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PubMed2000

Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction.

The key regulator of G(2)-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin. Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal. The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS). We exploited the yeast two-hybrid screen to find protein(s) potentially mediating localization of cyclin B1 and identified a novel interaction between cyclin B1 and cyclin F. We found that cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity. Cyclin B1 and cyclin F also colocalize through immunofluorescence studies. Additionally, deletion analysis revealed that each putative NLS of cyclin F is functional. Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus. The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function.

Animals↗
PubMed2001

Activation of the JNK/p38 pathway occurs in diseases characterized by tau protein pathology and is related to tau phosphorylation but not to apoptosis.

JNK and p38, two members of the MAP kinase family, are strongly induced by various stresses including oxidative stress and have been involved in regulation of apoptosis. As both kinases phosphorylate tau protein in vitro, we have investigated their immunohistochemical localization in a group of neurodegenerative diseases characterized by intracellular deposits of hyperphosphorylated tau. Cases included Alzheimer disease, Pick disease, progressive supranuclear palsy, corticobasal degeneration, Gerstmann-Sträussler-Scheinker disease-Indiana kindred, and frontotemporal dementia with parkinsonism linked to chromosome 17. In all tissue samples, strong immunoreactivity for both MAP kinases was found in the same neuronal or glial cells that contained tau-positive deposits. By double immunohistochemistry, JNK and p38 colocalized with tau in the inclusions. Analysis of apoptosis-related changes (DNA fragmentation, activated caspase-3) showed that the expression of JNK and p38 was unrelated to activation of an apoptotic cascade. Our data indicate that phospho-JNK and phospho-p38 are associated with hyperphosphorylated tau in a variety of abnormal tau inclusions, suggesting that these kinases may play a role in the development of degenerative diseases with tau pathology.

Adult↗
PubMed2006

Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus.

The expression of the purinergic receptor subtype P2X(7)R, a nonselective cationic channel activated by high levels of adenosine triphosphate (ATP), has been studied in adult microglia obtained from Alzheimer disease (AD) and nondemented (ND) brains, in fetal human microglia exposed to Abeta(1-42) peptide and in vivo in Abeta(1-42)-injected rat hippocampus. Semiquantitative reverse transcriptase-polymerase chain reaction showed enhanced expression (increase of 70%) of P2X(7)R in AD microglia compared with ND cells (analysis of 6 AD and 8 ND cases). Immunohistochemical analysis showed prominent P2X(7)R expression in association with Abeta plaques and localized to HLA-DR-immunoreactive microglia. In cultured fetal human microglia, cells exposed to Abeta(1-42) (5 microM for 18 hours) had significantly elevated levels of P2X(7)R (by 106%) compared with untreated cells. Amplitudes of Ca(2+) responses in these cells, induced by the selective P2X(7)R agonist BzATP, were increased by 145% with Abeta(1-42) pretreatment relative to control (no peptide pretreatment) and were largely blocked if the P2X(7)R inhibitor-oxidized ATP (oxATP) was added with peptide in pretreatment solution. In vivo, double immunostaining analysis showed considerable P2X(7)R colocalized with microglia after injection of Abeta(1-42) (1 nmol) into rat hippocampus. The overall results suggest roles of P2X(7)R in mediating microglial purinergic inflammatory responses in AD brain.

Adenosine Triphosphate↗
PubMed1997

Intergenic transcription and transinduction of the human beta-globin locus.

We have identified novel nuclear transcripts in the human beta-globin locus using nuclear run-on analysis in erythroid cell lines and in situ hybridization analysis of erythroid tissue. These transcripts extend across the LCR and intergenic regions but are undetectable in nonerythroid cells. Surprisingly, transient transfection of a beta-globin gene (epsilon, gamma, or beta) induces transcription of the LCR and intergenic regions from the chromosomal beta-globin locus in nonerythroid cell lines. The beta-globin genes themselves, however, remain transcriptionally silent. Induction is dependent on transcription of the globin gene in the transfected plasmid but does not require protein expression. Using in situ hybridization analysis, we show that the plasmid colocalizes with the endogenous beta-globin locus providing insight into the mechanism of transinduction.

Animals↗
PubMed1992

Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is co-localized with tenascin during adult newt limb regeneration.

Using immunohistochemical techniques and mAb MT2, we describe here a novel extracellular matrix (ECM) molecule that is developmentally regulated during limb regeneration in adult newts. The MT2 antigen appears during preblastema stages, is most abundant during blastema stages, and persists, near undifferentiated cells, until digit stages. The MT2 antigen is located in an acellular layer under the wound epithelium and throughout the ECM of the undifferentiated mesenchyme as a thick, cord-like component. In unamputated limbs mAb MT2 reactivity is restricted to tendons, myotendinous junctions, periosteum and to a layer of material beneath the epidermis. In both unamputated limbs and regenerating limbs, the reactivity to mAb MT2 colocalizes closely with urodele tenascin. Immunoblot analysis of blastema extracts showed that the unreduced form of the MT2 antigen is a large, polydispersed protein of approximately the same size as tenascin. However, based upon (a) molecular weights of reduced subunits, (b) competition experiments on tissue sections, and (c) analysis of molecules immunoprecipitated by mAb MT2, we conclude that the MT2 substance is unrelated biochemically to tenascin. The results from immunoblots, enzyme digestions and DEAE-Sephacell binding studies suggest that the unreduced MT2 antigen is a large protein composed of subunits which are connected by disulfide bonds. Reduction of the MT2 antigen results in three components recognized by mAb MT2. The largest of these reduced components is a chondroitin sulfate-like glycoprotein with a molecular weight (Mr) of 310-325 x 10(3). A second component (Mr, 285-300 x 10(3)) is the core protein of the 310-325 x 10(3) glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Functional segregation of synaptic GABAA and GABAC receptors in goldfish bipolar cell terminals.

The transmission of light responses to retinal ganglion cells is regulated by inhibitory input from amacrine cells to bipolar cell (BC) synaptic terminals. GABA(A) and GABA(C) receptors in BC terminals mediate currents with different kinetics and are likely to have distinct functions in limiting BC output; however, the synaptic properties and localization of the receptors are currently poorly understood. By recording endogenous GABA receptor currents directly from BC terminals in goldfish retinal slices, I show that spontaneous GABA release activates rapid GABA(A) receptor miniature inhibitory postsynaptic currents (mIPSCs) (predominant decay time constant (tau(decay)), 1.0 ms) in addition to a tonic GABA(C) receptor current. The GABA(C) receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) has no effect on the amplitude or kinetics of the rapid GABA(A) mIPSCs. In addition, inhibition of the GAT-1 GABA transporter, which strongly regulates GABA(C) receptor currents in BC terminals, fails to reveal a GABA(C) component in the mIPSCs. These data suggest that GABA(A) and GABA(C) receptors are highly unlikely to be synaptically colocalized. Using non-stationary noise analysis of the mIPSCs, I estimate that GABA(A) receptors in BC terminals have a single-channel conductance (gamma) of 17 pS and that an average of just seven receptors mediates a quantal event. From noise analysis of the tonic current, GABA(C) receptor gamma is estimated to be 4 pS. Identified GABA(C) receptor mIPSCs exhibit a slow decay (tau(decay), 54 ms) and are mediated by approximately 42 receptors. The distinct properties and localization of synaptic GABA(A) and GABA(C) receptors in BC terminals are likely to facilitate their specific roles in regulating the transmission of light responses in the retina.

Animals↗
PubMed2005

Relocalization of the Mre11-Rad50-Nbs1 complex by the adenovirus E4 ORF3 protein is required for viral replication.

Adenovirus replication is controlled by the relocalization or modification of nuclear protein complexes, including promyelocytic leukemia protein (PML) nuclear domains and the Mre11-Rad50-Nbs1 (MRN) DNA damage machinery. In this study, we demonstrated that the E4 ORF3 protein effects the relocalization of both PML and MRN proteins to similar structures within the nucleus at early times after infection. These proteins colocalize with E4 ORF3. Through the analysis of specific viral mutants, we found a direct correlation between MRN reorganization at early times after infection and the establishment of viral DNA replication domains. Further, the reorganization of MRN components may be uncoupled from the ability of E4 ORF3 to rearrange PML. At later stages of infection, components of the MRN complex disperse within the nucleus, Nbs1 is found within viral replication centers, Rad50 remains localized with E4 ORF3, and Mre11 is degraded. The importance of viral regulation of the MRN complex is underscored by the complementation of E4 mutant viruses in cells that lack Mre11 or Nbs1 activity. These results illustrate the importance of nuclear organization in virus growth and suggest that E4 ORF3 regulates activities in both PML nuclear bodies and the MRN complex to stimulate the viral replication program.

Acid Anhydride Hydrolases↗
PubMed1990

p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.

Antibodies, Monoclonal↗
PubMed1997

Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.

The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.

Actins↗
PubMed1996

Latent transforming growth factor-beta 1 and its binding protein are components of extracellular matrix microfibrils.

We studied the localization of latent transforming growth factor-beta 1 (TGF-beta 1) and its binding protein (LTBP-1) in the extracellular matrix of cultured human fibroblasts by immunofluorescence and immunoelectron microscopy. Immunofluorescence of confluent fibroblast cultures indicated that LTBP-1 localizes to extracellular fibrillar structures resembling fibronectin-collagen matrix. Similar fibrillar structures were detected in cells stained with antibodies specific for TGF-beta 1 propeptide (beta 1-LAP). Both LTBP-1 and beta 1-LAP colocalized with fibronectin in double immunofluorescence analysis. These fibrillar structures were resistant to extraction with sodium deoxycholate, which is further evidence that LTBP-1 and large latent TGF-beta 1 complexes are integral components of the extracellular matrix. SV-40-transformed human fibroblasts lacked extracellular LTBP-1 fibers. EM analysis revealed approximately 10-nm-thick microfibrils that were labeled by anti-LTBP at 90-140-nm intervals. In addition, LTBP-1 was found in structures that were heavily labeled for fibronectin. The accumulation of LTBP-1 in the fibronectin matrix could be reconstituted in vitro. When isolated matrix components were immobilized on nitrocellulose and incubated with fibroblast conditioned medium, LTBP-1 from the medium associated with cellular fibronectin but not with heparan or chondroitin sulfate, vitronectin, tenascin, laminin, or collagen I or IV. The association of LTBP-1 with cellular fibronectin was abolished by treatment of the medium with plasmin, which cleaves LTBP-1 and inhibits its assembly to matrix. The present results indicate that latent TGF-beta 1 complexes are components of the extracellular matrix and suggest that alterations of the pericellular matrix could result in aberrant TGF-beta signaling.

Carrier Proteins↗
PubMed2002

Miniglucagon (glucagon 19-29): a novel regulator of the pancreatic islet physiology.

Miniglucagon, the COOH-terminal (19-29) fragment processed from glucagon, is a potent and efficient inhibitor of insulin secretion from the MIN 6 beta-cell line. Using the rat isolated-perfused pancreas, we investigated the inhibitory effect of miniglucagon on insulin secretion and evaluated the existence of an inhibitory tone exerted by this peptide inside the islet. Miniglucagon dose-dependently inhibited insulin secretion stimulated by 8.3 mol/l glucose, with no change in the perfusion flow rate. A concentration of 1 nmol/l miniglucagon had a significant inhibitory effect on a 1 nmol/l glucagon-like peptide 1 (7-36) amide-potentiated insulin secretion. A decrease in extracellular glucose concentration simultaneously stimulated glucagon and miniglucagon secretion from pancreatic alpha-cells. Using confocal and electron microscopy analysis, we observed that miniglucagon is colocalized with glucagon in mature secretory granules of alpha-cells. Perfusion of an anti-miniglucagon antiserum directed against the biologically active moiety of the peptide resulted in a more pronounced effect of a glucose challenge on insulin secretion, indicating that miniglucagon exerts a local inhibitory tone on beta-cells. We concluded that miniglucagon is a novel local regulator of the pancreatic islet physiology and that any abnormal inhibitory tone exerted by this peptide on the beta-cell would result in an impaired insulin secretion, as observed in type 2 diabetes.

Animals↗
PubMed2006

Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation.

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.

Actins↗
PubMed1999

Thrombin induces surface and intracellular secretion of amyloid precursor protein from human endothelial cells.

Thrombin, a major coagulant and inflammatory mediator, was shown to regulate amyloid precursor protein (APP) secretion. APP is the protein from which the amyloid beta peptide (A(beta)) is derived. A(beta) forms the core of vascular and cerebral plaques in Alzheimer's disease (AD). In this study, human umbilical vein endothelial cells (HUVEC) were used to examine the effects of thrombin on APP expression. Cell supernatants from thrombin-treated HUVEC were immunoblotted to measure secreted APP. Thrombin-induced secretion of APP peaks at approximately 30 min post-treatment. Immunohistochemical analysis found that APP is not colocalized with or secreted through the same pathway as coagulation factor VIII. The secretion of APP is thrombin receptor-mediated, since it is inhibited by the thrombin antagonist N-Acetyl-D-Phe-Pro-1-Amido-4-Guanidino-Butyl-1-Boronic Acid. It also is induced by treatment with a calcium ionophore. Moreover, APP secretion is protein kinase C (PKC)-dependent because it is blocked by the PKC inhibitor bisindolylmaleimide. APP secretion also occurs from the cell surface, possibly through direct cleavage by thrombin. Immunoreactivity on the surface of HUVEC decreased after thrombin treatment but not after treatment with a non-proteolytic thrombin receptor activator. These data suggest that thrombin induces APP secretion through a PKC-dependent mechanism, as well as from the cell surface. Our results are consistent with thrombin playing a role in AD pathology.

Amyloid beta-Protein Precursor↗
PubMed2001

Angiopoietin-1 upregulation by vascular endothelial growth factor in human retinal pigment epithelial cells.

PURPOSE: To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells. METHODS: Expression of VEGF, Ang1, and Ang2 in surgically removed human choroidal neovascular membranes (CNVMs) was analyzed by double-label confocal immunofluorescence microscopy. Total RNA was extracted from cultured human RPE cells treated with VEGF for mRNA analysis. Northern blot analysis was performed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear run-on analyses were performed. Secreted Ang1 and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis. RESULTS: Ang1 and Ang2 immunostaining colocalized with VEGF-positive stromal cells in human CNVMS: Ang1 and Ang2 mRNAs were expressed by cultured serum-starved RPE cells. VEGF upregulated Ang1 mRNA in a time- and dose-dependent manner without a significant change in Ang2 mRNA. Ang1 and Ang2 mRNAs in RPE cells were as stable as that of S18. VEGF stimulation further increased the half-life of Ang1 mRNA, but did not alter its transcription rate. VEGF increased the amount of Ang1, but not Ang2, protein secreted into the medium. CONCLUSIONS: The colocalization of Ang1 and Ang2 with VEGF in CNVM stromal cells and the upregulation of Ang1 expression by VEGF in cultured RPE cells suggest that VEGF may selectively modulate Ang expression during CNV.

Angiopoietin-1↗
PubMed2002

Beta-catenin interacts with low-molecular-weight protein tyrosine phosphatase leading to cadherin-mediated cell-cell adhesion increase.

Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

3T3 Cells↗
PubMedDate not supplied

Internalization and recycling of glycoprotein 280 in BN/MSV yolk sac epithelial cells: a model system of relevance to receptor-mediated endocytosis in the renal proximal tubule.

The processes of endocytosis and recycling have been well characterized in renal proximal tubule and yolk sac epithelia. We utilized a yolk sac teratocarcinoma cell line, BN/MSV, which expresses two glycoproteins, megalin/gp330 and gp280, also detected in renal proximal tubule and yolk sac epithelial cells. In this study, we further define the localization, internalization and intracellular trafficking of both proteins in BN/MSV cells. For this purpose, double indirect immunofluorescence and immunoelectron microscopy were performed on BN/MSV cells. In addition, antibodies against gp280 and gp330, coupled to colloidal gold particles, were used as tracers to follow the endocytosis and recycling of the two glycoproteins in BN/MSV cells. BSA and MOPC21 (a nonspecific monoclonal antibody) coupled to gold particles were used as controls. We have previously shown that gp280 and megalin/gp330 were localized in clathrin-coated pits; both proteins can also be detected in noncoated areas. Vesicular labeling has previously been seen in the cytoplasm of permeabilized BN/ MSV cells. The results of the present study revealed that the glycoproteins were colocalized in the same cells. Ultrastructural analysis of ultracryosections of BN/MSV cells revealed a localization of both proteins in coated invaginations and small and large endocytic vacuoles. In addition, gp280 and megalin/gp330 were found in the Golgi apparatus and in the granular endoplasmic reticulum. Furthermore, incubation of BN/MSV cells in the presence of colloidal gold particles labeled with antibodies to gp280 and gp330 demonstrated an internalization from the apical membrane through coated pits into small and large endocytic vacuoles. While anti-gp330 is predominantly localized in large endocytic vacuoles, the anti-gp280 gold is mainly concentrated in the tubulovesicular structures, which probably correspond to dense tubules known to enable membrane recycling in the epithelial cells of renal proximal tubules. Moreover, anti-gp330 gold particles are also found in lysosomes, but to a lesser extent than BSA and MOPC21 gold particles, which were highly concentrated in lysosomes. In conclusion, our results show that gp280 is internalized in BN/MSV cells and that anti-gp280 gold is accumulated in a recycling compartment. Thus, we suggest that gp280 is a receptor for endocytosis.

Animals↗
PubMed2000

Colocalization of multiple GABA(A) receptor subtypes with gephyrin at postsynaptic sites.

Clustering of gamma aminobutyric acid (GABA)(A) receptors to postsynaptic sites requires the presence of both the gamma2 subunit and gephyrin. Here, we analyzed by double-immunofluorescence staining the colocalization of gephyrin and major GABA(A)-receptor subtypes distinguished by the subunits alpha1, alpha2, alpha3, or gamma2 in adult rat brain. By using confocal laser scanning microscopy, GABA(A)-receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin-positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABA(A)-receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin-positive clusters were not colocalized with GABA(A)-receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High-resolution images (voxel size, 50 x 50 x 150 nm) were restored with an iterative image deconvolution procedure based on a measured point-spread function to analyze the colocalization between GABA(A)-receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro-organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin- and gamma2 subunit-positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were "embedded" in larger GABA(A)-receptor clusters. These findings show that gephyrin is associated with a majority of GABA(A)-receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy.

Animals↗
PubMed2006

Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells.

Observation and counting of gamma-H2AX foci in untreated cells as well as in cells exposed to cytotoxic agents is a widely used method for documenting the presence of double-strand breaks (DSBs) in the DNA and for analysis of their repair. Similar methods are employed to analyze formation of foci by a variety of proteins implicated in the cellular responses to DNA damage. Despite the wide application of the approach, the manual counting that is frequently used is prone to inaccuracies and investigator-related biases and artifacts. To alleviate this limitation, we developed and describe here personal computer-based algorithms, operating as utilities on available software, that allow an objective and quantitative analysis of foci from confocal images. The algorithms allow focus counting as well as size definition and correct for focus coincidence due to the overlap normally occurring with an increasing number of foci per nucleus. Furthermore, the software allows measurement of the integrated optical density (IOD) of each individual focus, which enables analysis of properties of foci as a function of time. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. A validation of the software is presented for radiation-induced gamma-H2AX foci in three widely used human cell lines and colocalization tested with RAD51 and gamma-H2AX foci. The computational methods presented extend to images generated by digital cameras.

Artificial Intelligence↗