Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.

KEY WORDS: CLEM, live-cell imaging, phototoxicity, photobleaching, dynamic range Controlled Light Exposure Microscopy is a novel and simple technology that strongly reduces phototoxicity and photobleaching in live-cell imaging without compromising image quality [1,2]. To minimize phototoxicity and photobleaching, CLEM reduces the excitation light dose only in parts of the image where full exposure is not needed (in background and bright foreground). In these parts of the image S/N can be reduced without loss of image quality. CLEM has been implemented on a standard fluorescence confocal microscope. Light is controlled by a feedback system consisting of an electronic circuit and a acoustic-optical modulator (AOM) placed in the excitation pathway. We show that CLEM reduces photobleaching by a factor of 7. In HeLa cells expressing chromatin associated H2B-GFP the production of reactive oxygen species (ROS) is reduced 8-fold causing a 6 times longer scanning time without noticeable cell damage [3]. We will present applications of CLEM in cell biology. For example, we will show how we monitor the dynamics of telomeres in human cells for prolonged imaging periods. Application of CLEM in this research leads to biological results that cannot be obtained with non-CLEM (conventional imaging). Finally, we will discuss quantitative imaging with CLEM and non-CLEM, correction procedures for photobleaching, and noise properties of CLEM.

Understanding how cells maintain genome integrity when challenged with DNA double-strand breaks (DSBs) is of major importance, particularly since the discovery of multiple links of DSBs with genome instability and cancer-predisposition disorders. Ionizing radiation is the agent of choice to produce DSBs in cells; however, targeting DSBs and monitoring changes in their position over time can be difficult. Here we describe a procedure for induction of easily recognizable linear arrays of DSBs in nuclei of adherent eukaryotic cells by exposing the cells to alpha particles from a small Americium source (Box 1). Each alpha particle traversing the cell nucleus induces a linear array of DSBs, typically 10-20 DSBs per 10 mum track length. Because alpha particles cannot penetrate cell-culture plastic or coverslips, it is necessary to irradiate cells through a Mylar membrane. We describe setup and irradiation procedures for two types of experiments: immunodetection of DSB response proteins in fixed cells grown in Mylar-bottom culture dishes (Option A) and detection of fluorescently labeled DSB-response proteins in living cells irradiated through a Mylar membrane placed on top of the cells (Option B). Using immunodetection, recruitment of repair proteins to individual DSB sites as early as 30 s after irradiation can be detected. Furthermore, combined with fluorescence live-cell microscopy of fluorescently tagged DSB-response proteins, this technique allows spatiotemporal analysis of the DSB repair response in living cells. Although the procedures might seem a bit intimidating, in our experience, once the source and the setup are ready, it is easy to obtain results. Because the live-cell procedure requires more hands-on experience, we recommend starting with the fixed-cell application.

The total variation of chromosome peak positions, in bivariate distributions of Hoechst 33258 and chromomycin A3 fluorescence of 19 healthy individuals, was compared with the experimental variation, determined from 23 bivariate distributions of chromosomes prepared separately from a single cell lineage. The experimental variation in Hoechst and chromomycin fluorescence and the relative chromosomal DNA content were determined from experiments performed over several days. The additional variance contributed by time was the same as the daily variance. The accuracy by which the relative chromosomal DNA content can be calculated from bivariate peak positions was investigated. A least squares method was used to fit the distributions of relative DNA content, obtained, respectively, from mono-and bivariate flow analyses of chromosomes from the same cell lineage. In general the DNA contents match quite well, but for a few chromosomes a difference was found, statistically discernible at the 5% level. The average relative chromosomal DNA content of the chromosomes from the 19 normal individuals, calculated from bivariate peak positions, showed a linear relation with the estimates published by other investigators.

Repair of potentially lethal damage (PLD) was investigated in cells with functional G1-phase arrest with wild-type TP53 and wild-type RB and in cells in which G1-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy gamma rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G1-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat antiBrdUrd monoclonal antibody from Seralab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti-BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and TexasRed conjugated goat antimouse.Key terms: Immunochemistry, double staining procedure, IdUrd, CldUrd, flow cytometry, cell kineticsThe study of experimental and human tumors has been facilitated by the recent introduction of immunocytochemical procedures for the detection of halogenated deoxyuridines incorporated into cellular DNA (1,2,(4)(5)(6)(7)(8)(9)(10)(11)13,15,17,18). These methods are rapid and easy to perform, and data from a single sample can provide several cell kinetic parameters (cf. cell cycle time, duration of S-phase, and the potential doubling time) (3). However, not all relevant cell cycle kinetic parameters can be studied by this technique. Recruitment of cells can only be studied when a combination of DNA labels is used that can be detected separately. Detection of a second label in the same cell population requires the development of a n immunocytochemical double staining procedure using a pair of monoclonal antibodies with high specificity for different halogenated deoxyuridines and with low cross-reactivity.Most commercially available antibodies show crossreactivity between the two most frequently used halogenated deoxyuridines: bromo-and iododeoxyuridine (BrdUrd and IdUrd). Monoclonal antibodies with different specificities were reported by Vanderlaan et al. (16): Br-3, which should only recognize BrdUrd, and IU-4, which recognizes both BrdUrd and IdUrd. Shibui et al. (14) recently developed a n immunocytochemical staining procedure using these antibodies for the detection of IdUrd and BrdUrd given to the same cell population. A major disadvantage of this procedure is that the IU-4 antibody recognizes both labels IdUrd and BrdUrd. Moreover, since this reaction is based on enzyme reactions, it is not suitable for cells in suspension, hence is unsuitable for flow cytometric application. We recently studied several commercially available monoclonal antibodies for their specificity for binding to bromo-, iodo-, and chlorodeoxyuridine. Although nearly all monoclonal antibodies examined reacted with all the different halogenated deoxyuridines, we were able to select a pair of antibodies that, under the chosen experimental conditions, showed a large difference in binding to IdUrd and CldUrd. Using these com...

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DNA double-strand breaks (DSBs) are among the most dangerous types of DNA damage. Unrepaired, DSBs may lead to cell death, and when misrejoined, they can result in potentially carcinogenic chromosome rearrangements. The induction of DSBs and their repair take place in a chromatin microenvironment. Therefore, understanding and describing the dynamics of DSB-containing chromatin is of crucial importance for understanding interactions among DSBs and their repair. Recent developments have made it possible to study ionizing radiation-induced foci of DSB repair proteins in vivo. In this chapter, we describe techniques that can be applied to visualize and analyze the spatio-temporal dynamics of DSB-containing chromatin domains in mammalian cell nuclei. Analogous procedures may also be applied to the analysis of mobility of other intranuclear structures in living cells.