whole mounts show the centrioles and a pericentriolar network of filaments. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle.

Fetal rat calvarial-derived osteoblasts, in vitro, undergo a developmental sequence of events leading to bone tissue-like organization and osteoblast differentiation. Previous studies have documented temporal expression of genes reflecting stages of osteoblast phenotype development in relation to tissue organization. Two steroid hormones are known to modify the developmental sequence; 1,25(OH)2D3 can block differentiation when added to proliferating cells, while glucocorticoid addition to proliferating cultures increases the population of cells competent to produce a bone-like matrix and accelerates the differentiation time course. We have addressed the mechanisms contributing to these observations at the single cell level by analysis of a growth-related gene (H4 histone which is coupled with DNA synthesis) and matrix-associated genes (collagen, osteopontin, and osteocalcin) in hormone-treated cells. Our results demonstrate (1) a window of responsiveness for modifications in phenotype development; (2) distinct morphological changes and selective modifications in gene expression in response to both hormones as a function of whether the cell is proliferating or differentiated; and (3) location of the cell with respect to the mineralized nodule was a contributing factor to the levels of gene expression and hormonal responses. In response to vitamin D, surface osteoblasts associated with the nodules became flattened, elongated, and aligned, reminiscent of a bone lining cell. In glucocorticoid-treated cultures, proliferating cells became cuboidal and nodule-associated differentiated cells were approximately one-third the size of control osteoblasts. We also find subsets of hormone-responsive cells in the proliferating cultures in response to glucocorticoid but not vitamin D. In postproliferative cultures, both hormones increased osteocalcin mRNA in the more differentiated osteoblasts associated with the mineralized matrix but no induction occurred in monolayer internodular cells. Osteopontin was induced by glucocorticoid in a larger population of cells. Thus, our studies at the single cell level show selective morphological changes and changes in the level of gene expression supporting the hypothesis that hormones have differential effects on osteoblasts in relation to their stage of phenotype development.

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.

A quantitative comparison of isolated lymphocytes prepared for SEM by the critical-point drying (CPD) and freeze drying (FD) methods revealed that the mean cellular diameter dropped from 7.9 micron after fixation to a final diameter 6.9 micron after FD, and to 5.7 micron after CPD. In addition to their larger sizes, FD lymphocytes were immediately distinguishable by their more complex surfaces, featuring wider microvilli which often emanated from ridges on the cell surface and branched extensively near their bases. The mean width of microvilli was 0.22 micron after FD and 0.12 micron after CPD. The number of microvilli per cell was essentially the same by the two methods. In view of these findings, a critical comparison of the CPD and FD methods using the particular cells or tissue to be investigated is an essential prelude to a rigorous SEM study.

When human lymphocytes are treated with concanavalin-A (con A) and hemocyanin, the hemocyanin marker, which demonstrates con A binding sites, can be visualized by scanning (SEM) and transmission electron microscopy (TEM) on both critical-point-dried and freeze-dried cells. The ability to visualize the hemocyanin marker by SEM, its quantity and distribution, were all similar in lymphocytes prepared by both drying procedures. By TEM, hemocyanin was seen adjacent to the plasma membrane on critical-point-dried lymphocytes. In contrast, freeze-dried cells showed hemocyanin labeling at some distance from the plasma membrane (40-70 nm) as well as adjacent to it. The distribution of hemocyanin corresponded to the thickness of the amorphous coat seen on fixed, freeze-dried cells. Therefore, the extracellular coat on freeze-dried lymphocytes is a carbohydrate-containing glycocalyx.

In rat uterine luminal epithelial cells, nuclear bodies occur in the euchromatin in varying numbers in relation to the nuclear concentration of the estrogen receptor (Clark et al., 1978; Padykula et al., 1981, 1982). This functional responsiveness indicates that nuclear bodies may be useful indicators of the degree of cellular estrogenization. Because these filamentous bodies vary in size (200-1200 nm), shape, and composition, quantitative analysis of frequency of their occurrence has been difficult. A fundamental division into 2 categories can be made by the following criteria: 1) simple nuclear bodies (200-500 nm) consisting of a protein mesh of microfilaments, and 2) complex nuclear bodies (200-1200 nm) composed of an outer filamentous protein capsule enclosing a lucent core that may contain granules. Previous quantitative analyses at the electron microscopic level has excluded "simple bodies" because they might actually be ultrathin sections through the filamentous capsule of complex bodies (Le Goascogne and Baulieu, 1977; Clark et al., 1978). To resolve this sampling problem, we have performed serial ultrathin section analysis of nuclear bodies in hyperestrogenized luminal epithelial cells. Ultrastructural evidence presented here demonstrates that simple and complex nuclear bodies are anatomically separate entities. Ultrathin sections through the capsule of complex nuclear bodies will be misidentified as profiles of simple bodies during quantitative analysis. This anatomic distinctness of simple and complex nuclear bodies correlates with their differing responses to estrogenic stimulation and withdrawal (Fitzgerald and Padykula, pp. 131-141, this volume). Thus the existence of these two major categories should be taken into consideration during quantitative analyses.

Surface coats can be demonstrated on human peripheral blood lymphocytes by staining with ruthenium red, alcian blue, Thorotrast, and cationized ferritin, which are similar in distribution to a 40- to 65-nm layer of amorphous extracellular material recently reported on fixed, freeze-dried lymphocytes. Several additional lines of evidence, including X-ray microanalysis, suggest that the latter is not a contaminant added by freeze-drying. Freeze-drying may provide the means for a morphological assessment of the lymphocyte surface, including the extracellular coat, which may give additional insight into the immune response.