DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution
Katharina Laurence Jost
Maria Cristina Cardoso
- Background: Methyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues.
Results: Using immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2−/y) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2−/y and Mecp2wt mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2−/y mice.
Conclusions: MECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2-null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.
Adaptive resistance to immunotherapy directed against p53 can be overcome by global expression of tumor-antigens in dendritic cells
- Immunotherapy of cancer utilizes dendritic cells (DCs) for antigen presentation and the induction of tumor-specific immune responses. However, the therapeutic induction of anti-tumor immunity is limited by tumor escape mechanisms. In this study, immortalized dendritic D2SC/1 cells were transduced with a mutated version of the p53 tumor suppressor gene, p53M234I, or p53C132F/E168G, which are overexpressed in MethA fibrosarcoma tumor cells. In addition, D2SC/1 cells were fused with MethA tumor cells to generate a vaccine that potentially expresses a large repertoire of tumor-antigens. Cellular vaccines were transplanted onto Balb/c mice and MethA tumor growth and anti-tumor immune responses were examined in vaccinated animals. D2SC/1-p53M234I and D2SC/1-p53C132F/E168G cells induced strong therapeutic and protective MethA tumor immunity upon transplantation in Balb/c mice. However, in a fraction of immunized mice MethA tumor growth resumed after an extended latency period. Analysis of these tumors indicated loss of p53 expression. Mice, pre-treated with fusion hybrids generated from D2SC/1 and MethA tumor cells, suppressed MethA tumor growth and averted adaptive immune escape. Polyclonal B-cell responses directed against various MethA tumor proteins could be detected in the sera of D2SC/1-MethA inoculated mice. Athymic nude mice and Balb/c mice depleted of CD4(+) or CD8(+) T-cells were not protected against MethA tumor cell growth after immunization with D2SC/1-MethA hybrids. Our results highlight a potential drawback of cancer immunotherapy by demonstrating that the induction of a specific anti-tumor response favors the acquisition of tumor phenotypes promoting immune evasion. In contrast, the application of DC/tumor cell fusion hybrids prevents adaptive immune escape by a T-cell dependent mechanism and provides a simple strategy for personalized anti-cancer treatment without the need of selectively priming the host immune system.
FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development
Daniel del Toro
Ellen Clare Border
E. Yvonne Jones
- FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.
M-free: scoring the reference bias in sub-tomogram averaging and
Achilleas S. Frangakis
- Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.
Correlative light- and electron microscopy with chemical tags
Margot P. Scheffer
Erin M. Schuman
Achilleas S. Frangakis
- Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
Chemically induced photoswitching of fluorescent probes - a general concept for super-resolution microscopy
Peter J. Verveer
- We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
Apomicts in the vegetation of Central Europe
- Summed frequency data of apomictic taxa derived from vegetation relevées from Central Europe were correlated with ecological traits – water content of soil, calcium and magnesium content of soil or water, nutrient content of soil or water, environmental dynamics, hemeroby, frequency of stress-tolerant species, salt tolerance, altitude, height, and frequency of annual taxa – of alliances of the Central Euro-pean vegetation. The partly problematic identification of taxa where apomixis is important for seed production was based on a literature review. In Central Europe, high altitudes favour apomictic taxa. Otherwise, no correlations between ecological factors attributed to the alliances of the European phyto-sociological system and frequencies of apomictic taxa in the alliances of the European phytosociological system were found. Analyses could not confirm theories on ecological preferences of apomictic taxa; e.g., that competition penalizes apomicts, apomicts have better colonizing abilities than sexual plants, or apomicts have larger ranges than their sexual relatives could not be confirmed.
What are the long-term effects of livestock grazing in steppic sandy grassland with high conservation value? Results from a 12-year field study
- To examine the necessity of livestock grazing for managing threatened sand vegetation so as to en-sure and develop its nature-conservation value, we investigated a grazing system in a model nature reserve (German upper Rhine valley) characterized by threatened steppic sandy grassland (Allio-Stipetum vegetation complex: Habitats Directive Annex I, priority type 6240, additionally Koelerion glaucae: priority type 6120). The area has been grazed by sheep and additionally by donkeys since late summer of the year 2000. We established ungrazed (Au) and grazed plots (Ag) in the Allio-Stipetum complex in a systematic grid-plot design before grazing impact started. We sampled phytosociological relevés yearly in the Au/Ag plots for 12 vegetation periods. Addi-tionally, we sampled relevés on former farmland adjacent to the Allio-Stipetum complex (systematic grid-plot design) over ten vegetation periods. These former fields (Fg) were integrated in the grazing system since the beginning of the study. A constancy table was produced for Au, Ag, Fg. To determine the portions of target species (Koelerio-Corynephoretea, Festuco-Brometea species) we calculated target-species ratios (proportion of target species in comparison with the total species number; qualita-tive or quantitative approach: TSRqual/quant). We tested the effects of grazing, year and interactions on structural and phytodiversity characteristics of the relevés by mixed linear models. The results of the long-term experiment confirmed significant beneficial effects of grazing on habi-tat-typical structure and phytodiversity: e. g. reduction of litter, support of species which are short in height (< 20 cm), reduction of taller species (> 50 cm), support of the diversity of all plant species, of non-graminoid herbs and of target plant species, reduction of Calamagrostis epigejos, positive effects on TSRqual and TSRquant values. Similar developments, e.g. for phytodiversity and Calamagrostis epigejos cover, were observed on the former fields, but there are still seed limitations and high ruderal-plant dominances. In general TSRqual/quant show remarkable increases, but do not reach the values of the Ag plots. Concerning aims of nature conservation, the habitat-typical vegetation structure and phytodiversity of the Allio-Stipetum shows an excellent development as a consequence of the used grazing manage-ment. However, occasionally small populations of target species did not increase (e.g. Koeleria glauca). The former fields show a development towards valuable sandy grassland.
Zur pflanzensoziologischen Eingliederung von Fritillaria meleagris-Beständen im Grenzbereich von Mittel- und Südosteuropa
- Im südöstlichen Österreich, im angrenzenden Westungarn und Nordostslowenien wurden insgesamt 31 Bestände mit Fritillaria meleagris pflanzensoziologisch erfasst, ausgewertet und durch Bodendaten ergänzt. In folgenden Pflanzengesellschaften konnte die Schachblume nachgewiesen werden: Pruno-Fraxinetum, Pseudostellario-Carpinetum, Galio palustris-Caricetum ripariae, Lysimachio vulgaris-Filipenduletum caricetosum acutiformis, Iridetum sibiricae, Serratulo-Festucetum commutatae, Silaetum pratensis, Festuco pratensis-Alopecuretum pratensis. Die Auswertung zeigt, dass F. meleagris im Untersuchungsgebiet eine breite phytocoenologische Amplitude hat und nicht als Charakterart einer bestimmten Assoziation aufgefasst werden kann. Alle Standorte weisen Eingriffe in den Bodenwasser-haushalt in Form von Entwässerungsmaßnahmen auf, die sich aufgrund der sand- und schluffreichen, meist hydromorphen Böden z. T. erst allmählich auf die Artenzusammensetzung der Flächen auswir-ken. Besonders die Vorkommen im Arrhenatherion sollten dringend einem Monitoring unterzogen werden, um einen weiteren Rückgang der Art erkennen und verhindern zu können.
Plant communities and environmental gradients in mires of the Ammergauer Alps (Bavaria, Germany)
- In this study we present the plant communities, their abiotic site conditions and the main environ-mental gradients underlying variation in species composition of mire sites in three montane mires in the Northern Limestone Alps. For the phytosociological description of the mires we sampled species com-position in 115 relevés. In order to characterize the site conditions of the most important plant commu-nities we measured humification as well as water table depth, pH and electrical conductivity at 46 sites during a large part of the growing season. The main environmental gradients were identified by fitting environmental vectors and smoothed surfaces onto NMDS ordination. The most frequent communities on subneutral and neutral sites were the Caricetum davallianae, the Amblystegio stellati-Caricetum dioicae and the Schoenetum ferruginei, whereas acidic sites were mainly covered by the Eriophoro-Trichophoretum cespitosi. The most important factor for floristic variation between sites was pH. How-ever, within bogs and fens, variation in species composition was mainly due to differences in water table depth.