Jan Malínský

Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ

T he organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated in this study in order to address the long-standing controversy with regard to the intranucleolar localization of these genes. Detailed analyses of HeLa cell nucleoli include direct localization of ribosomal genes by in situ hybridization and their indirect localization via nascent ribosomal transcript mappings. On the light microscopy (LM) level, ribosomal genes map in 10–40 fluorescence foci per nucleus, and transcription activity is associated with most foci. We demonstrate that each nucleolar focus observed by LM corresponds, on the EM level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). The EM data identify the DFC as the nucleolar subcompartment in which rRNA synthesis takes place, consistent with detection of rDNA within the DFC. The highly sensitive method for mapping nascent transcripts in permeabilized cells on ultrastructural level provides intense and unambiguous clustered immunogold signal over the DFC, whereas very little to no label is detected over the FC. This signal is strongly indicative of nascent “Christmas trees” of rRNA associated with individual rDNA genes, sampled on the surface of thin sections. Stereological analysis of the clustered transcription signal further suggests that these Christmas trees may be contorted in space and exhibit a DNA compaction ratio on the order of 4–5.5.

Fluorescent probing of membrane potential in walled cells: diS‐C3(3) assay in Saccharomyces cerevisiae

Membrane‐potential‐dependent accumulation of diS‐C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, λmax, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential‐reporting parameter, the λmax shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential‐dependent red λmax shift depends on relative cell‐to‐probe concentration ratio, a maximum shift (572→582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (≤5×106 cells/ml) and probe (10−7 M) concentrations at which a clearly defined relationship exists between the λmax shift and the potential‐dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10–30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0·2% glucose (cell wall thickness 0·175±0·015 μm, n=30) are stained much faster and the λmax is more red‐shifted than in cells grown in 2% glucose (cell wall thickness 0·260±0·043 μm, n=44). At a suitable cell and probe concentration and under standard conditions, the λmax shift of diS‐C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

Electron microscopy of DNA replication in 3-D: Evidence for similar-sized replication foci throughout S-phase†

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin‐16‐deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S‐phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid‐ and late‐S‐phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early‐S‐phase. Pulse‐chase experiments demonstrate that the RS of early‐S‐phase are maintained when chased through S‐phase and into the next cell generation. Stereologic analysis demonstrates that the relative number of smaller sized foci present at a given time remains constant from early through mid‐S‐phase with only a slight decrease in late‐S‐phase. 3‐D reconstruction of serial sections reveals a network‐like organization of the RS in early‐S‐phase and confirms that numerous smaller‐sized replication foci comprise the larger RS characteristic of late‐S‐phase.

Spatio‐temporal dynamics at rDNA foci: Global switching between DNA replication and transcription

We have investigated the in situ organization of ribosomal gene (rDNA) transcription and replication in HeLa cells. Fluorescence in situ hybridization (FISH) revealed numerous rDNA foci in the nucleolus. Each rDNA focus corresponds to a higher order chromatin domain containing multiple ribosomal genes. Multi‐channel labeling experiments indicated that, in the majority of cells, all the rDNA foci were active in transcription as demonstrated by co‐localization with signals to transcription and fibrillarin, a protein involved in ribosomal RNA processing. In some cells, however, a small portion of the rDNA foci did not overlap with signals to transcription and fibrillarin. Labeling for DNA replication revealed that those rDNA foci inactive in transcription were restricted to the S‐phase of the cell cycle and were replicated predominantly from mid to late S‐phase. Electron microscopic analysis localized the nucleolar transcription, replication, and fibrillarin signals to the dense fibrillar components of the nucleolus and at the borders of the fibrillar centers. We propose that the rDNA foci are the functional units for coordinating replication and transcription of the rRNA genes in space and time. This involves a global switching mechanism, active from mid to late S‐phase, for turning off transcription and turning on replication at individual rDNA foci. Once all the rRNA genes at individual foci are replicated, these higher order chromatin domains are reprogrammed for transcription.

Monitoring of membrane potential changes inSaccharomyces cerevisiae by diS-C3(3) fluorescence

Attempt was made to measure the membrane potential in yeast cells by the electrochromic probe di-4-ANEPPS (dibutylaminonaphthylethylene pyridinium propyl sulfonate) which has previously been used for measuring action potentials in neurons [1, 2]. This probe is believed to provide fluorescent response to changes in transmembrane electric field in nanoseconds by changing its fluorescence intensity due to an underlying wavelength shift of emission maximum. The requirements for successful measurement are (1) defined dependence of the fluorescence response on change in membrane potential, (2) low probe toxicity at the concentrations used, (3) reproducible incorporation of the probe solely into the outer layer of the membrane lipid bilayer (incorporation into the inner layer would give rise to two probe pools whose respective responses to membrane potential changes would be mutually opposite, hampering the measurement), (4) absence of any penetration of the probe into the cell. The fluorescence of the electrochromic probe was measured in suspensions of intact cells, protoplasts and phosphatidylserine/phosphatidylcholine (20/80) liposomes. Tentative adjustment of membrane potential was done by incubating the samples in 3.5-150 mmol/L KC1, the overall molarity being adjusted in each case to 150 mmol/L by choline chloride. The effect of nonuniform staining of individual cells on the excitation spectrum of the probe was eliminated by measuring the ratio of fluorescence intensities at excitation wavelengths of 450 and 530 nm [3, 4]. The measurements showed that (1) the probe responds to membrane potential change by an electrochromic shift; (2) the cell wall hampers the penetration of the probe to the plasma membrane of yeast cells; (3) the actual equilibration of the probe in cell suspension should take 10-15 min but in fact the staining intensity keeps on rising even at longer intervals; (4) this is due to the fact that the probe is not incorporated solely into the plasma membrane but spreads gradually into the cells and liposomes, which causes persistent variations in fluorescence response to membrane potential change. This penetration brings about a fluorescence change mimicking a decrease in membrane potential, i.e. membrane depolarization. The probe is therefore suitable for monitoring membrane potential in yeast only over short periods of time (up to 30 min). Longer monitoring will require either a modified staining protocol or derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

In situ fluorescence visualization of bromouridine incorporated into newly transcribed nucleolar RNA.

Bromouridine-triphosphate is commonly used for in situ immunocytochemical labeling of newly synthesized RNA in living cells. While extranucleolar transcripts do not require special conditions for visualization, special treatment prior to fixation (e.g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in previous studies. We show in the present investigation that bromouridine-triphosphate is efficiently used by both extranucleolar and nucleolar RNA polymerases in living cultured cells. The failure to detect incorporated bromouridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluorescence signals within nucleoli can be routinely found.

Speed of Accumulation of the Membrane Potential Indicator diS-C3(3) in Yeast Cells

The carbocyanine dye diS-C3(3) (3,3’- dipropylthiacarbocyanine iodide), whose the steady-state fluorescence spectra were measured in yeast cell suspensions, belongs to the group of slow (Nernstian, or redistribution) dyes which report on membrane potential by their voltage-sensitive partition between the extracellular medium and the cytosol.1–3 Since the emission spectrum shifts and the quantum yield of fluorescence increases upon binding of the dye in the cell, two fluorescence parameters,4–5 the wavelength of emission maximum and the intensity of fluorescence at this wavelength, were used to monitor the redistribution of the dye inside/outside the cells. To demonstrate that the dye accumulation in cells, as revealed by observed fluorescence changes, is actually membrane-potential-driven we used the uncoupler CCCP (carbonyl cyanide 3-chlorophenylhydrazone) which drastically increases membrane permeability for protons and depolarizes the cell membrane.6

Study of membrane potential changes of yeast cells caused by killer toxin K1.

transport of acetic acid and other weak organic acids in the strain Zygosaccharomyces bailii IGC 1307 and its regulation by several carbon sources. When transport was measured in cells grown in a medium with glucose or fructose, with labelled acetic acid at concentrations from 0.1 to 12 mmol/L, pH 5.0, the Lineweaver-Burk plot of the initial uptake rates was linear and consistent with a Michaelis-Menten kinetics. Acetic acid uptake (pH 5.0) was accompanied by the disappearance of extracellular protons, the uptake rates of which also followed Michaelis-Menten kinetics as a function of the acid concentration. The results indicated a proton symport of the anion form of the acid. Transport of labelled acetic acid at pH 5.0 was accumulative, the accumulation ratio in terms of free acid being about 50. Furthermore, the accumulated acid flew out of the cells after the addition of cold acetic acid as well as of benzoic, sorbic or pentanoic acids. The results suggested that all these acids may use the same carrier. Accordingly, benzoic, sorbic or pentanoic acids were competitive inhibitors of the acetic acid transport at pH 5.0. As expected, their transport was also associated with proton uptake that followed Michaelis-Menten kinetics. Apparently, neither propionic acid nor lactic acid used this transport system since they were not competitive inhibitors of acetic acid transport. Furthermore, when either acid was added to a cell suspension no transient external alkalinization indicative of proton uptake was observed. Cells of Z. bailii grown in a medium with either acetic acid or ethanol as the carbon and energy source were also analyzed for their capacity to transport acetic acid and the other weak organic acids mentioned above. The data indicated that, under these growth conditions, a mediated transport system for acetic was present and probably an acetate proton symport was again involved. However, the carrier appeared to be less specific, being able to accept not only benzoic, sorbic and pentanoic acids but also propionic and formic acids.

Cell volume changes as revealed by fluorescence microscopy: Global vs local approaches

BACKGROUND Several techniques for cell volume measurement using fluorescence microscopy have been established to date. In this study, we compare the performance of three different approaches which allow for estimations of the cell volume changes in biological samples containing individual fluorescently labeled cells either in culture or in the tissue context. The specific requirements, limitations and advantages of individual approaches are discussed. NEW METHOD Global morphometric data are quantitatively compared with local information about the overall cell volume, represented by the concentration of a mobile fluorophore accumulated within the monitored cell. RESULTS Volume changes induced by variations in the extracellular osmolarity in murine fibroblasts and astrocytes either in the culture or in the acute brain slices were registered by the three- and two-dimensional morphometries and by local fluorescence intensity measurements. The performance of the latter approach was verified using FRAP assessment of the fluorophore mobility. Significantly lower amplitudes of the cortical astrocytes swelling were detected by three-dimensional morphometry, when compared to the other two approaches. Consequently, it failed to detect temperature-induced cell volume changes. COMPARISON WITH EXISTING METHOD(S) The three most popular methods of cell volume measurement are compared to each other in this study. CONCLUSIONS We show that the effectivity of global morphometry-based volumetric approaches drops with the increasing cell shape complexity or in the tissue context. In contrast to this, the performance of local fluorescence intensity monitoring, which is also fully capable of reflecting the instant cell volume variations remains stable, independent of the system used and application.