An improved method for nanogold in situ hybridization visualized with environmental scanning electron microscopy

This paper is not subject to U.S. copyright. The definitive version was published in Journal of Microscopy 236 (2009): 5-10, doi:10.1111/j.1365-2818.2009.03207.x.An important goal in geomicrobiology is the identification of microbes associated with specific mineral surfaces. Yet, simultaneously collecting phylogenetic and mineral information remains methodologically challenging. Recently, whole-cell in situ hybridization techniques using oligonucleotide rRNA probes bound to nanogold particles have been used to detect microbes with scanning electron microscopy (SEM) for geomicrobiological applications (Gerard et al., 2005; Kenzaka et al., 2005). These techniques rely on backscattered electron images or energy dispersive X-ray spectroscopy to map the presence and distribution of nanogold, and to identify areas of rRNA hybridization within cells and on mineral surfaces. Although these nanogold hybridization techniques have been successful for pure cultures of Bacteria and Archaea (Gerard et al., 2005) and for natural microbial communities associated with river sediment particles (Kenzaka et al., 2005) and basalt surfaces (Menez et al., 2007), their application to other metal-rich geomicrobiological systems is problematic. First, metallic substrates and surfaces can obscure detection of nanogold-labelled cells imaged with backscattered electron microscopy (Richards et al., 2001). Second, metallic surfaces can interfere with the hybridization reaction by causing non-specific precipitation of nanogold (Humbel et al., 1995; Weipoltshammer et al., 2000). Because many geomicrobiological systems of interest have a high concentration of metal substrates (i.e. hydrothermal vents, acid mine drainage) a new technique is needed to identify microbes found in these types of environments. In this work, we present a new nanogold in situ hybridization method that increases the concentration of nanogold probes bound to rRNA targets within the cell and makes individual hybridization events directly visible with secondary electron SEM imaging.This research was supported by National Science Foundation MRI, Ecology and Microbial Observatories programmes (MCB-0406999 and MCB-0534879, to RMH, PAH and SMS); the U.S. DOE NABIR programme; the U.S. EPA STAR programme

c-erbB-3: a nuclear protein in mammary epithelial cells

c-erbB receptors are usually located in cell membranes and are activated by extracellular binding of EGF-like growth factors. Unexpectedly, using immunofluorescence we found high levels of c-erbB-3 within the nuclei of MTSV1-7 immortalized nonmalignant human mammary epithelial cells. Nuclear localization was mediated by the COOH terminus of c-erbB-3, and a nuclear localization signal was identified by site-directed mutagenesis and by transfer of the signal to chicken pyruvate kinase. A nuclear export inhibitor caused accumulation of c-erbB-3 in the nuclei of other mammary epithelial cell lines as demonstrated by immunofluorescence and biochemical cell fractionation, suggesting that c-erbB-3 shuttles between nuclear and nonnuclear compartments in these cells. Growth of MTSV1-7 on permeable filters induced epithelial polarity and concentration of c-erbB-3 within the nucleoli. However, the c-erbB-3 ligand heregulin β1 shifted c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. The subcellular localization of c-erbB-3 obviously depends on exogenous stimuli and on the stage of epithelial polarity and challenges the specific function of c-erbB-3 as a transmembrane receptor protein arguing for additional, as yet unidentified, roles of c-erbB-3 within the nucle(ol)us of mammary epithelial cells

Expression patterns of protein C inhibitor in mouse development

Proteolysis of extracellular matrix is an important requirement for embryonic development and is instrumental in processes such as morphogenesis, angiogenesis, and cell migration. Efficient remodeling requires controlled spatio-temporal expression of both the proteases and their inhibitors. Protein C inhibitor (PCI) effectively blocks a range of serine proteases, and recently has been suggested to play a role in cell differentiation and angiogenesis. In this study, we mapped the expression pattern of PCI throughout mouse development using in situ hybridization and immunohistochemistry. We detected a wide-spread, yet distinct expression pattern with prominent PCI levels in skin including vibrissae, and in fore- and hindgut. Further sites of PCI expression were choroid plexus of brain ventricles, heart, skeletal muscles, urogenital tract, and cartilages. A strong and stage-dependent PCI expression was observed in the developing lung. In the pseudoglandular stage, PCI expression was present in distal branching tubules whereas proximal tubules did not express PCI. Later in development, in the saccular stage, PCI expression was restricted to distal bronchioli whereas sacculi did not express PCI. PCI expression declined in postnatal stages and was not detected in adult lungs. In general, embryonic PCI expression indicates multifunctional roles of PCI during mouse development. The expression pattern of PCI during lung development suggests its possible involvement in lung morphogenesis and angiogenesis

Histochemistry and Cell Biology / Nucleolus and chromatin

The nucleolus as site of ribosome biogenesis holds a pivotal role in cell metabolism. It is composed of ribosomal DNA (rDNA), which is present as tandem arrays located in nucleolus organizer regions (NORs). In interphase cells, rDNA can be found inside and adjacent to nucleoli and the location is indicative for transcriptional activity of ribosomal genesinactive rDNA (outside) versus active one (inside). Moreover, the nucleolus itself acts as a spatial organizer of non-nucleolar chromatin. Microscopy-based approaches offer the possibility to explore the spatially distinct localization of the different DNA populations in relation to the nucleolar structure. Recent technical developments in microscopy and preparatory methods may further our understanding of the functional architecture of nucleoli. This review will attempt to summarize the current understanding of mammalian nucleolar chromatin organization as seen from a microscopists perspective.(VLID)359162

Morphology of nuclear transcription

Gene expression control is a fundamental determinant of cellular life with transcription being the most important step. The spatial nuclear arrangement of the transcription process driven by RNA polymerases II and III is nonrandomly organized in foci, which is believed to add another regulatory layer on gene expression control. RNA polymerase I transcription takes place within a specialized organelle, the nucleolus. Transcription of ribosomal RNA directly responds to metabolic requirements, which in turn is reflected in the architecture of nucleoli. It differs from that of the other polymerases with respect to the gene template organization, transcription rate, and epigenetic expression control, whereas other features are shared like the formation of DNA loops bringing genes and components of the transcription machinery in close proximity. In recent years, significant advances have been made in the understanding of the structural prerequisites of nuclear transcription, of the arrangement in the nuclear volume, and of the dynamics of these entities. Here, we compare ribosomal RNA and mRNA transcription side by side and review the current understanding focusing on structural aspects of transcription foci, of their constituents, and of the dynamical behavior of these components with respect to foci formation, disassembly, and cell cycle.(VLID)340415