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Results for “Subcellular localization”

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PubMed1998

Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule.

To better understand the genetic mechanisms that regulate the formation of the temporal bone, we have characterized the developmental expression pattern of the mouse gene, Brn4/Pou3f4, which plays a central role in bony labyrinth formation. Expression of this gene is initially detected in the ventral aspect of the otic capsule at 10.5 days post coitus (dpc), and correlates with the onset of mesenchymal condensation in the otic capsule. As the otic capsule condenses further and surrounds the entire otic vesicle, the Brn4 gene product is detected throughout the inner ear in the mesenchyme of both the cochlear and vestibular aspects. Early in otic embryogenesis, the Brn4 gene product is localized to the nucleus of the vast majority of cells in which it is expressed. The Brn4 gene product remains nuclear in those regions of the otic capsule that eventually give rise to the mature bony labyrinth. However, the subcellular localization of the Brn4 gene product shifts from strictly nuclear to perinuclear in those regions of the otic capsule that will cavitate to form acellular regions in the temporal bone, such as the scala tympani, scala vestibuli, and the internal auditory meatus. These data provide a detailed analysis of the expression pattern of the Brn4 gene, and provide insight into the role of the Brn4 gene product and its regulation during otic capsule formation.

Animals↗

PubMed2001

An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization.

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein beta-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.

ADP-Ribosylation Factor 1↗

PubMed1991

Subcellular localization of prostaglandin-E2 in rat heart tissue.

It has been reported that isolated rat heart myocytes and cardiac mesenchymal cells convert arachidonic acid mainly into three types of prostaglandins (PGs): PGE2, PGF2 alpha, and PGI2. In addition, we have demonstrated that fresh atrial slices of patients with heart-valve disease contain appreciable quantities of PGE2 and PGF2 alpha. However, there have been no reports on the subcellular localization of the prostaglandin system in heart muscle tissue. The present study was performed to define the distribution of PGE2 in mitochondrial, microsomal, and cytosolic fractions, isolated by differential centrifugation from homogenates of fresh normal rat atrium and ventricle slices. In addition, we determined whether differences exist in PGE2 levels between atrial subcellular fractions and those of ventricular fractions. The results showed that PGE2 was located mainly in the high-speed cytosolic supernatant fraction of the heart homogenates analyzed. Furthermore, PGE2 concentrations (ng/mg protein) were significantly higher in fractions obtained from atrial than those of ventricular tissue.

Animals↗

PubMed1975

The subcellular localization of labelled tyrosine in the vitelline cells of Schistosoma mansoni.

Autoradiographic studies at light and electron microscope level showed that tritiated tyrosine injected into the peritoneal cavity of infected mice localised in the cells of adult Schistosoma mansoni within one hour. The amino acid was avidly taken up by vitelline cells compared to other tissues. Subcellular localization was in the granular endoplasmic reticulum and in the vitelline droplets. The label was also present in the tissues of the tegument and gut but the route of entry into the parasite was not determined.

Autoradiography↗

PubMed2003

Subcellular localization of collapsin response mediator proteins to lipid rafts.

Collapsin response mediator proteins (CRMPs) are involved in signal transduction after exposure of neural cells to the axon guidance molecule Semaphorin 3A/collapsin. All five known CRMPs are expressed in the developing cerebral cortex and neocortical neurons are responsive to Semaphorin 3A. Here, we examine the expression and subcellular localization of CRMPs in neocortical neurons and in neonatal rat brain. In neocortical neurons CRMP-4 was detected in the perikaryon with a diffuse cytosolic distribution. In neurites and at growth cones punctate staining patterns were observed. Extraction of neuron cultures with methyl-beta-cyclodextrin to deplete cholesterol caused rapid redistribution of the punctate CRMP-4 staining into larger patches and abundant growth cone collapse. Western blotting of brain extracts demonstrated for all CRMPs the existence of soluble, detergent-extractable, and Triton X-100-resistant forms. Furthermore, sucrose density gradient centrifugation after solubilization of brain membranes with Triton X-100 revealed that CRMP-1, -3, -5, and to a lower extent CRMP-4 are associated with a detergent-resistant fraction with low buoyant density, but CRMP-2 was not detectable in this fraction. Thus, we propose that lipid rafts form sites for the compartmentalization of signaling events involving specific CRMPs and that the integrity of these membrane microdomains is essential for the maintenance of growth cones.

Actin Cytoskeleton↗

PubMed2004

Subcellular localization of EEN/endophilin A2, a fusion partner gene in leukaemia.

EEN (extra eleven nineteen), also known as EA2 (endophilin A2), a fusion partner of the MLL (mixed-lineage leukaemia) gene in human acute leukaemia, is a member of the endophilin A family, involved in the formation of endocytic vesicles. We present evidence to show that EEN/EA2 is localized predominantly in nuclei of various cell lines of haemopoietic, fibroblast and epithelial origin, in contrast with its reported cytoplasmic localization in neurons and osteoclasts, and that EEN/EA2 exhibits nucleocytoplasmic shuttling. During the cell cycle, EEN/EA2 shows dynamic localization: it is perichromosomal in prometaphase, co-localizes with the bipolar spindle in metaphase and anaphase and redistributes to the midzone and midbody in telophase. This pattern of distribution coincides with changes in protein levels of EEN/EA2, with the highest levels being observed in G2/M-phase. Our results suggest that distinct subcellular localization of the endophilin A family members probably underpins their diverse cellular functions and indicates a role for EEN/EA2 in the cell cycle.

Animals↗

PubMed1996

Annexins I and II show differences in subcellular localization and differentiation-related changes in human epidermal keratinocytes.

The annexins are a family of calcium-dependent phospholipid-binding proteins whose in vitro properties have led to a number of hypotheses suggesting their cellular functions, including membrane fusion in exocytosis and endocytosis. To investigate the topography and possible functions of these proteins we compared the subcellular localization of annexins I, II, IV and VI in skin sections and in cultured epidermal keratinocytes by immunostaining. We found that annexin I staining was in a granular pattern in the monolayer epithelial cells but in an envelope pattern in the stratified keratinocytes. This finding corroborates previous reports that annexin I crosslinks to form cornified envelopes in the mid-epidermis and explains the absence of staining above that level. It is unlikely that this protein is related to exocytosis in the granular layer of the epidermis. In comparison, annexin II staining was also granular and was detected in all nucleated epidermal cells as bands at the cell periphery. However, only annexin II was detected extracellularly among the top layer of cultured cells. The intracellular linear envelope pattern of annexin I and the intercellular pattern of annexin II suggest their interactions with the membrane cytoskeleton in other biological functions. Taken together, both annexins undergo different differentiation-related changes. While methanol fixation enhanced staining of annexin I, it diminished staining of annexin II. Their opposite responses to methanol fixative suggests a different molecular organization of the two annexins with phospholipid in the cell membrane. Annexins IV and VI were predominantly confined to dermal cells including ductal and myoepithelial cells and were not detected in cultured keratinocytes using either cold methanol fixative or prefixation labeling.

Annexin A1↗

PubMedDate not supplied

Subcellular localization of phosducin in rod photoreceptors.

Phosducin (Pd) is a 28-kD phosphoprotein whose expression in retina appears limited to photoreceptor cells. Pd binds to the beta,gamma subunits of transducin (Gt). Their binding affinity is markedly diminished by Pd phosphorylation. While Pd has long been regarded as a candidate for the regulation of Gt, the molecular details of Pd function remain unclear. This gap in understanding is due in part to a lack of precise information concerning the total amount and subcellular localization of rod Pd. While earlier studies suggested that Pd was a rod outer segment (ROS) protein, recent findings have demonstrated that Pd is distributed throughout the rod. In this report, the subcellular distribution and amounts of rat Pd are quantified with immunogold electron microscopy. After light or dark adaptation, retinal tissues were fixed in situ and prepared for ultrathin sectioning and immunogold labeling. Pd concentrations were analyzed over the entire length of the rod. The highest Pd labeling densities were found in the rod synapse. Less intense Pd staining was observed in the ellipsoid and myoid regions, while minimal labeling densities were found in the ROS and the rod nucleus. In contrast with rod Gt, no evidence was found for light-dependent movement of Pd between inner and outer segments. There is a relative paucity of Pd in the ROS as compared with the large amounts of Gt found there. This does not support the earlier idea that Pd could modulate Gt activity by controlling its concentration. On the other hand, the presence of Pd in the nucleus is consistent with its possible role as a regulator of transcription. The functions of Pd in the ellipsoid and myoid regions remain unclear. The highest concentration of Pd was found at the rod synapse, consistent with a suggested role for Pd in the regulation of synaptic function.

Animals↗

PubMed1987

Monoclonal antibody study of the subcellular localization and DNA-stimulating activity of murine sarcoma virus-activated transformation-associated proteins.

Previously, we reported the monoclonal antibody detection of transformation-associated proteins (TAP) in ts110 murine sarcoma virus-transformed normal rat kidney (6M2) cells (Chan et al., 1986). In this study, we used the same monoclonal antibody to investigate the subcellular localization, the fate and the mitogenic activity of TAP, as well as the correlationship between TAP synthesis and the expression of transformation properties of 6M2 cells. It was found that TAP were localized in the cytoplasm (probably the Golgi apparatus) of 6M2 cells. TAP were found as three intracellular polypeptides (mol wt of 66K, 63K, and 60K, respectively), and were rapidly released into extracellular medium. Upon release, TAP changed to two extracellular polypeptides (mol wt of 68K and 64K, respectively). Furthermore, the synthesis of TAP was temperature sensitive and correlated closely with the expression of transformation properties of the 6M2 cells. TAP have been purified by monoclonal antibody-affinity column chromatography and found to have a synergistic effect with insulin in stimulating the DNA synthesis of normal rat kidney cells.

Animals↗

PubMed2005

Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein.

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

Amino Acid Sequence↗

PubMed2003

Developmental expression and subcellular localization of mouse MATER, an oocyte-specific protein essential for early development.

We reported previously that Mater is a maternal effect gene that is required for early embryonic development beyond the two-cell stage in mice. Here we show the expressional profile of Mater and its protein during oogenesis and embryogenesis as well as its subcellular localization in oocytes. Mater mRNA was detectable earliest in oocytes of type 2 follicles, whereas MATER protein appeared earliest in oocytes of type 3a primary follicles. Both mRNA and protein accumulated during oocyte growth. In situ hybridization showed that Mater mRNA appeared progressively less abundant in oocytes beyond type 5a primary follicles. By ribonuclease protection assay, Mater mRNA was abundant in germinal vesicle oocytes, but was undetectable in all stages of preimplantation embryos. In contrast, the protein persisted throughout preimplantation development. Immunogold electron microscopic analysis revealed that MATER was located in oocyte mitochondria and nucleoli, and close to nuclear pores. Taken together, our data indicate that Mater gene transcription and protein translation are active during oogenesis, but appear inactive during early embryogenesis. Thus, Mater and its protein are expressed in a manner typical of maternal effect genes. The presence of MATER protein in mitochondria and nucleoli suggests that it may participate in both cytoplasmic and nuclear events during early development.

Amino Acid Sequence↗

PubMed2006

The intricacies of p21 phosphorylation: protein/protein interactions, subcellular localization and stability.

p21 was originally described as functioning as a cell cycle regulator via inhibition of both cyclin-dependent kinases and processive DNA replication. Nowadays it is recognized to play other fundamental roles including transcriptional regulation and the modulation of apoptosis. Each of these functions of p21 is achieved through direct p21/protein interactions and the subcellular localization of p21 plays an important part in dictating the binding partners to which p21 is exposed. Over recent years, a number of phosphorylation sites in p21 have been identified, these being targeted by several important intracellular signalling protein kinases. Here we review the state of our knowledge of p21 phosphorylation with respect to the kinases involved and the molecular biological effects of each phosphorylation event.

Animals↗

PubMed2005

Spatial and temporal changes in the subcellular localization of the nuclear protein-tyrosine kinase, c-Fes.

Tyrosine phosphorylation has emerged as a mechanism to control cellular events in the nucleus. The c-Fes protein-tyrosine kinase is an important regulator of cell growth and differentiation in several cell types, and is found in the nucleus of hematopoietic cells. In this study, we showed nuclear localization of c-Fes in both hematopoietic (K562, TF-1, HEL, U937, and HL-60) and nonhematopoietic cell lines (293T, CaOv3, TfxH, MG-63, HeLa, DU-145) by immunofluorescence and confocal microscopy. c-Fes showed striking changes in subcellular localization at specific stages of mitosis. In interphase cells, the intranuclear distribution of c-Fes was diffuse with occasional bright foci. Some c-Fes was present in the cytosol after breakdown of the nuclear membrane, in prometaphase. At prometaphase and metaphase c-Fes was also associated with the chromosomes, in a punctate pattern that partially overlapped with the centromere. Further comparison with proteins that are known components of the kinetochore suggested that some c-Fes protein was located at the centromeric alpha-satellite DNA, between the kinetochores. At anaphase and telophase, c-Fes was entirely cytoplasmic and no protein was found associated with the chromosomes. The timing of c-Fes' appearance at the centromere coincides with the period of kinetochore assembly. These data suggest that c-Fes is recruited to the kinetochore during mitosis.

Animals↗

PubMed2006

Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphorylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I.

Normal fibroblast subpopulations have differential surface expression of the GPI-linked raft protein Thy-1, which correlates with differences in cellular adhesion and migration in vitro. Thrombospondin-1 (TSP-1) induces an intermediate state of adhesion in fibroblasts and other cells which facilitates migration. TSP-1 and the hep I peptide derived from the amino-terminal/heparin-binding domain of TSP-1 induce disassembly of cellular focal adhesions. Our lab previously reported that the induction of focal adhesion disassembly in fibroblasts by TSP-1 or by hep I requires surface expression of Thy-1, as well as lipid raft integrity and Src family kinase (SFK) signaling. We now report that TSP-1/hep I-induced fibroblast migration requires Thy-1 expression and FAK phosphorylation, and that following TSP-1/hep I stimulation, Thy-1 associates with FAK and SFK in a lipid raft-dependent manner. Furthermore, the GPI anchor of Thy-1, which localizes the protein to specific lipid raft microdomains, is necessary for hep I-induced FAK and SFK phosphorylation, focal adhesion disassembly, and migration. This is the first report of an association between Thy-1 and FAK. Thy-1 modulates SFK and FAK phosphorylation and subcellular localization, promoting focal adhesion disassembly and migration in fibroblasts, following exposure to TSP-1/hep I.

Animals↗

PubMed2004

Differential subcellular localization of CD86 in human PBMC-derived macrophages and DCs, and ultrastructural characterization by immuno-electron microscopy.

We have previously reported the presence of a discrete reservoir of the costimulatory molecule CD86 in the cytoplasm of human monocytes freshly isolated from peripheral blood mononuclear cells (PBMC). In the current study, we have extended analysis of the subcellular localization of this molecule to in vitro PBMC-derived dendritic cells (DCs) and macrophages. In a sub-population of DCs, we observed by confocal microscopy an intracellular focal concentration of CD86 that bore striking similarities to that previously reported in monocytes. Further analyses revealed that this intracellular CD86 was not localized to the Golgi apparatus, MHC II compartments or endocytic structures, and required intact microtubules to retain structural integrity. A similar concentration of CD86 was not present in PBMC-derived macrophages. Electron microscopy revealed two distinct DC phenotypes containing either sparse or abundant cytoplasmic vesicles, and CD86 was found to be concentrated within the vesicular compartment of this latter phenotype. Collectively, these data not only identify and characterize a novel CD86-containing cytoplasmic compartment in human PBMC-derived DCs, but also define micro-structurally distinct DC subsets that differentially concentrate CD86 within cytoplasmic vesicles. Although the functional significance of these observations remains to be established, available evidence supports the conclusion that the focal concentration of CD86 is a storage reservoir that facilitates rapid deployment of this molecule to the DC surface when increased costimulatory capacity is required.

Antigens, CD↗

PubMed2003

NOS 3 subcellular localization in the regulation of nitric oxide production.

Endothelium-derived nitric oxide (NO) is a key signalling molecule in the maintenance of cardiovascular health. Endothelial NO synthase (NOS 3), which catalyses the formation of NO, is targeted to the plasma membrane by dual acylation. In vitro studies suggest that membrane localization of NOS 3 is an important regulatory element of NO production. Dysfunction of the vascular endothelium and a decrease in NO bioavailability is associated with the development and progression of a number of cardiovascular diseases, including hypertension. Our laboratory has previously published that in salt-dependent hypertension there is an altered localization of NOS 3, with an increase in cytosolic expression. These data have led us to question whether the increased cytosolic NOS 3 expression is a form of compensation for endothelial dysfunction in hypertension, or an indicator and contributing factor to endothelial dysfunction. This review will outline the importance of subcellular localization in the regulation of NOS 3 in vitro, the role of NOS 3 in endothelial dysfunction associated with salt-dependent hypertension, and the potential physiological consequences of altered NOS 3 localization in vivo.

Acylation↗

PubMedDate not supplied

Acid mucopolysaccharide metabolism in leprosy. 2. Subcellular localization of hyaluronic acid and beta-glucuronidase in leprous infiltrates suggestive of a host-Mycobacterium leprae metabolic relationship.

Electron- and light microscopic analyses were conducted on leprosy skin biopsies relative to the origin of hyaluronic acid, which has previously been observed to be distributed inversely in ratio to the degree of cell- mediated immunity. The present study investigated the subcellular localization of hyaluronic acid and its degrading enzyme in various types of leprosy. Hyaluronic acid in some lepromatous leprosy cases was shown to be accumulated in the limiting membranes of the phagosomes of lepra cells and Myco-bacteria leprae have beta-glucuronidase which plays a role in the degradation of hyaluronic acid. Contrariwise, in tuberculoid leprosy, beta-glucuronidase was detected in the lysosomes of epithelioid cells and giant cells. This result suggests that the origin of hyaluronic acid is in histiocytes and at the same time it might suggest that M. leprae is in competition with enzymes of epithelioid cells for hyaluronic acid, whereas reduced or absent beta-glucuronidase in lepra cells enable bacilli to utilize the AMPS as a nutrient.

Acetylglucosaminidase↗

PubMed1985

Subcellular localization of thyroxine 5'-deiodinase activity in bovine anterior pituitary.

Bovine anterior pituitary glands were fractionated by differential centrifugation. T4 5'-monodeiodination to T3 was found predominantly in microsomal fractions (M2; 105,000 . g pellet) enriched in glucose-6-phosphatase and 5'-nucleotidase activities. T4 5'-deiodinase activity in M2 fraction was 85.2 fmol T3/min X mg protein and represented an 8.5-fold enrichment over homogenate specific activity (10.6 fmol T3/min . mg protein). Further subcellular localization of the T4 5'-deiodinase was effected by discontinuous sucrose density gradient centrifugation. Maximum T4 5'-deiodinase activity was found in fraction P5 at the interface of densities 1.18/1.20 (200 fmol T3/min . mg protein) and correlated with the profile of glucose-6-phosphatase and not with that of 5'-nucleotidase, the maximum activity of which was recovered in fraction P1 at the interface of densities 1.03/1.12. Electron microscopic examination of the fractions confirmed that P5 contained in excess of 90% rough membranes in contrast to 10% or less in P1. Characterization of T4 5'-deiodinase activity was carried out in M2 preparations. The reaction was thiol dependent, requiring the presence of 50 mM dithiothreitol or more (Km, 38 mM), with a maximum velocity of 55-150 fmol T3/min . mg protein (n = 8). Enzyme activity was substrate dependent, with a Km for T4 between 35-70 nM. 5'-Monodeiodination of T4 was abolished by heating to 70 C for 30 min and was unaffected by EDTA. Propylthiouracil and methimazole did not inhibit T3 generation. Iopanoic acid, on the other hand, was a competitive inhibitor of the 5'-monodeiodination reaction, abolishing T3 production in a dose-dependent manner with a Ki of 3 microM. These data indicate that the bovine anterior pituitary contains significant T4 5'-deiodinase activity, which shares many properties of the type II 5'-deiodinase of the rat. Bovine anterior pituitary T4 5'-deiodinase appears to be predominantly localized in the rough endoplasmic reticulum.

Animals↗