Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. V C 2014International Society for Advancement of Cytometry

Background: Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell "redox activity." The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture. Methods: In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential. Results: Only 25-45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occu-

The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H 2 O 2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty-to 60-min exposure of cells to 100 or 200 lM H 2 O 2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% O 2 ) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 lM) had a minor effect on CHP and CAA, though it decreased the level of H 2 O 2 -induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage. ' International Society for Analytical CytologyKey terms H2AX phosphorylation; ATM activation; reactive oxygen species; hypoxia; hydrogen peroxide; 3-bromopyruvate; dichloroacetate; celecoxib; aging; caloric restriction; mitochondria IN living cells, nuclear DNA is constantly being damaged by endogenous reactive oxidants generated during aerobic respiration in mitochondria (1-6). Both, the firstand the second-generation reactive intermediates, the byproducts of oxidative phosphorylation reactions, can diffuse from mitochondria, reach the nuclear DNA, and induce its damage (7,8). It has been estimated that DNA exposure to endogenous oxidants during a single cell cycle of about 24-h duration results in~5,000 DNA single-strand lesi...

DNA-binding dyes are useful in contrasting cell nuclei and chromatin for live cell fluorescence microscopy; however, they may interfere with nuclear and DNA structure and function. We investigated the influence exerted by two DNA dyes on chromatin and nuclear structure, as well as histone-DNA interactions in live HeLa cells. A membranepermeant fluorescent DNA intercalator DRAQ5 (anthracycline derivative), at a concentration of 1 lM, caused microscopically detectable changes of nuclear architecture. Following DRAQ5 intercalation into DNA, chromatin aggregated into distinct areas and foci. The loss of 3D chromatin distribution was exerted via interference with a dynamic exchange of a linker histone (H1), which is a known chromatin stabilizing factor. At higher concentrations (3 and 7.5 lM), DRAQ5 interfered with binding of H2B core histones to DNA. Similar effects resulted from intercalation of chemotherapeutic drugs, adriamycin and daunomycin, but were not observed after binding to DNA of a minor groove binder, Syto17. ' 2008 International Society for Advancement of Cytometry Key terms chromatin structure; histone exchange; DNA intercalator; minor groove binder; DRAQ5; Syto17; fluorescence confocal microscopy CHROMATIN-a complex of DNA, histones, and nonhistone proteins-yields poor contrast in optical microscopy. In standard transmitted light mode, or even in DIC observation, chromatin is barely detectable. Throughout the cell division cycle, chromatin becomes clearly distinguishable only for a relatively short time, when chromosomes condense for mitosis. Thus, chromatin of live cells is commonly visualized using specific fluorescent probes that exhibit affinity to DNA. Most of these probes are intercalators or minor groove binders. A fluorescent probe bound to chromatin reveals the presence of spatial distribution and a local concentration of DNA in nuclei of live cells. However, binding of a probe to DNA may cause changes in chromatin or nuclear structure, which are dependent on the chemical nature and concentration of the probe. In the absence of an independent way of visualizing chromatin, such structural changes remain unknown. Availability of GFP-tagged histones for noninvasive visualization of chromatin in vivo opens a new window of opportunity for investigation of the influence of various DNA-binding dyes on nuclear and chromatin structure.We used HeLa cells expressing eGFP-tagged linker (H1) and core (H2B) histones to investigate the influence of a membrane-permeant DNA intercalator, DRAQ5 (1), and a minor groove binder Syto17, on nuclear and chromatin structure. We describe concentration-dependent aggregation of chromatin and altered interactions between histones and DNA, induced by the bound DRAQ5. We also demonstrate that a minor