Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice

There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance. The Agouti viable yellow (A^vy) allele is the best-studied example of this phenomenon in mice. The A^vy allele is the result of a retrotransposon insertion upstream of the Agouti gene. Expression at this locus is controlled by the long terminal repeat (LTR) of the retrotransposon, and expression results in a yellow coat and correlates with hypomethylation of the LTR. Isogenic mice display variable expressivity, resulting in mice with a range of coat colours, from yellow through to agouti. Agouti mice have a methylated LTR. The locus displays epigenetic inheritance following maternal but not paternal transmission; yellow mothers produce more yellow offspring than agouti mothers. We have analysed the DNA methylation in mature gametes, zygotes, and blastocysts and found that the paternally and maternally inherited alleles are treated differently. The paternally inherited allele is demethylated rapidly, and the maternal allele is demethylated more slowly, in a manner similar to that of nonimprinted single-copy genes. Interestingly, following maternal transmission of the allele, there is no DNA methylation in the blastocyst, suggesting that DNA methylation is not the inherited mark. We have independent support for this conclusion from studies that do not involve direct analysis of DNA methylation. Haplo-insufficiency for Mel18, a polycomb group protein, introduces epigenetic inheritance at a paternally derived A^vy allele, and the pedigrees reveal that this occurs after zygotic genome activation and, therefore, despite the rapid demethylation of the locus.     

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.     

Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F‐actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax‐7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination. J. Cell. Physiol. 223: 376–383, 2010. © 2010 Wiley‐Liss, Inc.     

Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor binding site of the protein

Inducible costimulator (ICOS) ligand (ICOSL), a B7-related transmembrane glycoprotein with extracellular IgV and IgC domains, binds to ICOS on activated T cells and delivers a positive costimulatory signal for optimal T cell function. Toward determining the structural features of ICOSL crucial for its costimulatory function, the present study shows that ICOSL displays a marked oligomerization potential, resembling more like B7-1 than B7-2. Use of ICOSL constructs lacking either the IgC or IgV domain demonstrates that receptor binding is mediated solely by the IgV domain but requires the IgC domain for maintaining the structural integrity of the protein. To map further the receptor recognition surface on ICOSL, a homology-based protein structure model of the ICOS:ICOSL complex was constructed. Based on predictions from the model, a series of mutations were generated targeting the potential receptor binding surface on ICOSL, and the mutants were tested for their biological function in terms of ICOS binding and T cell costimulation ability. The results provide experimental validation of the model and show that the receptor binding site on ICOSL is constituted chiefly by aromatic/hydrophobic residues. Critical ICOSL residues essential for ICOS binding map to the GFCC'C' beta-sheet face of the IgV domain and approximately overlap with the B7-1/B7-2 motif(s) that recognize CD28/CTLA-4. Altogether, similar structural features of ICOSL and B7 isoforms suggest a close evolutionary relationship between these costimulatory ligands, yet differences at the same time explain their unique specificity for the cognate binding partners, ICOS and CD28/CTLA-4, respectively.     

Differential T cell-mediated regulation of CD23 (Fc epsilonRII) in B cells and follicular dendritic cells

Differences in murine follicular dendritic cells (FDC)-CD23 expression under Th1 vs Th2 conditions prompted the hypothesis that T cells help regulate the phenotype of FDCs. FDCs express CD40, suggesting that T cell-CD40L and lymphokines may be involved in regulating FDC-CD23. To test this, highly enriched FDCs were incubated with CD40L trimer or anti-CD40 to mimic T cell signaling in the presence of IFN-gamma or IL-4. Surface expression of CD23 was determined by flow cytometry, whereas mRNA levels of CD23 and its isoforms CD23a and CD23b were independently measured by quantitative PCR. When FDCs were incubated with either CD40L trimer or agonistic anti-CD40 Ab, the expression of FDC-CD23 was increased both at the mRNA and protein levels. Moreover, engagement of FDC-CD40 enhanced mRNA levels for both CD23a and CD23b isoforms. In addition, IFN-gamma substantially enhanced CD23a and CD23b mRNA levels in CD40-stimulated FDCs. Curiously, IL-4 could also up-regulate FDC-CD23a but not -CD23b. Anti-IFN-gamma dramatically inhibited FDC-CD23 in mice immunized with CFA, whereas anti-IL-4 had only a modest inhibitory effect. In contrast with FDCs, IFN-gamma inhibited surface expression of murine B cell-CD23 as well as mRNA for B cell CD23a and -CD23b, whereas IL-4 dramatically enhanced message for both isoforms as well as protein expression. In short, CD23 was regulated very differently in FDCs and B cells. Previous studies suggest that high levels of FDC-CD23 inhibit IgE production, and this IFN-gamma and CD40L-mediated up-regulation of FDC-CD23 may explain, at least in part, why Th1 responses are associated with low IgE responses in vivo.     

Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

Membrane composition of jetted lipid vesicles: a Raman spectroscopy study

Microfluidic jetting is a promising method to produce giant unilamellar phospholipid vesicles for mimicking living cells in biomedical studies. We have investigated the chemical composition of membranes of vesicles prepared using this approach by means of Raman scattering spectroscopy. The membranes of all jetted vesicles are found to contain residuals of the organic solvent decane used in the preparation of the initial planar membrane. The decane inclusions are randomly distributed over the vesicle surface area and vary in thickness from a few to several tens of nanometers. Our findings point out that the membrane properties of jetted vesicles may differ considerably from those of vesicles prepared by other methods and from those of living cells. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl₂ or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.     

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.