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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.
The focal distribution of regulatory macromolecules within the nucleus can effectively support the integration of regulatory networks and establish threshold levels of factors for positive and negative control in a broad spectrum of biological contexts that include development and tissue remodeling. Equally important, changes in the composition and organization of regulatory machinery in nuclear microenvironments provides insight into perturbed mechanisms that relate to human disease which is strikingly illustrated by, but not restricted to, skeletal disorders and tumorigenesis (1-5). Examples are modifications in the size, number, and composition of intranuclear sites that support transcription, replication, repair and altered regulatory domains that are causally associated with cleidocranial dysplasia and competency for metastatic breast cancer cells to form osteolytic lesions in bone.
Our understanding of the location of regulatory machinery for gene expression, replication, and repair and its role in functional outcome of various biological processes is increasingly evident. However, it is important to define and develop techniques that provide both specific and quantitative insight into various parameters of nuclear architecture. Development and deployment of such approaches is essential for establishing the biological relevance of subnuclear organization as well as necessary for diagnosing disease or providing a platform for development of targeted therapies.
Traditionally, compartmentalization of regulatory machinery has been identified and characterized by subnuclear fractionation followed by biochemical and molecular analyses. These are informative approaches, but with limitations. During the past several years, advances in microscopy, together with the development of highly specific antibodies and epitope tags have allowed to examine the assembly and activities of regulatory machinery at single cell level in both the fixed as well as live cell preparations. Thus, the combined use of high resolution cellular, biochemical and molecular approaches maximizes the extent to which regulatory mechanisms can be defined.
We will focus on visualization of nuclear microenvironment using Runx transcription factors as an example for compartmentalization of regulatory machinery within nuclei of osteoblastic cells. We will present approaches for imaging of focally localized regulatory complexes in interphase nuclei as well as throughout mitosis. Specificity and quantitation of regulatory complexes that are visualized by microscopy are required to informatively relate cell morphology with regulatory mechanisms. In addition, we will describe a recently developed approach in our laboratory, designated “Intranuclear Informatics”, that quantitatively assimilates multiple parameters of regulatory protein localization within the nucleus into contributions towards skeletal gene expression from a temporal/spatial perspective (6).
Metaphase chromosome spreads are traditionally used for identification of chromosomal abnormalities (translocation, deletions, and insertions) in patients. It has been recently observed that some lineage-specific proteins, such as Runx transcription factors, retain association with chromosomes during mitosis (Figure 1A). In such cases, metaphase chromosome spreads provide powerful means for the identification of chromosomes where Runx transcription factors reside during mitosis. For example, Runx proteins associate with nucleolar organizing regions (NORs) on metaphase chromosomes. NORs can be visualized in situ by immunolabeling metaphase chromosome spread preparations for Upstream Binding Factor (UBF), a known regulator of ribosomal RNA transcription (Figure 1B). Below is a protocol used in our laboratory for preparation of metaphase chromosome spreads (7-9).
It is becoming increasingly evident that regulatory proteins are organized in highly specialized compartments within the mammalian nucleus. The biological activity of proteins often correlates with their presence or absence in these nuclear microenvironments (1). The subnuclear organization of regulatory proteins can be assessed by the sequential removal of soluble proteins and chromatin from the mammalian cell (Figure 2) followed by either in situ immunofluorescence or western blot analysis. Below is an optimized protocol that we routinely use for in situ assessment of parameters of gene expression.
Fluorescence microscopy provides a powerful tool to assess subcellular and subnuclear localization of regulatory proteins as well as nucleic acids. A variety of microscopes are available; each microscope has its own set of unique features. Below are the instructions specific for the Zeiss Axioplan 2 Microscope.
Our lab has characterized the Runx family of transcription factors, describing spatial distribution, subnuclear architectural scaffolding and relationships with coregulatory factors. Much of this work was done with fixed cells on an epifluorescence microscope with verification using a confocal microscope. This naturally led to an interest in documenting the Runx protein dynamics using live cell imaging; looking at mobility, mitotic labeling and protein-protein interactions.
The laser scanning confocal microscope coupled with a Bioptechs micro observation system offers us higher image resolution of live cells with the ability to capture images that are sharply defined optical sections produced by the elimination of out of focus light and background information from which three dimensional renderings can be recreated. This coupled with the live cell stage allow us to verify the location of Runx foci throughout dynamic processes, for example, mitosis and to assess the mobility of Runx foci in interphase and during mitosis (Figure 3B) using Fluorescence Recovery After Photobleaching (FRAP) techniques.
A common problem occurs while live cell imaging GFP transfected cells. Cells become extremely sensitive when exposed to blue filtered UV light (green fluorescence) and die while viewing over long periods of time. For example, in a dynamic process like mitosis, cells permanently stall in Metaphase. Using the scanning laser confocal microscope relieves this situation. Red fluorescence proteins (RFP) are not as sensitive to UV light, thus we transfected a RFP mitotic stage marker, found the stage of mitosis we were interested in, then used the lasers to image the spatial localization of GFP Runx labeled protein through mitosis. Another clear advantage to laser scanning confocal microscopy is the elimination of possible bleed through from double labeling by turning off one of the lasers to confirm specific localization.
The Focht’s Closed System Chamber (FCS) allows the microscopic observation of living cells by duplicating conditions of an incubator on the microscope. Temperature is controlled by a slide heater and an objective heater. The slide heater works in conjunction with a microaqueduct slide that has a thermally conductive coating which the slide heater arms rest on. The temperature is set by a controller unit. The objective heater’s temperature is also set by a controller unit and has an adjustable loop which surrounds the objective lens. These heaters are designed to eliminate heat-sink loss. Over time, cells under microscopic observation must be fed, pH level maintained and waste eliminated. A micro peristaltic pump working in conjunction with a microaqueduct slide allows medium to perfuse across the coverslip at a precise, very slow rate, feeding the cells, maintaining pH and eliminating waste.
A powerful approach for measuring the dynamics of nuclear microenvironments is to track fluorescently tagged molecules in living nuclei. There are always at least two pools of molecules in nuclear microenvironments: free and bound. Molecules will move at diffusion rates when free and at the same rate as the structure when bound. Taking advantage of this difference, photobleaching-recovery techniques can help characterize the binding equilibriums for molecules docking on a simple, stable structure. The analysis becomes complicated if the protein has multiple and heterogeneous interactions. Several studies have examined the dynamics of cellular and nuclear GFP-fusion proteins by Fluorescence Recovery After Photobleaching (FRAP) (13-16).
In FRAP, fluorescence in a Spot of Interest (SOI) or Region Of Interest (ROI) inside the cell is irreversibly bleached with a high intensity focused laser (Figure 3C, left panel). This results in non-fluorescent molecules in the bleached areas surrounded by fluorescent molecules outside the bleached region. Since the binding of these molecules is dynamic, bleached molecules will unbind and diffuse away (10-11). Molecules that are still fluorescent and are bound in the unbleached region will unbind and diffuse into the bleached zone where they can bind. Photobleach recovery rates are determined by unbinding, diffusion, and binding rates (13,14). After bleaching, a series of images of the bleached cell are taken to measure the recovery of fluorescence in the bleached spot (Figure 3C, right panel). Most papers in the biological literature report FRAP recovery rates in terms of half time of recovery or T1/2. Some others report “apparent diffusion coefficients” even though FRAP recovery rates are dependent on binding but not on diffusion (14). Therefore, measurement of binding and unbinding constants is very important in understanding FRAP recovery rates, especially for nuclear proteins (13,14). After FRAP, data from the confocal system is exported into a spreadsheet software package, corrected for the loss of fluorescence in the whole cell and normalized for pre- and immediate post-bleach fluorescence in the bleached zone. Post-normalization, full recovery of the fluorescence in the bleached zone might be expected. If there is no full recovery, then a fraction of the protein is immobile, which means the protein is tightly bound to the subcellular structure and exchanges too slowly to be measured in the post bleach session. FRAP has been valuable in many applications including, but not limited to, measurement of the binding of Histone proteins to DNA (14,17,18), the binding of the estrogen receptor to promoters (19), the binding of the glucocorticoid receptor to promoters (20), and the dynamic binding of Exon Junction Complex proteins to RNA splicing speckled domains (15,16).
Intranuclear informatics is a mathematical algorithm that is designed to identify and assign unique quantitative signatures that define regulatory protein localization within the nucleus (6); Figure 4). Quantitative parameters that can be assessed include nuclear size and variability in domain number, size, spatial randomness, and radial positioning (Figure 4, top panel).
The significance and implication of Intranuclear Informatics can be shown by two distinct biological examples (Figure 4). Regulatory proteins with different activities can be subjected to Intranuclear Informatics analysis that assigns each protein a unique architectural signature. The overlap between the architectural signatures of different proteins is often correlated to their functional overlap. Alternatively, the subnuclear organization of a protein domain can be linked with subnuclear targeting, biological function, and disease. For example, biologically active Runx2 and its inactive subnuclear targeting defective mutant (mSTD) show distinct architectural signatures, indicating that the biological activity of a protein can be defined and quantified as subnuclear organization.
These architectural signatures have the potential to discriminate between intranuclear localization of proteins in normal and cancer cells. Intranuclear informatics can be combined with proteomics (changes in protein-DNA and protein-protein interactions) and genomics (altered gene expression profiles) to develop a novel platform for identification and targeting of perturbed regulatory pathways in cancer cells.
Studies reported in this article were in part supported by grants from NIH (5PO1CA82834-05, 5PO1AR048818-05, 2R01GM32010, 5R01AR049069).
1If the metaphase chromosomes are highly condensed, use a lower concentration of Colcemid and decreased time of Colcemid treatment.
2Appropriate hypotonic treatment is vital to the quality of metaphase spreads. The concentration of KCl can be changed according to the quality of chromosome spread.
3Drop the cell suspension on cold, slightly moist slides. Chromosomes will spread poorly on dry slides. If necessary, breathe on the slide before dropping the suspension.