Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “colocalization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources
PubMed2006

Interacting molecule of AT1 receptor, ATRAP, is colocalized with AT1 receptor in the mouse renal tubules.

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2006

TCF-4 isoforms absent in TCF-4 mutated MSI-H colorectal cancer cells colocalize with nuclear CtBP and repress TCF-4-mediated transcription.

TCF-4 is the main effector of the Wnt/Wingless signalling pathway. As with other TCF/LEF factors, numerous alternative splicings at its 3' end affect its expression. Such a mechanism leads to the synthesis of numerous TCF-4 isoforms among which some contain binding domains for CtBP, an ubiquitous transcriptional corepressor. Of interest, we described a frequent TCF-4 frameshift mutation in mismatch-repair deficient colorectal cancers (MSI-H cancers) that leads to the selective loss of TCF-4 isoforms with CtBP binding abilities. We provide here data that argue for a partial colocalization of CtBP with TCF-4 isoforms containing CtBP binding domains in cellulo, and for a functional role of CtBP in repressing TCF-4 mediated transcription. We also demonstrate that such a colocalization is not observed in MSI-H colorectal cancer cells that harbour the TCF-4 frameshift mutation, and that CtBP is not able to repress TCF-4-mediated transcription in this context. Taken together, our results strongly suggest that CtBP would play a role in regulating TCF-4 mediated transcription upon its binding with some TCF-4 isoforms encoded by alternatively spliced mRNA. They also suggest a role for TCF-4 frameshift mutation during MSI-H colorectal tumour progression, by regulating the relative proportion of the different TCF-4 isoforms.

Alcohol Oxidoreductases↗
PubMed2001

Increased colocalization of corticotropin-releasing factor and arginine vasopressin in paraventricular neurones of the hypothalamus in lactating rats: evidence from immunotargeted lesions and immunohistochemistry.

In lactating female rats, tonically elevated glucocorticoid secretion is accompanied by blunted stress responsiveness, reduced expression of hypothalamic corticotropin-releasing factor (CRF) mRNA and modest increases in arginine vasopressin (AVP) expression in the paraventricular nucleus (PVN). To determine the relative contribution of CRF and AVP to parvocellular function, we performed selective CRF (CRF-Tx) or AVP (AVP-Tx) lesions in the PVN neurones of ovariectomized virgin or lactating females (day 2 of lactation) by using ricin A associated with monoclonal antibodies directed towards CRF or AVP. We also performed double immunohistochemical labelling of CRF and AVP in the PVN of control rats injected with immunoglobulin (Ig)Gs associated with the ricin A (IgG-Tx). Brains were collected 12 days after the lesion and processed for in situ hybridization of CRF and AVP mRNA or for double fluorescence CRF and AVP immunohistochemistry. We found that lactating females exhibit a high degree of CRF and AVP colocalization in parvocellular PVN neurones, hypothalamic processes and median eminence terminals compared to virgins. While CRF mRNA is significantly reduced in lactating rats, AVP mRNA and protein levels are greatly enhanced in parvocellular PVN neurones during lactation. Hypothalamic CRF or AVP ricin-A lesions significantly reduced both CRF and AVP expression (15-35% decrease) as well as peptide immunoreactivity in PVN neurones in both groups of females. The specificity of the lesions varied between virgins and lactators since in virgin females, AVP-Tx did not affect CRF mRNA expression whereas in lactating females, this same lesion significantly reduced CRF mRNA expression, suggesting that parvocellular PVN neurones are more sensitive to the effects of the lesions during lactation. In both virgins and lactators, lesion with CRF-Tx tended to increase AVP mRNA expression; however, in virgins, parvocellular PVN neurones were possibly compensating for the loss of CRF synthesis by increasing AVP expression and immunoreactivity. We conclude that lactation is associated with a high degree of CRF and AVP colocalization in parvoPVN neurones and that the increased AVP production in these neurones increases their sensitivity to immunotargeted lesions. The opposite regulation of CRF and AVP gene expression during lactation might provide a useful model to study differential sensitivity to glucocorticoid feedback or hypothalamic activation of transcription factors.

Animals↗
PubMed1998

Brain Ac39/physophilin: cloning, coexpression and colocalization with synaptophysin.

Physophilin is an oligomeric protein that binds the synaptic vesicle protein synaptophysin constituting a complex that has been hypothesized to form the exocytotic fusion pore. Microsequencing of several physophilin peptides putatively identified this protein as the Ac39 subunit of the V-ATPase. Ac39 has recently been shown to be present in a synaptosomal complex which, in addition to synaptophysin, includes the bulk of synaptobrevin II, and subunits c and Ac115 of the V0 sector of the V-ATPase. We have cloned physophilin from mouse brain and found a differential region of 12 amino acids when compared with the previously reported sequence of Ac39 from bovine adrenal medulla. RT-PCR cloning from the bovine adrenal medulla demonstrates that sequencing errors occurred in the previous cloning study, and shows that the amino acid sequences of physophilin and Ac39 are completely identical. In situ hybridization in rat brain reveals a largely neuronal distribution of Ac39/physophilin mRNA which spatio-temporally correlates with those of subunit c and synaptophysin. Immunohistochemical analysis shows that Ac39/physophilin is mostly concentrated in the neuropil with a pattern identical to subunit A and very similar to synaptophysin. Double-labelling immunofluorescence shows a complete colocalization of Ac39/physophilin with subunit A and a partial colocalization with synaptophysin in the neuropil. Our findings bring anatomical support for the in vivo occurrence of the synaptophysin-Ac39/physophilin interaction and further suggest a coordinated transcription of V-ATPase and synaptophysin genes. A putative role of Ac39/physophilin in the inactivation of the V-ATPase by disassembly of its V1 sector is also discussed.

Amino Acid Sequence↗
PubMed1996

Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells: evidence for a role of GLP-1 receptor agonists as an inhibitory signal for food and water intake.

This study was designed to determine the possible role of brain glucagon-like peptide-1 (GLP-1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP-1 receptors, glucokinase, and GLUT-2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose-sensitive neurons mediating feeding behavior. Because GLP-1 receptors, GLUT-2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic beta cells, the colocalization of specific GLP-1 receptors and glucose sensing-related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP-1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP-1 (7-36) amide did not modify food and water intake, although a dose-dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP-1 (7-36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP-1 (1-37), a peptide with lower biological activity than GLP-1 (7-36) amide, did not change feeding behavior in food-deprived animals. Exendin-4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP-1 (7-36) amide. It decreased, in a dose-dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP-1 receptor antagonist [exendin (9-39); 2,500 ng] reversed the inhibitory effects of GLP-1 (7-36) amide (1,000 ng dose) and exendin-4 (25 ng dose) on food and water ingestion. These findings suggest that GLP-1 (7-36) amide may modulate both food and drink intake in the rat through a central mechanism.

Animals↗
PubMed1999

Immunohistochemical distribution of RGS7 protein and cellular selectivity in colocalizing with Galphaq proteins in the adult rat brain.

Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins alphai/o and aq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Galphaq/11, an important G protein-coupled receptor signal transducer for phospholipase Cbeta-mediated activity. In general, both RGS7 and Galphaq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Galphaq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Galphaq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Galphaq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Galphaq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.

Age Factors↗
PubMed2000

Adenosine A(1) receptor in cultured neurons from rat cerebral cortex: colocalization with adenosine deaminase.

Adenosine A(1) receptors (A(1)Rs) have been characterized in primary cultures of neurons from cerebral cortex. The specific adenosine A(1) antagonist 8-cyclopentyl-1,3-[(3)H]dipropylxanthine bound to both membranes and intact cells. When saturation experiments were performed in membranes, a K(D) value of 0.76 nM and a B(max) of 57 fmol/mg of protein were obtained. Competition assays revealed a pharmacological profile characteristic of A(1)Rs. The presence of this receptor was further confirmed by RT-PCR analysis. The expression of the receptor showed no significant changes during the period of culture studied, up to 12 days in vitro. A(1)R agonist inhibited forskolin-stimulated adenylyl cyclase, showing the functional coupling of these receptors with the effector. alphaG(i1, 2) protein level, detected by immunoblot, presented an increase during the period of culture. This increase correlated with an increase in the mRNA level of alphaG(i1) but not alphaG(i2). By immunochemical assays, it is shown that these receptors are expressed in both the neuronal cell body and the proximal dendrites. Colocalization of A(1)Rs with microtubule-associated protein 2 and cell surface adenosine deaminase was shown by confocal microscopy. The high degree of colocalization observed between A(1)Rs and ectoadenosine deaminase in neurons could suggest an important role of the enzyme in adenosine-mediated neuromodulation.

Adenosine Deaminase↗
PubMed2003

Cystatin C colocalizes with amyloid-beta and coimmunoprecipitates with amyloid-beta precursor protein in sporadic inclusion-body myositis muscles.

Cystatin C (CC), an endogenous cysteine protease inhibitor, is accumulated within amyloid-beta (A beta) amyloid deposits in Alzheimer's disease (AD) brain and was proposed to play a role in the AD pathogenesis. Because the chemo-morphologic muscle phenotype of sporadic inclusion-body myositis (s-IBM) has several similarities with the phenotype of AD brain, including abnormal accumulation of A beta deposits, we studied expression and localization of CC in muscle biopsies of 10 s-IBM, and 16 disease- and five normal-control muscle biopsies. Physical interaction of CC with amyloid-beta precursor protein (A beta PP) was studied by a combined immunoprecipitation/immunoblotting technique in the s-IBM muscle biopsies and in A beta PP-overexpressing cultured human muscle fibers. In all s-IBM muscle biopsies, CC-immunoreactivity either colocalized with, or was adjacent to, the A beta-immunoreactive inclusions in 80-90% of the vacuolated muscle fibers, mostly in non-vacuolated regions of their cytoplasm. Ultrastructurally, CC immunoreactivity-colocalized with A beta on 6-10 nm amyloid-like fibrils and floccular material. By immunoblotting, CC expression was strongly increased in IBM muscle as compared to the controls. By immunoprecipitation/immunoblotting experiments, CC coimmunoprecipitated with A beta PP, both in s-IBM muscle and in A beta PP-overexpressing cultured normal human muscle fibers. Our studies (i) demonstrate for the first time that CC physically associates with A beta PP, and (ii) suggest that CC may play a novel role in the s-IBM pathogenesis, possibly by influencing A beta PP processing and A beta deposition.

Amyloid beta-Peptides↗
PubMed2004

Genetic inactivation of prohormone convertase (PC1) causes a reduction in cholecystokinin (CCK) levels in the hippocampus, amygdala, pons and medulla in mouse brain that correlates with the degree of colocalization of PC1 and CCK mRNA in these structures in rat brain.

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

Amygdala↗
PubMed2001

Colocalization of ANCA-antigens and fibrinoid necrosis in ANCA-associated vasculitis.

A variety of antineutrophil cytoplasmic auto-antibodies (ANCAs) are known to be associated with small vessel vasculitides such as Wegener's granulomatosis and microscopic polyangiitis. To visualize colocalization patterns of the fibrinoid necrotic lesions and ANCA-antigens more accurately, we have developed a double staining technique in which an immunohistochemical staining is followed by a histological staining. Instead of using sequential biopsy slides of histologically and immunohistochemically stained sections, which may lead to an underestimation of the number and size of the lesions, our technique permits the visualization of the colocalized patterns of fibrinoid necrosis with an ANCA-antigen in a single slide. The double staining procedure is presented in this Technical Note.

Adult↗
PubMed2006

Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster.

Regulation of gene expression is a highly complex process that requires the concerted action of many proteins, including sequence-specific transcription factors, cofactors, and chromatin proteins. In higher eukaryotes, the interplay between these proteins and their interactions with the genome still is poorly understood. We systematically mapped the in vivo binding sites of seven transcription factors with diverse physiological functions, five cofactors, and two heterochromatin proteins at approximately 1-kb resolution in a 2.9 Mb region of the Drosophila melanogaster genome. Surprisingly, all tested transcription factors and cofactors show strongly overlapping localization patterns, and the genome contains many "hotspots" that are targeted by all of these proteins. Several control experiments show that the strong overlap is not an artifact of the techniques used. Colocalization hotspots are 1-5 kb in size, spaced on average by approximately 50 kb, and preferentially located in regions of active transcription. We provide evidence that protein-protein interactions play a role in the hotspot association of some transcription factors. Colocalization hotspots constitute a previously uncharacterized type of feature in the genome of Drosophila, and our results provide insights into the general targeting mechanisms of transcription regulators in a higher eukaryote.

Animals↗
PubMed2000

Ultrahigh-resolution multicolor colocalization of single fluorescent probes.

An optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described in this paper. It relies on the use of two unique families of fluorophores, namely energy-transfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A multicolor sample-scanning confocal microscope was constructed that allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps with a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread function of the microscope. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3-10 nm in diameter) and demonstrate distance-measurement accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near- and far-field imaging, spanning a range of a few nanometers to tens of micrometers.

Color↗
PubMed1990

Colocalization of galanin and luteinizing hormone-releasing hormone in a subset of preoptic hypothalamic neurons: anatomical and functional correlates.

Colocalization of neurotransmitters, including neuropeptides and amines, in the same neuron of certain areas or well-defined nuclei of the central and peripheral nervous systems appears to be the rule rather than the exception. The coexistent neurotransmitters can be coreleased and interact at pre- and postsynaptic levels in a synergistic or antagonistic manner. Galanin is a recently isolated and characterized "gut-brain" peptide. It is colocalized with many neurotransmitters in both the central and the peripheral nervous systems. Among other regions in the central nervous system, galanin is present in neuronal perikarya of the septum and the hypothalamus. The dense accumulation of nerve terminals in the external zone of the median eminence suggests that galanin is an important peptide regulating neuroendocrine functions. Although most galanin and luteinizing hormone-releasing hormone (LHRH) neurons have a distinctly different morphology, a subset of galanin-immunoreactive perikarya in the diagonal band of Broca and the medial preoptic area, near the organum vasculosum of the lamina terminalis, have morphological features similar to those of LHRH neurons. By using double-labeling immunocytochemistry, we have found that in the preoptic region of the male rat brain approximately 15-20% of these "LHRH-like" galanin-immunoreactive neurons are also immunopositive for LHRH. Moreover, in the medial preoptic area and the diagonal band of Broca, some of the single-labeled LHRH cells are surrounded with galanin-immunoreactive nerve terminals, suggesting that LHRH perikarya have synaptic contacts with galanin-immunoreactive terminals. Additional studies indicated that galanin can readily enhance in vitro release of LHRH from nerve terminals in the median eminence. The observations that (i) galanin is coexpressed with LHRH, (ii) galanin seems to innervate LHRH-producing neurons, and (iii) galanin acts as a putative neurotransmitter to enhance the release of LHRH suggest that galanin should be considered an important regulator of LHRH-containing neurons and, therefore, of reproductive functions.

Animals↗
PubMed1991

Colocalization in pericentral hepatocytes in adult mice and similarity in developmental expression pattern of ornithine aminotransferase and glutamine synthetase mRNA.

In situ hybridization showed that the mRNA for ornithine aminotransferase (OAT; ornithine-oxo-acid aminotransferase; L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13) colocalized with glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] in pericentral hepatocytes of the adult mouse liver. In addition to an identical distribution in adult hepatocytes, OAT and GS have very similar expression patterns in fetal and neonatal liver. As was earlier described for GS, there is a low level of OAT mRNA in fetal cells and increasing pericentral levels in neonates that reach adult patterns within 2 weeks. These results suggest that the transcriptional regulation of the two genes is similar in the liver. However, there was a lack of colocalization of the mRNAs for the two enzymes in cells of the kidney, intestine, and brain, suggesting different regulatory decisions for the OAT and GS genes in the cells of these different tissues. The metabolic consequences of these localized expression patterns favor ammonia clearance from the blood by the liver and urea synthesis by the kidney.

Age Factors↗
PubMed1992

Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain.

The rodent and primate basal forebrain is a target of a family of endogenous peptide signaling molecules, the neurotrophins--nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3--and of the gonadal steroid hormone estrogen, both of which have been implicated in cholinergic function. To investigate whether or not these ligands may act on the same neurons in the developing and adult rodent basal forebrain, we combined autoradiography with 125I-labeled estrogen and either nonisotopic in situ hybridization histochemistry or immunohistochemistry. We now report colocalization of intranuclear estrogen binding sites with the mRNA and immunoreactive protein for the low-affinity nerve growth factor receptor, which binds all three neurotrophins, and for the cholinergic marker enzyme choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Colocalization of estrogen and low-affinity nerve growth factor receptors implies that their ligands may act on the same neuron, perhaps synergistically, to regulate the expression of specific genes or gene networks that may influence neuronal survival, differentiation, regeneration, and plasticity. That cholinergic neurons in brain regions subserving cognitive functions may be regulated not only by the neurotrophins but also by estrogen may have considerable relevance for the development and maintenance of neural substrates of cognition. If estrogen-neurotrophin interactions are important for survival of target neurons, then clinical conditions associated with estrogen deficiency could contribute to the atrophy or death of these neurons. These findings have implications for the subsequent decline in those differentiated neural functions associated with aging and Alzheimer disease.

Animals↗
PubMed1994

Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.

We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.

Animals↗
PubMed1996

Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein.

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

Base Sequence↗
PubMed1996

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

Animals↗