Immunogold Labeling Electron Microscopy Research Articles

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia

X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an ∼35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP.

Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1.

The multifunctional glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a novel macrophage lactoferrin receptor1This article is part of Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Several proteins with limited cell type distribution have been shown to bind lactoferrin. However, except in the case of hepatic and intestinal cells, these have not been definitively identified and characterized. Here we report that the multifunctional glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a novel receptor for lactoferrin (Lf) in macrophages. GAPDH is a well-known moonlighting protein, and previous work from our laboratory has indicated its localization on macrophage cell surfaces, wherein it functions as a transferrin (Tf) receptor. The KDvalue for GAPDH–lactoferrin interaction was determined to be 43.8 nmol/L. Utilizing co-immunoprecipitation, immunoflorescence, and immunogold labelling electron microscopy we could demonstrate the trafficking of lactoferrin to the endosomal compartment along with GAPDH. We also found that upon iron depletion the binding of lactoferrin to macrophage cell surface is enhanced. This correlated with an increased expression of surface GAPDH, while other known lactoferrin receptors CD14 and lipoprotein receptor-related protein (LRP) were found to remain unaltered in expression levels. This suggests that upon iron depletion, cells prefer to use GAPDH to acquire lactoferrin. As GAPDH is an ubiquitously expressed molecule, its function as a receptor for lactoferrin may not be limited to macrophages.

A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.

Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was ∼100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chain-mOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFP-Flot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

A double, long polar fimbria mutant of Escherichia coli O157:H7 expresses Curli and exhibits reduced in vivo colonization.

Escherichia coli O157:H7 causes food and waterborne enteric infections that can result in hemorrhagic colitis and life-threatening hemolytic uremic syndrome. Intimate adherence of the bacteria to intestinal epithelial cells is mediated by intimin, but E. coli O157:H7 also possess several other putative adhesins, including curli and two operons that encode long polar fimbriae (Lpf). To assess the importance of Lpf for intestinal colonization, we performed competition experiments between E. coli O157:H7 and an isogenic ΔlpfA1 ΔlpfA2 double mutant in the infant rabbit model. The mutant was outcompeted in the ileum, cecum, and midcolon, suggesting that Lpf contributes to intestinal colonization. In contrast, the ΔlpfA1 ΔlpfA2 mutant showed increased adherence to colonic epithelial cells in vitro. Transmission electron microscopy revealed curli-like structures on the surface of the ΔlpfA1 ΔlpfA2 mutant, and the presence of curli was confirmed by Congo red binding, immunogold-labeling electron microscopy, immunoblotting, and quantitative real-time reverse transcription-PCR (qRT-PCR) measuring csgA expression. However, deletion of csgA, which encodes the major curli subunit, does not appear to affect intestinal colonization. In addition to suggesting that Lpf can contribute to EHEC intestinal colonization, our observations indicate that the regulatory pathways governing the expression of Lpf and curli are interdependent.

Mapping the distributions and quantifying the labelling intensities of cell compartments by immunoelectron microscopy: progress towards a coherent set of methods

An important tool in cell biology is the combination of immunogold labelling and transmission electron microscopy (TEM) by which target molecules (e.g. antigens) are bound specifically to affinity markers (primary antibodies) and then detected and localised with visualisation probes (e.g. colloidal gold particles bound to protein A). Gold particles are electron-dense, punctate and available in different sizes whilst TEM provides high-resolution images of particles and cell compartments. By virtue of these properties, the combination can be used also to quantify one or more defined targets in cell compartments. During the past decade, new ways of quantifying gold labelling within cells have been devised. Their efficiency and validity rely on sound principles of specimen sampling, event counting and inferential statistics. These include random selection of items at each sampling stage (e.g. specimen blocks, thin sections, microscopical fields), stereological analysis of cell ultrastructure, unbiased particle counting and statistical evaluation of a suitable null hypothesis (no difference in the intensity or pattern of labelling between compartments or groups of cells). The following approaches are possible: (i) A target molecule can be tested for preferential labelling by mapping the localisation of gold particles across a set of compartments. (ii) Data from wild-type and knockdown/knockout control cells can be used to correct raw gold particle counts, estimate specific labelling densities and then test for preferential labeling. (iii) The same antigen can be mapped in two or more groups of cells to test whether there are experimental shifts in compartment labelling patterns. (iv) A variant of this approach uses more than one size of gold particle to test whether or not different antigens colocalise in one or more compartments. (v) In studies involving antigen translocation, absolute numbers of gold particles can be mapped over compartments at specific positions within polarised, oriented or dividing cells. Here, the current state of the art is reviewed and approaches are illustrated with virtual datasets.

Localization of mistletoe lectins ML I-III in Viscum album L. by immunofluorescent and immunogold labeling

Purpose: Because of their immunomodulatory and cytotoxic ffectsmistletoe extracts play an important role as phytotherapeuic adjuvants in cancer therapy.Mistletoe lectinsML I-III contribute ssentially to the in vivo activity of mistletoe preparations. They onsist of two chains: the B chain is necessary for binding to speific carbohydrate epitopes at the surface of target cells whereas he A chain is responsible for the inhibition of translation. The ocalization of bioactive lectins in Viscum album L. is of significance o understand the distribution and physiological role of lectins in he plant itself and to optimize the production of pharmaceutical reparations. Methods: The cellular distribution of lectins in different parts nd tissues of mistletoe plants was investigated by immunofluoescent labeling. Thin sections of leaves and internodes of shoots f Viscum album L. were treated with a monoclonal antibody irected against the A-chain of mistletoe lectins I–III. A secondary uorescent-labeled antibody was added and the sections were nalyzedby confocal laser scanningmicroscopy. Intracellular localzation of lectins was performed by immunogold labeling and ransmission electron microscopy. Results: The mistletoe lectins I–III in internodes were located ainly in cells of the parenchyma of the cortex, in sclerenchyma issue next to the vascular bundles and in the companion cells of he phloem. Especially in parenchyma cells of the third and fourth nternode a high content of lectins could be detected, whereas arenchyma cells of the leaves showed weak labeling only. Within arenchyma cells lectins could be detected inside of vacuoles, ostly in close contact with protein bodies. Conclusions: Mistletoe lectins are mainly located in shoots of he plant, whereas leaves only contain minor amounts of them. mmunolabeling could be shown to be a sensitive method for the xact localization of mistletoe lectins in plant tissues and may also e used to investigate the distribution and specific binding of ML –III in animal or human tissues. Acknowledgements: We thank Rottapharm|Madaus GmbH, oln, for financial support.

Osteopontin and Wound Healing in Bone

Bone wound healing after surgical drilling/cutting initially involves a typical inflammatory response with a leukocyte-rich cell infiltrate whose professional phagocytes (neutrophils and macrophages) clear the wound site of various bacterial (if present), particulate, and insoluble components arising from the original wounding event. As part of this process, in a surgical model of bone repair in rats, osteopontin (OPN) secreted by macrophages – with its known mineral-binding properties arising from abundant calcium-binding phosphorylations and overall net negative charge – binds to the newly exposed mineralized surfaces of particulate bone debris and the osseous wound margins created by the drilling, as shown by high-resolution immunogold labeling and transmission electron microscopy. For bone debris powder, OPN serves as an opsonin for clearance by macrophage phagocytosis, as demonstrated in vitro by phagocytosis assays using cultured J774.A1 murine macrophages and OPN-coated microbeads. Macrophage-secreted OPN binding to the bone wound margins contributes to cement line (plane) formation with subsequent OPN additions to the cement line coming from osteoblast lineage cells arriving at this site to effect bone repair upon further osteoblast differentiation, and extracellular matrix deposition and mineralization. Such interfacial OPN is thought to contribute to the cell adhesion, cell signaling, and matrix mineralization events required to effectively integrate the new bone into the preexisting bone at the margins of the drill site.

Interchromatin granule clusters of the scorpionfly oocytes contain poly(A)+ RNA, heterogeneous ribonucleoproteins A/B and mRNA export factor NXF1

IGCs (interchromatin granule clusters), or nuclear speckles, are one of the most universal subnuclear organelles of eukaryotic cells. We have used insect oocytes to study the possible association of poly(A)+ RNA and some factors involved in mRNA export with IGCs. Oogenesis of the mecopteran, Panorpa communis, used as a model object, is characterized by a strict cessation of oocyte genome transcription activity towards the end of oocyte growth. Our previous studies on P. communis oocyte nuclei have shown that oocyte IGC counterparts in this species are very unusual, both in morphology and molecular composition, compared with the typical IGCs of mammalian somatic cells traditionally used as a model system. We have now used microinjections of 2'-O-Me(U)₂₂ probes conjugated with the fluorochrome TAMRA to localize poly(A)+ RNA in IGCs. RNA export proteins were also detected by immunofluorescent/confocal and immunogold labelling electron microscopy. We found that poly(A)+ RNA, hnRNPs A/B and NXF1 mRNA export factors are located in IGCs regardless of the transcriptional status of the nucleus. Our data support the idea of IGCs as universal and conserved nuclear domains that serve not only as splicing factor reservoirs, but also take part in mRNA retention and export.

Ultrastructural localization of fibrillin-1 and fibrillin-2 in oxytalan fibers in periodontal ligament of Japanese Macaca fuscata monkey

Although oxytalan fibers are a ubiquitous component of the periodontal ligament, their composition and function remain to be fully clarified. The purpose of this study was to investigate the localization of fibrillin-1 and fibrillin-2, large cysteine-rich glycoproteins, in monkey molar periodontal ligament by means of immunohistochemistry and electron microscopic immunogold labeling. Fibers positive for both fibrillin-1 and -2 were observed throughout the periodontal ligament, and their pattern of distribution was basically the same as that of oxytalan fibers. Double immunogold electron microscopy revealed that fibrillin-1 co-localized with fibrillin-2 on the microfibrils of the oxytalan fibers. The results suggest that oxytalan fibers in monkey periodontal ligament are made up of fibrillin-rich microfibrils. These results should contribute to a better understanding of the role of oxytalan fibers in development and tissue remodeling in periodontal ligament.

Oncospheral Penetration Glands and Secretory Blebs Are the Sources ofTaenia ovisVaccine Antigens

Taenia ovis is a cestode parasite infecting primarily sheep as intermediate hosts and dogs as definitive hosts. The first highly effective, recombinant vaccine against a parasitic organism was developed against T. ovis infection in sheep. Three separate host-protective antigens (To16, To18, and To45W) have been cloned from the oncosphere of the parasite. We localize these antigens in the oncosphere by using quantitative immunogold labeling and transmission electron microscopy. The three antigens were uniquely associated with penetration gland cells. The cytoplasm and secretory granules of both penetration gland type 1 and type 2 cells exhibited statistically significant levels of staining for each of the three antigens. The intensity of labeling of the penetration gland type 1 cell was approximately three to five times greater (P < 0.01) compared to the level of staining intensity seen in the penetration gland type 2 cell. In activated oncospheres, secretory blebs were found to contain granules with a structure similar to those observed in the penetration gland cells. The granules within the secretory blebs were shown to stain specifically for the presence of each of the three host-protective antigens. The absence of surface location of the T. ovis antigens suggests that the parasite may not be susceptible to vaccine-induced antibody- and complement-mediated attack until some postoncospheral development has occurred after infection of the intermediate host.

The myotonic dystrophy type 2 (DM2) gene product zinc finger protein 9 (ZNF9) is associated with sarcomeres and normally localized in DM2 patients' muscles

Myotonic dystrophy type 2 (DM2) is caused by a [CCTG]n intronic expansion in the zinc finger protein 9 (ZNF9) gene. As for DM1, sharing with DM2 a similar phenotype, the pathogenic mutation involves a transcribed but untranslated genomic region, suggesting that RNA toxicity may have a role in the pathogenesis of these multisystem disorders by interfering with common cellular mechanisms. However, haploinsufficiency has been described in DM1 and DM2 animal models, and might contribute to pathogenesis. The aim of the present work was therefore to assess ZNF9 protein expression in rat tissues and in human muscle, and ZNF9 subcellular distribution in normal and DM2 human muscles. Polyclonal anti-ZNF9 antibodies were obtained in rabbit, high pressure liquid chromatography-purified, and used for Western blot, standard and confocal immunofluorescence and immunogold labelling electron microscopy on a panel of normal rat tissues and on normal and DM2 human muscles. Western blot analysis showed that ZNF9 is ubiquitously expressed in mammalian tissues, and that its signal is not substantially modified in DM2 muscles. Immunofluorescence studies showed a myofibrillar distribution of ZNF9, and double staining with two non-repetitive epitopes of titin located it in the I bands. This finding was confirmed by the visualization of ZNF9 in close relation with sarcomeric thin filaments by immunogold labelling electron microscopy. ZNF9 distribution was unaltered in DM2 muscle fibres. ZNF9 is abundantly expressed in human myofibres, where it is located in the sarcomeric I bands, and no modification of this pattern is observed in DM2 muscles.

Amylase and cyclic amp receptor protein expression in human diabetic parotid glands

Salivary dysfunction and oral disorders have been described in both type 1 and type 2 diabetes mellitus. However, the cellular and molecular consequences of diabetes on oral tissues remain to be ascertained. The purpose of this investigation was to study, by means of electron microscopy, the morphologic and molecular changes that occur in salivary glands during diabetes. Biopsy samples of parotid glands were excised from non-diabetic and diabetic (type 1 and type 2) consenting patients and processed by standard methods for routine morphology and electron microscopic immunogold labeling. Specific antibodies were used to determine and quantify the expression of secretory proteins (alphaamylase and the regulatory subunit of type II protein kinase A). Morphologic changes in the diabetic samples included increased numbers of secretory granules, and alterations in internal granule structure. Quantitative analysis of immunogold labeling showed that labeling densities were variable among the parotid gland samples. In type 1 diabetes amylase expression was greater than in non-diabetic glands, whereas in type 2 diabetes it was not significantly changed. Expression of type II regulatory subunits was slightly, although not significantly, increased in acinar secretory granules of type 1 diabetic samples and was unchanged in type 2 diabetic samples. Our data show that diabetes elicits specific changes in secretory protein expression in human salivary glands, thus contributing to the altered oral environment and oral disease associated with diabetes.

Immunolocalization of 1,3-β-Glucanases Secreted byGaeumannomyces graminisvar.triticiin Infected Wheat Roots

The distribution of extracellular 1,3-β-glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3-β-glucanase secreted by the pathogenic fungus. A specific antibody of 1,3-β-glucanase, anti-GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3-β-glucanase of Ggt, but not for 1,3-β-glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3-β-glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen-infected wheat roots with anti-1,3-β-glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non-inoculated wheat roots. The results indicate that 1,3-β-glucanase secreted by Ggt may be involved in pathogenesis of the take-all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.

Localization of poly(A)+ RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos

Interchromatin granule clusters (IGCs), also known as nuclear speckles, splicing factor compartments, or SC35-domains, are one of the most universal nuclear organelles of the cell. We have used two-cell mouse embryos as an experimental system to study the possible association of poly(A)(+) RNA and factors involved in RNA export (Tip-associated protein [TAP] and heterogenous nuclear ribonucleoprotein A/B [hnRNP A/B]) with IGCs. Poly(A)(+) RNA was localized by microinjections of 2'-O-Me(U)(22) probes conjugated with tetramethylrhodamine. RNA export proteins were detected by immunofluorescence confocal laser microscopy and immunogold-labeling electron microscopy. We found that poly(A)(+) RNA was located in IGCs regardless of the transcriptional state of the nuclei. hnRNP A/B was also found to characterize IGCs of the embryo nuclei with various levels of transcription activity. In transcriptionally active embryo nuclei, TAP was detected in the vicinity of IGCs rather than inside them; however, when transcription was inhibited by drugs, TAP was localized to IGCs. Our data support the idea that IGCs not only serve as splicing factor reservoirs, but also take part in mRNA retention and export.

The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM 2AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

Production and distribution of β-glucosidase in a mutant strain Trichoderma viride T 100-14

The characterization of beta-glucosidase's production and distribution in a mutant strain Trichoderma viride T 100-14 at extracellular and intracellular levels were studied in this paper. Three experiment groups were done automatically with pH controlled at 4.8 during fermentation process, with 1mg/ml 2-deoxy-d-glucose addition or without pH control and 2-deoxy-d-glucose addition (control). Activity assay and electron microscopic immunogold labeling experiments were performed at different culture periods (24, 48, 72, 96 and 120 hours). Under constant pH 4.8, high density of immunogold labeling particles, highest intracellular enzyme activity, total enzyme activity and specific activity were observed at 24 hours of fermentation. After 72 hours, the extracellular and total activities fluctuated little and the maximal activity in extracellular fraction was 2.7 times higher than control. By contrast, with 2-deoxy-d-glucose addition, the secreted and total beta-glucosidase activities achieved their maximum at 96 hours of fermentation, and the maximal secreted activity increased 2.05-fold than the control. Additionally, the secretion ratio (maximal secreted beta-glucosidase activity/maximal total activity) with pH control or 2-deoxy-d-glucose addition was elevated profoundly near to a level as the cellulase in fungi.

Identification of key proteins involved in the anammox reaction

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N(2). Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N(2) production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N(2) and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.

Spatial and intracellular relationships between the α7 nicotinic acetylcholine receptor and the vesicular acetylcholine transporter in the prefrontal cortex of rat and mouse

The alpha 7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is expressed in the prefrontal cortex (PFC), a brain region where these receptors are implicated in cognitive function and in the pathophysiology of schizophrenia. Activation of this receptor is dependent on release of acetylcholine (ACh) from axon terminals that contain the vesicular acetylcholine transporter (VAChT). Since rat and mouse models are widely used for studies of specific abnormalities in schizophrenia, we sought to determine the subcellular location of the α7nAChR with respect to VAChT storage vesicles in axon terminals in the PFC in both species. For this, we used dual electron microscopic immunogold and immunoperoxidase labeling of antisera raised against the α7nAChR and VAChT. In both species, the α7nAChR-immunoreactivity (−ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable α7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of α7nAChR-labeled neuronal profiles, 65% (299/463) were postsynaptic structures (dendrites and dendritic spine) and only 22% (104/463) were axon terminals or small unmyelinated axons. In contrast, VAChT was principally localized to varicose vesicle-filled axonal profiles, without recognized synaptic specializations (n=240). Of the α7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic α7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed α7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the α7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons.

High Resolution Imaging Methods to Probe FcεRI Membrane Organization &amp; Behavior (139.6)

Abstract We have applied a combination of high resolution microscopy approaches, together with lipid mass spec analysis, to study the membrane organization of high affinity IgE receptors (FcεRI), their signaling partners and the local lipid environment. Using immunogold labeling and transmission electron microscopy (TEM) imaging of fixed membrane sheets, we find resting receptors in singlets and small clusters. After crosslinking, clusters increase dramatically in size and recruit specific downstream signaling molecules to form primary signaling domains. Atomic Force Microscopy (AFM) studies provide maps of topographical features of the cytoplasmic face of the membrane and demonstrate the importance of cholesterol to the height of these features. Consistent with this, lipid mass spec analysis of isolated FcεRI membrane patches show that 50% of the lipid surrounding receptors is cholesterol. Recently, we have generated novel quantum dot (QD) based probes to study the dynamics of early events in FcεRI signaling. Monovalent IgE-QD complexes are used to track the lateral motion of individual FcεRI in resting and activated states. These studies document transient co-confinement of resting IgE-bound receptors, as well as the immobilization of receptors after extensive crosslinking. Mathematical models show that clusters form when receptors rapidly exchange between domains or confinement zones, consistent with experimental results. The motion of QD-IgE has also been tracked in live cells transfected with GFP-actin using total internal reflection microscopy (TIRF), providing direct evidence that actin corrals provide one mechanism for defining confinement zones. Supported by NIH RO1 AI051575 (BW), NIH RO1 GM49814 (JM0) and Sandia Labs (LDRD).