Vladana Vukojević

Study of molecular events in cells by fluorescence correlation spectroscopy

Abstract.To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

Protein oligomerization induced by oleic acid at the solid–liquid interface – equine lysozyme cytotoxic complexes

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self‐assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid–liquid interface within an ion‐exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein α‐lactalbumin, known as humanα‐lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4–30 lysozyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8‐anilinonaphthalene‐1‐sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin‐T dye, shows a spherical morphology, assembles into ring‐shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well‐populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

Quantitative single-molecule imaging by confocal laser scanning microscopy

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Quantitative study of synthetic Hox transcription factor–DNA interactions in live cells

Transcription factor–DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein–DNA interactions in live cells that enables us to “titrate” the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide–DNA interactions, and estimated their corresponding dissociation constants (Kd) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain–DNA interactions in live cells using numerical simulations.

Genomic DNA Hypomethylation by Histone Deacetylase Inhibition Implicates DNMT1 Nuclear Dynamics

ABSTRACT Histone deacetylase inhibitors (HDACi) are promising antitumor drugs acting through reactivation of silenced tumor suppressor genes. Several HDACi are currently in clinical trials both for hematological and solid tissue malignancies. Cooperative action of HDACi and DNA methylation inhibitors (DNMTi) has been reported, making combined treatment an attractive choice for cancer therapy. There is some evidence that synergistic effects of HDACi and DNMTi are achieved by their action on common targets, including DNA methyltransferase 1 (DNMT1). To further analyze this interaction, we investigated the effect of the HDACi trichostatin A on global and gene-specific DNA methylation and applied methods with single molecule sensitivity, confocal laser scanning microscopy with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), to study its effect on the nuclear dynamics of DNMT1 in live cells. Our data show that trichostatin A treatment reduces global DNA methylation and the DNMT1 protein level and alters DNMT1 nuclear dynamics and interactions with chromatin. The mechanisms underlying these effects are apparently distinct from the mechanisms of action of the DNMT inhibitor 5-azacytidine. Our study sheds light on the molecular mechanisms underlying the synergistic action of HDACi and DNMTi and may also help to define improved policies for cancer treatment.

Membrane leakage induced by dynorphins

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors and induce non‐opioid excitotoxic effects. Here we show that big dynorphin and dynorphin A, but not dynorphin B, cause leakage effects in large unilamellar phospholipid vesicles (LUVs). The effects parallel the previously studied potency of dynorphins to translocate through biological membranes. Calcein leakage caused by dynorphin A from LUVs with varying POPG/POPC molar ratios was promoted by higher phospholipid headgroup charges, suggesting that electrostatic interactions are important for the effects. A possibility that dynorphins generate non‐opioid excitatory effects by inducing perturbations in the lipid bilayer of the plasma membrane is discussed.

Localization of cholesterol, amyloid and glia in Alzheimer’s disease transgenic mouse brain tissue using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and immunofluorescence imaging

The spatial distributions of lipids, amyloid-beta deposits, markers of neurons and glial cells were imaged, at submicrometer lateral resolution, in brain structures of a mouse model of Alzheimer’s disease using a new methodology that combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) and confocal fluorescence microscopy. The technology, which enabled us to simultaneously image the lipid and glial cell distributions in Tg2576 mouse brain structures, revealed micrometer-sized cholesterol accumulations in hippocampal regions undergoing amyloid-beta deposition. Such cholesterol granules were either associated with individual amyloid deposits or spread over entire regions undergoing amyloidogenesis. Subsequent immunohistochemical analysis of the same brain regions showed increased microglial and astrocytic immunoreactivity associated with the amyloid deposits, as expected from previous studies, but did not reveal any particular astrocytic or microglial feature correlated with cholesterol granulation. However, dystrophic neurites as well as presynaptic vesicles presented a distribution similar to that of cholesterol granules in regions undergoing amyloid-beta accumulation, thus indicating that these neuronal endpoints may retain cholesterol in areas with lesions. In conclusion, the present study provides evidence for an altered cholesterol distribution near amyloid deposits that would have been missed by several other lipid analysis methods, and opens for the possibility to study in detail the putative liaison between lipid environment and protein structure and function in Alzheimer’s disease.

Lipoprotein complex of equine lysozyme with oleic acid (ELOA) interactions with the plasma membrane of live cells.

Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying PC12 cell death caused by ELOA oligomers. ELOA, a lipoprotein complex formed by equine lysozyme (EL) and oleic acid (OA), induces cell death in all tested cell lines, but the actual mechanism of its action is not known. We have used methods with single-molecule sensitivity, fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and confocal laser scanning microscopy (CLSM) imaging by avalanche photodiodes (APD), so-called APD imaging, to study ELOA interactions with the plasma membrane in live PC12 cells. We detected ELOA accumulation in the cell surroundings, observed ELOA interactions with the plasma membrane, and local changes in plasma membrane lipid dynamics in the vicinity of ELOA complexes. These interactions resulted in plasma membrane rupture, followed by rapid influx and distribution of ELOA inside the already dead cell. In order to probe the ELOA-plasma membrane interaction sites at the molecular and atomic levels, the ELOA complexes were further studied by photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, nuclear magnetic resonance (NMR) and atomic force microscopy (AFM). We observed a novel mechanism of oligomer toxicity-cell death induced by continuous disturbance of the plasma membrane, eventually causing permanent plasma membrane damage and identified the sites in ELOA that are potentially involved in the interactions with the plasma membrane.

Determination of Cl–, Br–, I–, Mn2+, malonic acid and quercetin by perturbation of a non-equilibrium stationary state in the Bray–Liebhafsky reaction

A new method applying a non-linear chemical system under conditions far from thermodynamic equilibrium in microvolume/microconcentration quantitative analysis is described. The chemical system used as a matrix is the Bray–Liebhafsky reaction in a non-equilibrium stationary state close to a bifurcation point. The method is based on monitoring the response of this system to perturbations by Cl–, Br–, I–, Mn2+, malonic acid and quercetin analyte solutions, which are followed potentiometrically either by an Ag+/S2– ion-sensitive or by a Pt electrode. A linear response of the potential shift versus the logarithm of the analyte concentrations is found in the following ranges: 1.3 × 10–6 mol dm–3 ≤ [Cl–] ≤ 1.6 × 10–4 mol dm–3, 1.0 × 10–6 mol dm–3 ≤ [Br–] ≤ 8.3 × 10–5 mol dm–3, 2.0 × 10–6 mol dm–3 ≤ [I–] ≤ 1.0 × 10–4 mol dm–3, 8.4 × 10–7 mol dm–3 ≤ [Mn2+] ≤ 8.3 × 10–5 mol dm–3, 3.8 × 10–7 mol dm–3 ≤ [malonic acid] ≤ 2.1 × 10–5 mol dm–3 and 1.5 × 10–8 mol dm–3 ≤ [quercetin] ≤ 3.7 × 10–5 mol dm–3. Under the investigated conditions an improved detection limit for all halides tested is obtained. Unknown concentrations of the analytes can be determined from a standard series of calibration curves to an accuracy within ±5%. In addition, the application of potentiometric measurements in microvolume/microconcentration quantitative analysis is diversified.

Telomerase-Dependent and Independent Telomere Maintenance and its Clinical Implications in Medullary Thyroid Carcinoma

Context: Telomere maintenance via telomerase activation and the alternative lengthening of telomeres (ALT) mechanism was assessed in medullary thyroid carcinoma. Setting and Design: In total, 42 medullary thyroid carcinomas (MTC) were studied including 24 rearranged during transfection (RET)- mutated cases. Relative telomerase reverse transcriptase (TERT) expression, splice forms, and telomere length were determined by PCR-based methods, and telomerase activity by ELISA. The ALT mechanism was detected by Southern blot analysis and immunofluorescence. Results: TERT expression and telomerase activity were detected in 21/42 tumors (50%), and was independent of the common somatic M918T RET mutation. Mean telomere length was shorter in MTCs compared with thyroids. Telomerase activation was associated with large tumor size (P = .027), advanced clinical stage (P = .0001), and short survival (P = .0001). Full-length TERT and the α− and β−-deletion forms were revealed, and the full-length form was associated with short survival (P = .04). A subset of cases without telomerase activation showed involvement of the ALT mechanism, which was associated with a low MIB-1 proliferation index (P = .024). Conclusions: Stabilization of telomeres by telomerase activation occurs in half of the MTCs and by the ALT mechanism in a subset of cases. Telomerase activation may be used as an additional prognostic marker in medullary thyroid carcinoma.