Dynamic functional and structural analysis of living cells: New tools for vital staining of nuclear DNA and for characterisation of cell motion

Increasing interest has been paid to applications of fluorescence measurements to analyze physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization as well as cell motion during the cell cycle. This approach involves both optimal conditions for DNA staining and cell tracking methods. In this context, this report describes a stoichiometric method for nuclear DNA specific staining, using the bisbenzimidazole dye Hoechst 33342 associated with verapamil, a calcium membrane channel blocker. This method makes it possible to correlate variations of nuclear DNA content with cell motion in cells that are maintained alive. Motion measurement is the second goal of this paper and it explains the snake-spline method, and the associated cell following method.     

Tandem use of immunofluorescent and DNA staining assays to validate nested PCR detection of mycoplasma

Mycoplasma contamination in cell culture is a serious setback to cell culturists across the world with a very high rate of reported occurrence particularly because of difficult early detection. Out of a variety of detection methods known, the double-step nested polymerase chain reaction (PCR)-based detection of mycoplasma in cell culture has been critically viewed upon because of chances of producing reliable results. A nested PCR technique, described to detect a large range of cell-culture-contaminating mycoplasma species, with greater sensitivity to detect as low a contamination as a few organisms, was compared with the results from two cytological techniques employed in tandem. These are DNA staining using Hoechst, the gold standard, and an immunofluorescent assay using a highly specific monoclonal antibody. The study undertaken on randomly collected cell cultures revealed a false-negative and several false-positive results in comparison to the cytological methods employed. The observations were particularly more unambiguous with the immunofluorescent assay employed in the study while simultaneously employed Hoechst staining serving as an indicator of bacterial contamination. There is a general apprehension that genus-specific PCR approaches could be associated with inaccurate outcome and only species-specific PCRs may be satisfactory in routine screening for mycoplasma contamination in cell cultures. At this juncture, it may be suggested that caution must be exercised while adopting the two-step nested PCR-based detection approaches, and the simultaneous employment of cytological methods used in this investigation could prove to be practicable in the proper interpretation of results.     

Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation

The techniques for inducing the death of specific cells in tissue has attracted attention as new methodologies for studying cell function and tissue regeneration. In this study, we show that a sequential process of targeted cell death and removal can be triggered by short-term exposure of near-infrared femtosecond laser pulses. Kinetic analysis of the intracellular accumulation of trypan blue and the assay of caspase activity revealed that femtosecond laser pulses induced immediate disturbance of plasma membrane integrity followed by apoptosis-like cell death. Yet, adjacent cells showed no sign of membrane damage and no increased caspase activity. The laser-exposed cells eventually detached from the substrate after a delay of >54 min while adjacent cells remained intact. On the base of in vitro experiments, we applied the same approach to ablate targeted single cardiac cells of a live zebrafish heart. The ability of inducing targeted cell death with femtosecond laser pulses should find broad applications that benefit from precise cellular manipulation at the level of single cells in vivo and in vitro.     

Identification of a novel population of adrenal-like cells in the mammalian testis

Steroidogenic cells of the adrenal and gonad are thought to be derived from a common primordium that divides into separate tissues during embryogenesis. In this paper, we show that cells with mixed adrenal and Leydig cell properties are found dispersed in the insterstitium of the embryonic and adult mouse testis. They express the adrenal markers Cyp11b1 and Cyp21 and respond to ACTH. Consistent with these properties, we show that the embryonic testis produces the adrenal steroid corticosterone. These cells also express Cyp17 and respond to hCG stimulation but do not express the Leydig specific marker Insl3 showing that they are a population of steroidogenic cells distinct from Leydig cells. Based on their properties, we refer to these cells as adrenal-like cells of the testis and propose that they are the mouse equivalent of the precursors of human adrenal rests, tumors found primarily in male patients with congenital adrenal hyperplasia. Organ culture studies show that ACTH-responsive cells are present at the gonad/mesonephros border and seem to migrate into the XY but not the XX gonad during development. Consistent with this, using transgenic Cyp11a1 reporter mice, we definitively show that steroidogenic cells can migrate from the mesonephros into the XY gonad. We also show that the region between the mesonephros and the gonad harbors steroidogenic cell precursors that are repressed by the presence of the mesonephros. We propose that this region is the source of the adrenal-like cells that migrate into the testis as it develops and are activated when Leydig cells differentiate. These studies reveal the complex nature of steroidogenic cell differentiation during urogenital development.     

Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

Fluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-α (TNF-α)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage.     

Correlation between cell cycle phase of adherent Chinese hamster ovary cells and laser phase shift determined by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent Chinese hamster ovary cells using a fluorescence microscope after staining with 4′,6-diamidine-2′-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by a phase-shifting laser microscopy revealed that the laser phase shift of cells in the G2/M phase was markedly higher than that of cells in the G1 and S phases.     

Lithium induces dorsal-type migration of mesodermal cells in the entire marginal zone of urodele amphibian embryos

Summary The effect of lithium (Li⁺) on gastrulation movements was investigated during the development of the urodele amphibianPleurodeles waltl. Attention was focused on mesodermal cell migration. Under conditions of Li⁺ treatment providing a maximal enhancement of dorsoanterior structures, it was found that the dorsoventral polarity of gastrulation was abolished. In particular, vital staining and scanning electron microscopy observations on embryo fractures showed that mesodermal cells migrated radially after Li⁺ treatment, which led to the formation of rounded embryos. Epiboly movements thus were accelerated. Nevertheless, contrasting with the precocious disappearance of the early-formed yolk plug, archenteron invagination was constantly retarded and commenced with a delay of several hours as compared to control gastrulae. Cell-lineage analysis of the progenies from ventral or dorsal equatorial blastomeres of 32-cell-stage embryos provided evidence that both dorsal and ventral mesoderm contributed to notochordal tissue after Li⁺ treatment. Dorsalization of the entire marginal zone was confirmed by the ability of the entire mesoderm rudiment to behave as a dorsal organiser after Li⁺ treatment. Comparison of the migratory behaviour of isolated animal hemispheres from Li⁺-treated or control embryos cultured on fibronectin-coated substrate indicated that all marginal cells acquired the autonomous capacity for migration of dorsal marginal cells under the action of lithium.     

Effect of different fluorescent dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon Aeropyrum pernix

The interactions of DNA-binding dyes (Hoechst 33258, DAPI, acridine orange) and DiBAC₄(3) with hyperthermophilic archaeon Aeropyrum pernix cells were investigated by the combination of calorimetric, spectroscopic and microscopic techniques. All of the dyes, studied here, affect the thermal stability of DNA in vivo and in vitro. Hoechst 33258 is highly DNA-specific probe, which does not affect the thermal transitions of other cellular components as can be detected by differential scanning calorimetry (DSC). Due to this unique property, it can be used as a potential DNA marker for in vivo DSC studies. The localization of the dyes in the cells and viability assay was revealed by fluorescence microscopy. Hoechst 33258, DAPI and acridine orange did not distinguish between viable and non-viable cells of Aeropyrum pernix. Only with the commercially available Live/Dead BacLight^TM kit we were able to discriminate viable and non-viable Aeropyrum pernix cells.     

Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy

The correction of genetically based disorders by the introduction of a therapeutic genetic construct into the appropriate cell type (“gene therapy”), has become a distinct possibility in recent years. In order for gene therapy to be a practical alternative to more conventional pharmaceutical approaches to treatment, it must be administrable in vivo. This demands that a system be developed that can specifically target the DNA to the desired cell type once introduced into the patient. Among the procedures that are currently being pursued, the delivery of DNA to cells by receptor mediated endocytosis (RME), comes closest to fulfilling this crucial requirement. The natural physiological process of RME can be exploited to deliver genetic material to cells. An antibody or ligand to a cell surface receptor that is known to undergo endocytosis, is complexed with DNA through a covalently linked polycationic adjunct (e.g., polylysine, protamines). Such complexes retain their binding specificity to the cell surface and are taken up into the cell where they enter the endosomal compartment via normal endocytotic processes. In addition, steps must be taken to avoid degradation of the DNA within the endosome-lysosome. Cells can be treated with the lysosomatropic agent chloroquine during the transfection procedure. Alternatively, the components of viruses that enter cells by endocysis and possess an endosomal “break out” capacity can be used. Replication defective adenovirus coupled to the ligand-DNA complex gives transfection efficiencies of virtually 100% on tissue culture cells in vitro. Synthetic peptides that mimic the membrane fusing region of influenza virus hemagglutinin, have also been successfully used as part of the ligand-DNA complex to bring about endosomal escape. Preliminary studies have demonstrated the potential of this method to specifically target DNA to the cell type of choice in vivo. Delivery of genes by receptor-mediated endocytosis offers the greatest hope that gene therapy can be an inexpensive, easily applicable, widespread technology.     

Fluorometric quantification of DNA in cells and tissue

The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNAase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.     

CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy

A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized.     

DNA damage response is suppressed by the high cyclin-dependent kinase 1 activity in mitotic mammalian cells

DNA damage response (DDR) is vital for genomic stability, and its deficiency is linked to tumorigenesis. Extensive studies in interphase (G(1)-S-G(2)) mammalian cells have revealed the mechanisms of DDR in great detail; however, how mitotic cells respond to DNA damage remains less defined. We report here that a full DDR is suppressed in mitotic mammalian cells until telophase/cytokinesis. Although early DDR markers such as the phosphorylations of ataxia telangiectasia mutated (ATM) and histone H2A.x (H2AX) can be readily detected, the ionizing radiation-induced foci (IRIF) formation of late DDR markers such as breast cancer type 1 susceptibility protein (BRCA1) and p53-binding protein 1 (53BP1) are absent until the telophase/cytokinesis stage. We further showed that the IR-induced ubiquitination cascade around DNA damage sites did not occur in mitotic cells, which explains, at least in part, why BRCA1 and 53BP1 cannot be recruited to the damaged sites. These observations indicate that DDR is suppressed in mitotic cells after the step of γH2AX formation. Not surprisingly, we found that the absence of a full DDR in mitotic cells was associated with the high cyclin-dependent kinase 1 (CDK1) activities. More 53BP1 IRIF could be detected when the irradiated mitotic cells were treated with a CDK1 inhibitor. Further, the activation of CDK5 in interphase cells impedes the formation of 53BP1 IRIF. Together, these results suggest that the DDR is suppressed by the high CDK1 activity in mitotic mammalian cells.     

Cell-cycle-dependent variations in FTIR micro-spectra of single proliferating HeLa cells: Principal component and artificial neural network analysis

We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.     

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.     

Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining

Human mitochondria DNA (mtDNA) is arranged within the mitochondria into discrete DNA-protein complexes, termed nucleoids. The size of the human mitochondrial genome is less than that of yeast and is more difficult to visualise by fluorescent DNA stains such as DAPI and Hoescht. We have developed a simple yet effective method to visualise mtDNA in situ within living cells using the fluorescent stain PicoGreen. Quantitative analysis shows that PicoGreen can be used to estimate the degree of mtDNA depletion within living cells. We have used this approach to study the arrangement and fluorescence of nucleoids in cells depleted of mtDNA by treatment with the anti-viral nucleoside analogue, 2',3'-dideoxycytidine. We also studied the distribution of mtDNA in fibroblasts cultured from patients with mitochondrial disease. Combining PicoGreen staining with histochemical and immunocytochemical approaches enabled us to examine the effects of mtDNA depletion on mtDNA-related components at the level of single cells. This method is able to detect an intermediate degree of mtDNA depletion in living cells, and can be used to detect mtDNA free cells (rho0 cells) in culture even at very low numbers. We have also adapted the technique to efficiently sort rho0 cells from populations of normal cells by fluorescent-assisted cell sorting (FACS), without the need for selection of respiratory competence. This should be useful for the construction of new trans-mitochondrial 'cybrid' cell lines.     

The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy

 A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)₂]^3– is much shorter than that of Ln³⁺, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4′-diisothiocyanato-2,2′-stilbenendisulfonate on the transport of [Ln(cit)₂]^3–. On the other hand, the transport of free Ln³⁺ might be attributed to the enhanced permeability of erythrocytes owing to Ln³⁺ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes. Received: 7 January 1999 / Accepted: 7 May 1999