Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

Nucleolar rDNA folds into condensed foci with a specific combination of epigenetic marks.

Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.

Evaluation of lipid peroxidation by the analysis of volatile aldehydes in the headspace of synthetic membranes using Selected Ion Flow Tube Mass Spectrometry, SIFT-MS.

RATIONALE: Oxidative stress of cell membranes leads to a number of pathological processes associated with some diseases and is accompanied by the release of volatile aldehydes, which, potentially, can be used as biomarkers. Thus, the aim was to investigate peroxidation of defined synthetic membranes by direct quantitative analysis of volatile aldehydes. METHODS: The concentration spectra of volatile compounds present in the headspace of synthetic membranes under peroxidation stress and following mechanical stress due to sonication were obtained using solid phase microextraction (SPME) in combination with Gas Chromatography Mass Spectrometry (SPME/GC/MS) and Selected Ion Flow Tube Mass Spectrometry (SIFT-MS). The focus was on the direct, real time quantification of volatile aldehydes. In addition, the total aldehydes in the aqueous membrane suspensions were quantified using the TBARS method. RESULTS: Propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde were detected and quantified in the humid headspace of the media containing the synthetic membranes following peroxidation. The composition and concentration of these saturated aldehydes strongly depend on the unsaturated fatty acids representation in the liposomes. Some protective effect of cholesterol was observed especially for membranes peroxidised by Fenton reagents and after application of a mechanical stress. CONCLUSIONS: This study demonstrates that peroxidation of model synthetic membranes in vitro can be tracked in real time using direct quantification by SIFT-MS of several specific aldehydes in the headspace of the membrane suspensions. Cholesterol plays an important role in retaining membrane structure and can indirectly protect membranes from lipid peroxidation.

Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH4.

Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.

Di- and tri-oxalkyl derivatives of a boron dipyrromethene (BODIPY) rotor dye in lipid bilayers.

The environment-sensitive fluorescent probes provide excellent tools for studying membranes in their native state. We have modified the BODIPY-based fluorescent molecular rotor by increasing the number of alkyl moieties from one to two or three to achieve a more defined and deeper positioning of the probe in membranes. Detailed characterisation of fluorescence properties and localisation/orientation of probes was performed using a variety of fluorescence techniques and model membranes composed of different lipids. As expected, additional alkyls attached to the fluorophore moiety led to a deeper and more defined localisation of the probe in the lipid bilayer. The results strongly indicate that fluorescence properties of such probes are influenced not only by lipid packing but also by the orientation of the probe in membranes. The orientation of rotors studied herein was significantly altered by changes in the lipid composition of membranes. Our observations demonstrate the limits of BODIPY-based molecular rotors as environmental sensors in cellular membranes with complex lipid composition. The results presented herein also underline the importance of the detailed characterisation of fluorescent membrane dyes and provide a guide for future testing.

Multi-Level Approach for Comprehensive Enamel Phenotyping.

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.

Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites.

Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Roughness of Transmembrane Helices Reduces Lipid Membrane Dynamics.

The dynamics of cellular membranes is primarily determined by lipid species forming a bilayer. Proteins are considered mainly as effector molecules of diverse cellular processes. In addition to large assemblies of proteins, which were found to influence properties of fluid membranes, biological membranes are densely populated by small, highly mobile proteins. However, little is known about the effect of such proteins on the dynamics of membranes. Using synthetic peptides, we demonstrate that transmembrane helices interfere with the mobility of membrane components by trapping lipid acyl chains on their rough surfaces. The effect is more pronounced in the presence of cholesterol, which segregates from the rough surface of helical peptides. This may contribute to the formation or stabilization of membrane heterogeneities. Since roughness is a general property of helical transmembrane segments, our results suggest that, independent of their size or cytoskeleton linkage, integral membrane proteins affect local membrane dynamics and organization.