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PubMed2001

Focal adhesion kinase is a key mediator of human trophoblast development.

Trophoblast differentiation during the first trimester of pregnancy involves cell proliferation and invasion and extracellular matrix (ECM) remodeling. Reports have indicated that, in a variety of cell types, processes such as proliferation, invasion, and ECM remodeling require the turnover of focal adhesions mediated by a cytoplasmic tyrosine kinase named focal adhesion kinase (FAK). Therefore, in the present study we examined the expression and spatial localization of FAK during early human placental development. Immunocytochemical and immunoblot analysis showed that FAK and a focal adhesion-associated protein named paxillin were highly expressed between the 5th and 8th weeks of gestation, specifically in villous cytotrophoblast and extravillous trophoblast (EVT) cells. Activated FAK, phosphorylated on Tyr-397, colocalized with alpha5 integrin and matrix metalloproteinase-2 (MMP2) expression in EVT cells within a previously characterized intermediate, invasive-restrained region. FAK and paxillin expression dramatically decreased after 10 to 12 weeks of gestation coincident with increasing pO(2) levels. Exposure of human villous explants of 5 to 8 weeks to a 3% O(2) environment resulted in increased trophoblast outgrowth, cell proliferation, and detection of alpha5 integrin and MMP2, as well as increased activation of FAK in EVT cells compared with explants grown in a 20% O(2) environment. To determine whether FAK was a key requisite for trophoblast differentiation, villous explants of 5 weeks gestation were grown in Matrigel in a 3% O(2) environment and incubated with 20-mer antisense FAK oligonucleotides. A dramatic reduction of trophoblast outgrowth was observed in antisense-treated explants compared with missense and control cultures, and, in addition, cell proliferation and MMP2 activity in antisense-treated explants were dramatically reduced. These data suggest that FAK is a key kinase involved in early trophoblast cell differentiation and plays a role in regulating cell proliferation and motility during early placental development.

Antisense Elements (Genetics)↗
PubMed2006

Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging.

Methods to record action potential (AP) firing in many individual neurons are essential to unravel the function of complex neuronal circuits in the brain. A promising approach is bolus loading of Ca(2+) indicators combined with multiphoton microscopy. Currently, however, this technique lacks cell-type specificity, has low temporal resolution and cannot resolve complex temporal firing patterns. Here we present simple solutions to these problems. We identified neuron types by colocalizing Ca(2+) signals of a red-fluorescing indicator with genetically encoded markers. We reconstructed firing rate changes from Ca(2+) signals by temporal deconvolution. This technique is efficient, dramatically enhances temporal resolution, facilitates data interpretation and permits analysis of odor-response patterns across thousands of neurons in the zebrafish olfactory bulb. Hence, temporally deconvolved Ca(2+) imaging (TDCa imaging) resolves limitations of current optical recording techniques and is likely to be widely applicable because of its simplicity, robustness and generic principle.

Action Potentials↗
PubMed2001

A tumor necrosis factor alpha- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase delta and proliferating cell nuclear antigen.

A cDNA encoding a protein of 36 kDa, polymerase delta-interacting protein 1 (PDIP1), that interacts with the small subunit (p50) of DNA polymerase delta (pol delta) was identified in a two-hybrid screen of a HepG2 cDNA library by using p50 as bait. The interaction of PDIP1 with p50 was confirmed by pull-down assays, and a similar assay was used to demonstrate that PDIP1 interacts directly with the proliferating cell nuclear antigen (PCNA). PCNA and p50 bound to PDIP1 simultaneously, and PDIP1 stimulated pol delta activity in vitro in the presence, but not the absence, of PCNA, suggesting that PDIP1 also interacts functionally with both p50 and PCNA. Subcellular localization studies demonstrated that PDIP1 is a nuclear protein that colocalizes with PCNA at replication foci. A putative PCNA-binding motif was identified within the C terminus of PDIP1, and a synthetic peptide containing this PCNA-binding motif was shown to bind PCNA by far-Western analysis. Northern analysis demonstrated that PDIP1 mRNA is present in a wide variety of human tissues. PDIP1 was found to be highly homologous to a previously identified protein, B12 [Wolf, F. W., Marks, R. M., Sarma. V., Byers, M. G., Katz, R. W., Shows, T. B. & Dixit, V. M. (1992) J. Biol. Chem. 267, 1317-1326], one of the early response genes induced by tumor necrosis factor alpha. PDIP1 synthesis can also be induced by tumor necrosis factor alpha and by IL-6, cytokines essential for liver regeneration after loss of hepatic tissue. It is suggested that PDIP1 provides a link between cytokine activation and DNA replication in liver as well as in other tissues.

Amino Acid Sequence↗
PubMed2001

Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase.

Myeloperoxidase uses hydrogen peroxide (H2O2) to generate hypochlorous acid (HOCl), a potent cytotoxic oxidant. We demonstrate that HOCl regulates the activity of matrix metalloproteinase-7 (MMP-7, matrilysin) in vitro, suggesting that this oxidant activates MMPs in the artery wall. Indeed, both MMP-7 and myeloperoxidase were colocalized to lipid-laden macrophages in human atherosclerotic lesions. A highly conserved domain called the cysteine switch has been proposed to regulate MMP activity. When we exposed a synthetic peptide that mimicked the cysteine switch to HOCl, HPLC analysis showed that the thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as sulfinic acid, sulfonic acid, and a dimer containing a disulfide bridge. In contrast, the peptide reacted slowly with H2O2, and the only product was the disulfide. Moreover, HOCl markedly activated pro-MMP-7, an MMP expressed at high levels in lipid-laden macrophages in vivo. Tandem mass spectrometric analysis of trypsin digests revealed that the thiol residue of the enzyme's cysteine switch domain had been converted to sulfinic acid. Thiol oxidation was associated with autolytic cleavage of pro-MMP-7, strongly suggesting that oxygenation activates the latent enzyme. In contrast, H2O2 failed to oxidize the thiol residue of the protein or activate the enzyme. Thus, HOCl activates pro-MMP-7 by converting the thiol residue of the cysteine switch to sulfinic acid. This activation mechanism is distinct from the well-studied proteolytic cleavage of MMP pro-enzymes. Our observations raise the possibility that HOCl generated by myeloperoxidase contributes to MMP activation, and therefore to plaque rupture, in the artery wall. HOCl and other oxidants might regulate MMP activity by the same mechanism in a variety of inflammatory conditions.

Amino Acid Sequence↗
PubMed1993

Differences in the endosomal distributions of the two mannose 6-phosphate receptors.

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.

Animals↗
PubMed2006

Heterogeneous nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation of A2 response element mRNAs.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

Adult↗
PubMed2002

Role of vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional permeability.

At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation.

Adenosine↗
PubMed1998

Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle.

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at approximately 40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle alpha-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

Adrenal Glands↗
PubMed1996

Localization and regulation by vitamin D of calcium transport proteins in rabbit cortical collecting system.

The 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced expression of Na+/Ca2+ exchanger, Ca(2+)-adenosinetriphosphatase (Ca(2+)-ATPase), and calbindin-D28k was investigated in the rabbit distal nephron. Immunocytochemical studies in rabbit kidney sections revealed colocalization of the three Ca2+ transport proteins in the majority of cells in the distal nephron, including connecting tubules and cortical collecting ducts. Subsequently, rabbit connecting and cortical collecting tubule cells were immunodissected and cultured on permeable supports. Immunocytochemical analysis of the cultured cells by confocal microscopy revealed that Na+/Ca2+ exchanger and Ca(2+)-ATPase were present at the basolateral membrane, whereas calbindin-D28k was evenly distributed throughout the cytosol. Concomitant with an increase in Ca2+ transport, 1,25(OH)2D3 increased calbindin-D28k protein and RNA content two- to threefold, as determined by Northern and Western blotting. By contrast, neither Na+/Ca2+ exchanger nor Ca(2+)-ATPase RNA or protein content was noticeably altered. Our findings suggest that 1,25(OH)2D3 stimulation of transcellular Ca2+ transport in primary cultures of rabbit cortical collecting system cells involves an increase in the gene expression of calbindin-D28k but not of Na+/Ca2+ exchanger and Ca(2+)-ATPase.

Animals↗
PubMed2007

Multimodality molecular imaging identifies proteolytic and osteogenic activities in early aortic valve disease.

BACKGROUND: Visualizing early changes in valvular cell functions in vivo may predict the future risk and identify therapeutic targets for prevention of aortic valve stenosis. METHODS AND RESULTS: To test the hypotheses that (1) aortic stenosis shares a similar pathogenesis to atherosclerosis and (2) molecular imaging can detect early changes in aortic valve disease, we used in vivo a panel of near-infrared fluorescence imaging agents to map endothelial cells, macrophages, proteolysis, and osteogenesis in aortic valves of hypercholesterolemic apolipoprotein E-deficient mice (30 weeks old, n=30). Apolipoprotein E-deficient mice with no probe injection (n=10) and wild-type mice (n=10) served as controls. Valves of apolipoprotein E-deficient mice contained macrophages, were thicker than wild-type mice (P<0.001), and showed early dysfunction detected by MRI in vivo. Fluorescence imaging detected uptake of macrophage-targeted magnetofluorescent nanoparticles (24 hours after injection) in apolipoprotein E-deficient valves, which was negligible in controls (P<0.01). Valvular macrophages showed proteolytic activity visualized by protease-activatable near-infrared fluorescence probes. Ex vivo magnetic resonance imaging enhanced with vascular cell adhesion molecule-1-targeted nanoparticles detected endothelial activation in valve commissures, the regions of highest mechanical stress. Osteogenic near-infrared fluorescence signals colocalized with alkaline phosphatase activity and expression of osteopontin, osteocalcin, Runx2/Cbfa1, Osterix, and Notch1 despite no evidence of calcium deposits, which suggests ongoing active processes of osteogenesis in inflamed valves. Notably, the aortic wall contained advanced calcification. Quantitative image analysis correlated near-infrared fluorescence signals with immunoreactive vascular cell adhesion molecule-1, macrophages, and cathepsin-B (P<0.001). CONCLUSIONS: Molecular imaging can detect in vivo the key cellular events in early aortic valve disease, including endothelial cell and macrophage activation, proteolytic activity, and osteogenesis.

Animals↗
PubMed2001

The p57Kip2 cyclin kinase inhibitor is expressed by a restricted set of amacrine cells in the rodent retina.

Amacrine cells in the vertebrate retina can be grouped according to morphology into distinct types, which are organized into characteristic mosaics. Each type is believed to perform a unique role in visual signal processing. Neurotransmitters and calcium binding proteins have served as important markers for amacrine cell populations, yet many types remain to be characterized at the molecular level. We have found that a cyclin kinase inhibitor, p57Kip2, is expressed in a restricted group of amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the rodent retina. Whole-mount antibody staining revealed that the p57Kip2 amacrine cells are evenly distributed across the retina with a density of 1654 +/- 63 cells/mm(2) in the INL and 994 +/- 26 cells/mm(2) in the GCL. These amacrine cells accumulate the major inhibitory neurotransmitter gamma-aminobutyric acid (GABA) but do not accumulate high levels of glycine. In addition, p57Kip2 immunoreactivity does not colocalize with any of the previously identified amacrine cell markers including calbindin, calretinin, parvalbumin, choline acetyltransferase, and tyrosine hydroxylase. To determine whether the p57Kip2 population of amacrine cells is organized into a regular or a random mosaic, nearest neighbor analysis was performed for both the INL and GCL populations. Results from this analysis demonstrated that the p57Kip2-immunoreactive amacrine cells are randomly organized and therefore they are likely to constitute two or more distinct populations. This new molecular marker will serve as a useful tool for future studies on the development and function of amacrine cells in the vertebrate retina.

Animals↗
PubMed1991

Immunolocalization of a cysteine protease within the lysosomal system of Trypanosoma congolense.

A cysteine protease has been purified from bloodstream forms of Trypanosoma congolense by affinity chromatography on cystatin-Sepharose. A polyclonal antibody was raised against the purified enzyme and used for immunocytochemical localization of the enzyme by electron microscopy. Antibody labeling of the cysteine protease, using colloidal gold-labeled protein A (PrA-Au), was observed over amorphous material within subcellular organelles which have the appearance of lysosome-like bodies. This intracellular labeling colocalized in organelles containing bovine serum albumin-gold (BSA-Au) that had been endocytosed by the living parasites. The PrA-Au/antibody also labeled the flagellar pocket and parasite cell surface, albeit less consistently. Volume density analysis showed that the organelles containing endocytosed BSA-Au, after 30 min incubation at 37 degrees C in BSA-Au, comprised approximately 22% of the total parasite cell volume. Under similar conditions, but employing horseradish peroxidase (HRP) as a fluid-phase marker of the lysosomal system, only 5.7% of the cell contained HRP. This value dropped to 3.6% after 60 min incubation. Volume density analysis showed that the amorphous material which was labeled by the antibody to the cysteine protease occupied 6.9% of the cell volume. This amorphous material was contained within a membrane-bound lysosome-like organelle that occupied 11.5% of the cell. Thus, the cysteine protease appears to be present in half, or less, of the lysosomal system of T. congolense.

Animals↗
PubMed1996

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes.

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.

3T3 Cells↗
PubMed1996

Novel mouse IgG-like immunoreactivity expressed by neurons in the moth Manduca sexta: developmental regulation and colocalization with crustacean cardioactive peptide.

Immunoglobulin-related molecules have been shown to play important roles in cell-cell recognition events during the development of both vertebrate and invertebrate nervous systems. In the moth, Manduca sexta, we report the presence of novel, mouse, immunoglobulin G (mIgG)-like immunoreactivity in a discrete population of identified neurosecretory neurons (the NS-Ls also known as the cell 27s) and interneurons (the IN-704s). A number of polyclonal anti-mIgG antibodies were used to immunostain these cells in wholemount. The mIgG-like-immunoreactive (IR) neurons were present during embryogenesis through the developing adult stages, but disappeared in the postemerged adult. Biochemical analysis of M. sexta ventral nerve cords revealed that the mIgG-like antigen is a membrane-associated 27-kDa protein which is likely responsible for the mIgG-like immunostaining observed. Unambiguous identification of the mIgG-like-IR neurons was based on neuronal morphology and our ability to demonstrate conclusively that these neurons expressed immunoreactivity to an antiserum against crustacean cardioactive peptide (CCAP). The NS-Ls and IN-704s were both shown to colocalize the CCAP and mIgG-like immunoreactivities. The mIgG-like and CCAP-IR neurons were identical to a subset of CCAP-IR neurons recently described by Davis et al. [(1993) J. Comp. Neurol., 338:612-627] in pupae. We found these CCAP-IR neurons, however, also to be present in larvae. The mIgG-like- and CCAP-IR neurons included the NS-L pair of the subesophageal maxillary neuromere, which projected anteriorly to the corpora cardiaca, and the NS-L of the labial neuromere whose axons projected out the dorsal nerve of the next posterior ganglion. The mIgG-like and CCAP-IR NS-Ls were also observed throughout the three thoracic ganglia, and all shared strikingly similar structural features. These cells exited out the dorsal nerve of the next posterior ganglion and eventually projected to the neurohemal release sites of the perivisceral organs. These neurons appear to be the homologues of the abdominal CCAP-IR NS-Ls, neurons that in the adult switch their neurotransmitter and release the neuropeptide bursicon. Our description of the distribution and developmental expression of this novel mIgG-like immunoreactivity may provide new insights into the regulation of neurotransmitter plasticity and/or recognition-signaling events involved in the embryonic and postembryonic assembly of the nervous system.

Animals↗
PubMedDate not supplied

Analysis of biofilm structure and gene expression using fluorescence dual labeling.

The use of biofilms for the degradation of recalcitrant environmental contaminants or for the production of secondary metabolites necessitates understanding and controlling gene expression. In this work, dual labeling with green fluorescent protein (GFP) variants was used to investigate inducible gene expression in a biofilm. Colocalization of GFP emissions was used to determine regions of attached cells and to correlate structure and activity within the biofilm. The labeling strategy reported here is unique in that the two GFP signals were distinguished by differential excitation rather than differential emission.

Biofilms↗
PubMed2004

Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1.

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

Alleles↗
PubMed1999

Sex hormone binding globulin expression and colocalization with estrogen receptor in the human Fallopian tube.

The detection of sex hormone binding globulin (SHBG) or SHBG mRNA in several sex steroid target tissues, has raised the possibility that SHBG modulates the action of sex steroids outside the vascular compartment. The presence of SHBG mRNA was investigated by RT-PCR in the poly (A+) RNA fraction of the human Fallopian tube. Human and rat liver were used as positive and negative control tissues, respectively. The electrophoretic analysis of the amplified PCR products showed bands at 219 bp, corresponding to the expected size of the SHBG cDNA, in the Fallopian tube and human liver but not in rat liver, indicating that SHBG might be synthesized by the Fallopian tube. The cellular localization of SHBG and of estrogen receptor (ER) was examined by immunohistochemistry in consecutive sections of Fallopian tube tissues for individual staining or double immunostaining in the same section. Specific immunostaining of SHBG was present in the epithelial, vascular and muscle cells of the ampullary and isthmic region. In epithelial cells, immunoreactive SHBG was present in the apical end with the highest concentration close to the luminal membrane. The ER was localized in the nuclei of epithelial, stromal and muscle cells of the ampulla and isthmus. Double immunostaining showed that SHBG and ER are colocalized principally in epithelial cells of the ampulla and in muscle cells of the isthmus. In conclusion, the detection of SHBG and SHBG mRNA and the localization of SHBG in estrogen target cells was shown. These findings support the hypothesis that SHBG might regulate sex steroid action at the tissue level.

Animals↗
PubMed1997

Direct cell/cell communication in the lymphoid germinal center: connexin43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes.

Direct cell/cell communication occurs through gap junctions (GJ). We mapped GJ expression in secondary lymphoid organs and found, for the first time, a high density of connexin43 (Cx43) GJ in follicular dendritic cells (FDC) in close association with lymphocytes (Krenacs T. and Rosendaal M., J. Histochem. Cytochem. 1995. 43: 1125-1137). In this work, we used a combination of ultrastructural, immunocytochemical, molecular methods, and functional dye transfer experiments to study which germinal center cells are involved in direct cell/ cell communication and how GJ expression is regulated during antigen responses. One week after injecting the footpad of mice with 50 micrograms lysozyme, Cx43 GJ were detected on elongated cells in the paracortex of their popliteal lymph nodes. Repeated challenge led to the formation of secondary follicles with enlarged FDC meshwork full of Cx43 GJ. This positive correlation may reflect an importance for GJ in the pattern formation of FDC and lymphoid follicles. In human tonsil, the density of GJ and FDC was highest in the light zone of germinal centers where the fate of B cells is thought to be decided. Cx43 colocalized with CD21 and CD35 antigens in the vicinity of desmosomal junctions on FDC embracing lymphocytes. Freeze-fracture hallmarks of GJ of 200-400 nm were also found on FDC in the vicinity of desmosomal plaques. Furthermore, Northern blot analysis showed the consistent presence of Cx43 mRNA in human tonsil and spleen. Most Cx43 message was localized in situ to cells with FDC morphology and some to a few germinal center lymphocytes. To investigate functional cell coupling, we set up FDC/B cell cultures from the low density cell fractions of human tonsils. Cx43 plaques associated with lymphocytes were detected both on elongated FDC processes in early cultures (up to 4 h) and in established FDC/B cell clusters (between 4 and 24 h). In early cultures, we injected FDC with Lucifer Yellow, a fluorescent dye which passes through GJ: the dye spread into adjacent FDC and occasionally from FDC into CD19+ B cells. Based on these results, we propose that direct cell/cell communication through Cx43 GJ is involved in FDC/FDC and in FDC/B cell interactions. The functionally coupled FDC meshwork may serve as a communication channel synchronizing germinal center events. FDC may also deliver crucial direct signals through GJ involved in the rescue of high-affinity B cell clones from apoptotic cell death.

Animals↗