Reiner Bleher

Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

Fertilization of a mammalian egg induces a series of ‘zinc sparks’ that are necessary for inducing the egg-to-embryo transition. Despite the importance of these zinc efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches to resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy dispersive spectroscopy, X-ray fluorescence microscopy, and 3D elemental tomography for high resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 106 zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.

A Directional Entropic Force Approach to Assemble Anisotropic Nanoparticles into Superlattices

Abstract : Not touching but sticking: By using cationic surfactant micelles as depletants, a directional entropic force approach (DEFA) assembles anisotropic nanoparticles into superlattices in solution. The micelles induce the face-to-face stacking of the nanoparticles to maximize the systems entropy. The shape of the nanoparticles determines the symmetry of the superlattice, the interparticle spacing is determined by the charged surfactant.

Thermoresponsive magnetic hydrogels as theranostic nanoconstructs.

We report the development of thermoresponsive magnetic hydrogels based on poly(N-isopropylacrylamide) encapsulation of Fe3O4 magnetic nanostructures (MNS). In particular, we examined the effects of hydrogels encapsulated with poly-ethylene glycol (PEG) and polyhedral oligomeric silsesquioxane (POSS) surface modified Fe3O4 MNS on magnetic resonance (MR) T2 (transverse spin relaxation) contrast enhancement and associated delivery efficacy of absorbed therapeutic cargo. The microstructural characterization reveal the regular spherical shape and size (∼200 nm) of the hydrogels with elevated hydrophilic to hydrophobic transition temperature (∼40 °C) characterized by LCST (lower critical solution temperature) due to the presence of encapsulated MNS. The hydrogel-MNS (HGMNS) system encapsulated with PEG functionalized Fe3O4 of 12 nm size (HGMNS-PEG-12) exhibited relaxivity rate (r2) of 173 mM–1s–1 compared to 129 mM–1s–1 obtained for hydrogel-MNS system encapsulated with POSS functionalized Fe3O4 (HGMNS-POSS-12) of the same size. Further studies with HGMNS-PEG-12 with absorbed drug doxorubicin (DOX) reveals approximately two-fold enhance in release during 1 h RF (radio-frequency) field exposure followed by 24 h incubation at 37 °C. Quantitatively, it is 2.1 μg mg–1 (DOX/HGMNS) DOX release with RF exposure while only 0.9 μg mg–1 release without RF exposure for the same period of incubation. Such enhanced release of therapeutic cargo is attributed to micro-environmental heating in the surroundings of MNS as well as magneto-mechanical vibrations under high frequency RF inside hydrogels. Similarly, RF-induced in vitro localized drug delivery studies with HeLa cell lines for HGMNS-PEG-12 resulted in more than 80% cell death with RF field exposures for 1 h. We therefore believe that magnetic hydrogel system has in vivo theranostic potential given high MR contrast enhancement from encapsulated MNS and RF-induced localized therapeutic delivery in one nanoconstruct.

Cytometric, morphologic and enzymatic characterisation of haemocytes in Anodonta cygnea

The haemocytes in bivalve mussels are involved in many processes such as lesion repair, shell repair, elimination of small particles and toxic substances. In Anodonta cygnea there are two categories of haemolymph cells, the granulocytes and hyalinocytes. Two groups of cells were identified by flow cytometry and morphological studies: one with larger size and granularity representing 75%, and another group of cells (25%) which were approximately half the size. The cytochemical reactions showed peroxidase activity in the larger cells and a weak prophenoloxidase activity in the smaller cells. These characteristics suggest that the most common haemocytes are granulocytes and hyalinocytes are less common. Enzymatic studies showed clear activities of few enzymes in different compartments of the mantle. Both haemocytes presented significant variations for alpha-manosidase and beta-glucurosidase activities depending on the acid or alkaline pH. Almost all were sensitive to the pH changes, mainly the beta-galactosidase in the haemolymph plasma. On the contrary, the same enzymatic analysis in the extrapallial elements showed more stabilised activities. The simulation of acidic and alkaline condition with the observation of significant morphological and enzymatic activity changes, allow us to speculate some functional role, mainly in the haemolymph elements. The granulocytes may be speculated to have intense involvement in the digestion of small residues with the formation of calcareous stores while the hyalinocytes are more responsible for the elimination of soluble cytotoxic compounds.

Multiple Correlative Immunolabeling for Light and Electron Microscopy Using Fluorophores and Colloidal Metal Particles

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscoopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric miscroscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and α-actinin bands of skeletal muscle tissue and also actin and α-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM. (J Histochem Cytochem 55: 983–990, 2007)

Imaging and elemental mapping of biological specimens with a dual-EDS dedicated scanning transmission electron microscope

A dedicated analytical scanning transmission electron microscope (STEM) with dual energy dispersive spectroscopy (EDS) detectors has been designed for complementary high performance imaging as well as high sensitivity elemental analysis and mapping of biological structures. The performance of this new design, based on a Hitachi HD-2300A model, was evaluated using a variety of biological specimens. With three imaging detectors, both the surface and internal structure of cells can be examined simultaneously. The whole-cell elemental mapping, especially of heavier metal species that have low cross-section for electron energy loss spectroscopy (EELS), can be faithfully obtained. Optimization of STEM imaging conditions is applied to thick sections as well as thin sections of biological cells under low-dose conditions at room and cryogenic temperatures. Such multimodal capabilities applied to soft/biological structures usher a new era for analytical studies in biological systems.

Expression and polarity reversal of V-type H+-ATPase during the mineralization-demineralization cycle in Porcellio scaber sternal epithelial cells

SUMMARY The formation and resorption of CaCO3 by epithelial cell layers require epithelial transport of protons. We used the anterior sternal epithelium of the terrestrial isopod Porcellio scaber as a model to study the expression pattern and immunolocalization of a V-type H+-ATPase during the mineralization and demineralization of intermittent CaCO3 deposits. Semiquantitative RT-PCR revealed that the expression of the V-type H+-ATPase increases from non Ca2+-transporting control stages to the stages of CaCO3 deposit formation and resorption. In the Ca2+-transporting stages the expression was larger in the anterior than in the posterior sternal epithelium, which is not involved in deposit formation and transports just moderate amounts of CaCO3. Immunocytochemistry of the B-subunit of the V-type H+-ATPase in the anterior sternal epithelium reveals an increase in the abundance of the protein within the basolateral membrane, from undetectable to strong signals in the control stage to the stages of CaCO3 deposit formation, respectively. From the stage of CaCO3 deposit formation to that of CaCO3 resorption the signal decreased within the basolateral plasma membrane and increased within the apical plasma membrane. For the first time the results indicate a contribution of a V-type H+-ATPase to CaCO3 deposition and a reversal of its polarity from the basolateral to the apical plasma membrane compartment within the same cells.

Nucleo-cytoplasmic translocation of histone H1 during the HeLa cell cycle.

Abstract.The distribution of unphosphorylated and phosphorylated isoforms of linker histone H1 protein was examined during the cell cycle of HeLa cells by quantitative light and electron microscopic immunocytochemistry. Immunolabeling with a monoclonal antibody directed against the globular domain of H1 (anti-H1), which recognized predominantly unphosphorylated H1, and a polyclonal antibody directed against hyperphosphorylated H1 (anti-H1P) revealed that: (1) H1 immunolabeling was lowest at the start of S phase (SS), and then increased progressively during the middle (Si) to end of S phase (Se), mitosis (M) and telophase (T) to reach the highest level in G1 phase, at which time there was a sudden reduction in H1 immunolabeling before the start of S phase; (2) H1P immunolabeling paralleled this progressive increase, but only until M phase, after which it abruptly disappeared and was virtually absent in G1; (3) H1P immunolabeling in S and M phase was found on both nuclear chromatin or chromosomes and in the cytoplasm, while H1 immunolabeling was found only on nuclear chromatin or chromosomes where it was predominantly localized on condensed chromatin. Our study indicates that H1 dissociates from the DNA to a large extent during replication and chromosome condensation, but not in interphase when cells are transcriptionally active.

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice.

Ribosomes in the squid giant axon

Ribosome clusters, referred to as endoaxoplasmic plaques, were documented and quantitatively analyzed in the squid giant axon at the light and electron microscopic levels. The methods included nonspecific high affinity fluorescence staining of RNA by YOYO-1, specific immunofluorescence labeling of ribosomal RNA, electron energy loss spectroscopic mapping of ribosomal phosphorus, and conventional transmission electron microscopy. The endoaxoplasmic plaques were sharply defined, oval in shape, and less than 2 microm in diameter. While they were very numerous in the postsynaptic axonal area of the giant synapse, the frequency of occurrence was much lower in the peripheral giant axon, with a density of about 1 plaque/1000 microm3. Their distribution was random within axoplasm, with no preferential localization near the membrane. The several thousand ribosomes in a plaque usually were not membrane bound, but vesicular structures were observed in or near plaques; plaques were often surrounded by mitochondria. We conclude that ribosomes, a requisite machinery for protein synthesis, are present in the squid giant axon in discrete configurations.