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PubMed1999

Subcellular localization of RhoA and ezrin at membrane ruffles of human endothelial cells: differential role of collagen and fibronectin.

Endothelial cells and the regulation of their migration are of prime importance in many physiological and pathological processes such as angiogenesis. RhoA, an important Rho family member known to trigger actin reorganization, has been shown to mediate the formation of focal adhesions and stress fibers in quiescent fibroblasts. However, recent studies have emphasized its functional diversity and its implication in migration or metastatic processes in different cell types other than fibroblasts. Its role in endothelial cells is little known. In this study, we were interested by analyzing in human endothelial cells the subcellular redistribution of endogenous RhoA and the reorganization of cytoskeletal actin induced by two important extracellular matrix proteins, collagen and fibronectin. This paper shows a translocation of RhoA and its association with cortical actin in focal contact domains at membrane ruffles and at lamellipodia of spread or migrating endothelial cells, in the absence of any soluble mitogen stimulation. Furthermore, RhoA was found colocalized with ezrin, a member of the ERM family proteins newly described as important membrane-actin cytoskeleton linkers, at early membrane ruffles of endothelial cells spread on collagen but not on fibronectin. The present study points out that extracellular matrix, depending on the nature of its components, may promote distinct assemblies of focal contact constitutive proteins and strongly suggests that endothelial RhoA, like Rac1, may be an important mediator of matrix signaling pathway regulating endothelial cell adhesiveness and motility, independently of growth factor stimulation.

Actins↗
PubMed2005

Cellular and subcellular localization of kainic acid in the marine red alga Digenea simplex.

Polyclonal antibodies specific for the excitatory amino acid, kainic acid (KA), were raised in rabbits. The antibody recognized KA but did not cross-react with other structurally related amino acids, including glutamate. We used this anti-KA antibody to localize KA immunohistochemically in the KA-producing red alga Digenea simplex. KA immunoreactivity was most dense in the fine cylindrical thallus, which covers the middle to upper part of the alga. The cortical cells, but not the inner layers of the main axis, and cells of the rhizoid were also stained with this antibody. The presence of KA in cells that cover the surface of the alga might reflect its role in chemical defense. At the subcellular level, KA immunoreactivity was most intense in the nucleus, pit plugs, and the electron-dense areas denoted as "granule bodies", which were found only in the pericentral cells of the thallus.

Immunohistochemistry↗
PubMed1978

The subcellular localization of the long-acting thyroid stimulator inhibitor in bovine thyroid gland.

To characterize further the nature and site of the receptor for the IgG stimulator in Graves' disease, we have developed a preparative scheme to provide a bovine thyroid subfraction rich in plasma membranes. Pellets (800 X g) obtained from bovine thyroid homogenates were layered on a 30-64% continuous sucrose gradient (SG). The centrifuged gradient was divided into four portions (SG1, 2, 3, and 4); and these along with the 800 X g pellet were characterized in terms of their capacity to neutralize (bind) the biological activity of LATS, their specific binding of [125I]TSH, and their subcellular marker content. Subfraction SG1 contained the highest adenyl cyclase specific activity, the highest [125I]TSH binding specific activity, and vied with the 800 X g pellet and SG3 for the highest specific activity of LATS neutralization. Electron microscopy of SG1 showed a predominance of plasma membrane structures contaminated with a modest amount of cellular debris. Adenyl cyclase activity in SG1 was enhanced by TSH, LATS, and sodium fluoride. Although a dose-response curve could be established for TSH, a similar relationship could not be established for LATS. We conclude that activation of plasma membrane-bound adenyl cyclase is associated with neutralization of the biological activity of LATS. Further, the difference between [125I]TSH binding and LATS neutralization activity observed in the various thyroid membrane subfractions suggests that either LATS and TSH interact at different sites or have different mechanisms of binding at a common site.

Adenylyl Cyclases↗
PubMed1989

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2.

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.

Amino Acid Sequence↗
PubMed2005

Subcellular localization determines MAP kinase signal output.

The Raf-MEK-ERK MAP kinase cascade transmits signals from activated receptors into the cell to regulate proliferation and differentiation. The cascade is controlled by the Ras GTPase, which recruits Raf from the cytosol to the plasma membrane for activation. In turn, MEK, ERK, and scaffold proteins translocate to the plasma membrane for activation. Here, we examine the input-output properties of the Raf-MEK-ERK MAP kinase module in mammalian cells activated in different cellular contexts. We show that the MAP kinase module operates as a molecular switch in vivo but that the input sensitivity of the module is determined by subcellular location. Signal output from the module is sensitive to low-level input only when it is activated at the plasma membrane. This is because the threshold for activation is low at the plasma membrane, whereas the threshold for activation is high in the cytosol. Thus, the circuit configuration of the module at the plasma membrane generates maximal outputs from low-level analog inputs, allowing cells to process and respond appropriately to physiological stimuli. These results reveal the engineering logic behind the recruitment of elements of the module from the cytosol to the membrane for activation.

Animals↗
PubMed2001

Subcellular localization of a glutathione-dependent dehydroascorbate reductase within specific rat brain regions.

Recently, we described the occurrence of a dehydroascorbate reductase within the rat CNS. This enzyme regenerates ascorbate after it is oxidized during normal aerobic metabolism. In this work, we describe the neuronal compartmentalization of the enzyme, using transmission electron microscopy of those brain areas in which the enzyme was most densely present when observed under light microscopy. In parallel biochemical studies, we performed immunoblotting and measured the enzyme activity of the cytoplasm and different nuclear fractions. Given the abundance of ascorbate in the caudate-putamen, we focused mostly on the occurrence of dehydroascorbate reductase at the striatal subcellular level. We also studied cerebellar Purkinje cells, hippocampal CA3 pyramidal cells and giant neurons in the magnocellular part of the red nucleus. In addition to neurons, immunolabeling was found in striatal endothelial cells, in the basal membrane of blood vessels and in perivascular astrocytes. In neuronal cytosol, the enzyme was observed in a peri-nuclear position and on the nuclear membrane. In addition, in both the striatum and the cerebellum, we found the enzyme within myelin sheets. Dehydroascorbate reductase was also present in the nucleus of neurons, as further indicated by measuring enzyme activity and by immunoblotting selected nuclear fractions. Immunocytochemical labeling confirmed that the protein was present in isolated pure nuclear fractions. Given the great amount of free radicals which are constantly generated in the CNS, the discovery of a new enzyme with antioxidant properties which translocates into neuronal nuclei appears to be a potential starting point to develop alternative strategies in neuroprotection.

Animals↗
PubMed1989

Subcellular localization of superoxide dismutases, glutathione peroxidase and catalase in developing rat cerebral cortex.

The levels of copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity have been assessed in a nuclear fraction (NF), a mitochondrial fraction (MF) and a non-mitochondrial, non-nuclear fraction (NMNNF) isolated from developing rat cerebral cortex. The NF showed increasing CuZn and MnSOD activities and static, low activities of GSH-Px and CAT during development. The MF had increased levels of MnSOD and GSH-Px activities and a rapid decrease in CAT activity associated with development. Histochemical methods have localized greater CAT activity in mitochondria isolated from 2-day-old rat brain when compared to 77-day-old animals. Development was associated with increasing CuZnSOD activity, a decrease in CAT activity and, after an initial fall at 19 days, increasing GSH-Px activity in the NMNNF. Measurable activity of MnSOD were found in the NMNNF and appeared to be static during the time period assessed. A distinct ontogenetic pattern of oxidative enzyme activities and subcellular locations is associated with development in rat cerebral cortex.

Age Factors↗
PubMed1996

Changing patterns of expression and subcellular localization of TrkB in the developing visual system.

Neurotrophins play important roles in the survival, differentiation, and maintenance of CNS neurons. To begin to investigate specific roles for these factors in the mammalian visual system, we have examined the cellular localization of the neurotrophin receptor trkB within the developing cerebral cortex and thalamus of the ferret using extracellular domain-specific antibodies. At prenatal ages (gestation is 41 d), trkB-immunostained fibers were observed in the internal capsule and as two distinct fascicles within the intermediate zone of the cerebral cortex. The staining of these fiber tracts declined with increasing age, whereas soma and dendrite staining of cortical neurons was first evident in early postnatal life and increased during subsequent development. Staining of subplate neurons [by prenatal day 5 (P5)] was followed by staining of cortical layer 5 neurons (at P10). By P31, trkB immunoreactivity was particularly prominent in layers 3 and 5 but was absent from subplate neurons. Staining included cells, especially pyramidal neurons, in all cortical layers by P45, and this pattern was maintained into adulthood. The optic tract and fibers within the lateral geniculate nucleus (LGN) were also strongly trkB immunoreactive at prenatal ages. Cellular staining of a subset of LGN neurons, those within the C-layers and perigeniculate nucleus, was apparent by P10 and maintained until P45, when the adult pattern of highly trkB-immunoreactive neurons in all layers of the LGN first appeared. The pattern of trkB immunoreactivity suggests that specific subsets of cortical and thalamic neurons may respond to neurotrophins such as brain-derived neurotrophic factor and/or NT-4/5 at discrete developmental times and locations. The appearance of trkB on axon fibers early in development and then on cell bodies and dendritic processes later is consistent with roles for both long-range and local, including autocrine and/or paracrine, delivery of neurotrophins in cell survival and maturation.

Aging↗
PubMed2004

Tissue- and developmental stage-specific changes in the subcellular localization of the 26S proteasome in the ovary of Drosophila melanogaster.

The intracellular localization of the 26S proteasome in the different ovarian cell types of Drosophila melanogaster was studied by means of immunofluorescence staining and laser scanning microscopy, with the use of antibodies specific for regulatory complex subunits or the catalytic core of the 26S proteasome. During the previtellogenic phase of oogenesis (stages 1-6), strong cytoplasmic staining was observed in the nurse cells and follicular epithelial cells, but the proteasome was not detected in the nuclei of these cell types. The subcellular distribution of the 26S proteasome was completely different in the oocyte. Besides a constant, very faint cytoplasmic staining, there was a gradual nuclear accumulation of proteasomes during the previtellogenic phase of oogenesis. A characteristic subcellular redistribution of the 26S proteasome occurred in the ovarian cells during the vitellogenic phase of oogenesis. There was a gradual decline in the concentration of the 26S proteasome in the nucleus of the oocyte, and in the stage 10 oocyte the proteasome could barely be detected in the nucleus. This was accompanied by a massive nuclear accumulation of proteasomes in the follicular epithelial cells. These results demonstrate that the subcellular distribution of the 26S proteasome in higher eukaryotes is strictly tissue- and developmental stage-specific.

Animals↗
PubMed1997

Calcium- and myristoyl-dependent subcellular localization of the neuronal calcium-binding protein VILIP in transfected PC12 cells.

Wild-type neuronal calcium-binding protein VILIP (visinin-like protein), and a myristoylation mutant of VILIP which lacks the consensus sequence for N-terminal myristoylation, have been stably transfected in PC12 cells. Immunocytochemical studies of VILIP-transfected PC12 cells have revealed the wild-type VILIP is strongly concentrated at the cell membrane, particularly at cell-cell contact sites, but is also distributed throughout the cytosol at moderate levels. In contrast, myristoylation-mutant VILIP shows a more even distribution, with significantly less association at cell-cell contact sites. Western blot analysis of subcellular fractions has shown that wild-type VILIP associates in a calcium-dependent manner with membrane fractions, whereas the myristoylation mutant only weakly associates with this fraction. Therefore, a calcium-myristoyl switch seems to be a major, but not sole determinant for the association of VILIP with membranes in living cells.

Animals↗
PubMed2003

Multiple luteinizing hormone receptor (LHR) protein variants, interspecies reactivity of anti-LHR mAb clone 3B5, subcellular localization of LHR in human placenta, pelvic floor and brain, and possible role for LHR in the development of abnormal pregnancy, pelvic floor disorders and Alzheimer's disease.

Distinct luteinizing hormone receptor (LHR) protein variants exist due to the posttranslational modifications. Besides ovaries, LHR immunoreactivity (LHRI) was also found in other tissues, such as the brain, fallopian tube, endometrium, trophoblast and resident tissue macrophages. The 3B5 mouse monoclonal antibody was raised against purified rat LHR. In rat, porcine and human ovaries, the 3B5 identified six distinct LHR bands migrating at approximately 92, 80, 68, 59, 52 and 48 kDa. Characteristic LHRI was detected in rat, human and porcine corpora lutea. During cellular differentiation, subcellular LHR distribution changed from none to granular cytoplasmic, perinuclear, surface, nuclear and no staining. There were also differences in vascular LHR expression--lack of LHRI in ovarian vessels and strong staining of vessels in other tissues investigated. In normal human term placentae, villous LHRI was associated with blood sinusoids and cytotrophoblast cells, and rarely detected in trophoblastic syncytium. In all abnormal placentae, the LHRI of sinusoids was absent, and syncytium showed either enhanced (immature placental phenotypes) or no LHRI (aged placental phenotype). LHRI in human brain was identified in microglial cells (CD68+ resident macrophages). Protein extracts from human vaginal wall and levator ani muscle and fascia showed strong approximately 92 and 68 kDa species, and LHRI was detected in smooth muscle cells, fibroblasts, resident macrophages and nuclei of skeletal muscle fibers. Our observations indicate that, in contrast to the theory on the role of vascular hormone receptors in preferential pick up of circulating hormones, there is no need to enhance selective pick up rather only prevent LH/CG transport to inappropriate sites. Abnormal placental LHR expression may play a role in the development of abnormal pregnancy. Expression of LHR in the pelvic floor compartments suggests that high LH levels in postmenopausal women may contribute to the pelvic floor relaxation and increased incidence of pelvic floor disorders. Since chorionic gonadotropin increases secretion of a variety of cytokines by monocytes, and induces their inflammatory reaction and phagocytic activity, high LH levels in aging individuals may also activate microglia (mononuclear phagocyte system in the central nervous system) and contribute to the development of Alzheimer's disease and other inflammation-mediated neurodegenerative diseases.

Alzheimer Disease↗
PubMed1980

Subcellular localization of the sedimentable form of mammary tumor glycoprotein to the tumor cell plasmalemma.

A form of mammary tumor glycoprotein (MTGP) has been observed to be associated with the sedimentable fraction of homogenates of human carcinomas of the breast. A MTGP-containing breast carcinoma cell line, HS-578T (AY-726), containing both soluble and sedimentable forms of MTGP, was fractionated by differential ultracentrifugation, discontinuous sucrose gradient and on a polyethylene glycol-dextran gradient. The separated crude nuclei fraction, crude mitochondrial fraction, disrupted organelles, ribosomes and rough microsomes, smooth microsomes, and plasmalemma were characterized by electron microscopy and enzymatic markers which confirmed that each fraction was highly enriched in the respective subcellular constituents. Analysis of insoluble MTGP demonstrated significant coisolation with the plasmalemma. On the basis of the present data showing that the sedimentable form of MTGP is firmly associated with the plasmalemma and previous evidence showing the specificity of MTGP for breast carcinoma cells and the surface exposure of MTGP on viable cells, we conclude that sedimentable MTGP is an integral membrane glycoprotein that could have biological significance in the behavior or immunobiology of these tumors.

Breast Neoplasms↗
PubMed1997

Subcellular localization of the alpha 7 nicotinic receptor in rat cerebellar granule cell layer.

The distribution of the alpha 7 nicotinic receptor subunit in the rat cerebellum was studied immunohistochemically at the electron microscope level using an alpha 7 subunit-specific antibody. The granule cell layer showed a much lower level of immunoreactivity for the alpha 7 subunit than the Purkinje cell layer. Granule cell somata were completely devoid of labeling; this appeared to be restricted to glomeruli exclusively located in the membranes of granule cell dendrites. The alpha 7 immunolabeling was located not at active synaptic areas but was mostly perisynaptic. This localization suggests that nicotinic receptors containing the alpha 7 subunit could have a modulatory function and/or play a direct role in the generation of synaptic currents.

Animals↗
PubMed1992

Dual subcellular localization of sialidase in cultured granule cells differentiated in culture.

A rapid small-scale procedure was set up to obtain highly purified preparations of lysosomes and plasma membranes from the homogenate of cerebellar granule cells differentiated in culture. It consisted in a centrifugation of the postnuclear fraction P2, on a Percoll gradient with formation of an upper and lower band. The upper band, upon centrifugation on 1 M sucrose, produced a light band lying on the top, that constituted the plasma membrane preparation. The upper band constituted the lysosome preparation. The plasma membrane preparation exhibited a 6-fold relative specific activity increase of Na+, K(+)-ATPase and 5'-nucleotidase, with negligible contamination by other subcellular markers; the lysosomal preparation exhibited a 30-fold relative specific activity increase of beta-galactosidase and beta-hexosaminidase, with virtually no contamination by other subcellular markers. Both the lysosome and plasma membrane preparations carried sialidase activity on MUB-NeuNAc and ganglioside GD1a. The sialidase activity on GD1a required the presence of Triton X-100 in both subcellular preparations; the sialidase activity on MUB-NeuNAc was markedly activated by albumin only in the lysosomes. The lysosomal sialidase had a unique optimal pH value, 3.9. The plasma membrane sialidase featured two values of optimal pH, one at 3.9, for both substrates and second at 5.4 and 6.0 for MUB-NeuNAc and GD1a, respectively. It is concluded that cerebellar granule cells differentiated in vitro possess one lysosomal sialidase and two plasma membrane sialidases, all of them active on ganglioside.

Animals↗
PubMed1999

Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms.

Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization.

Amino Acid Motifs↗
PubMed2002

Bio-distribution and subcellular localization of Hypericin and its role in PDT induced apoptosis in cancer cells.

The development of new-generation photosensitizers to improve photodynamic therapy (PDT) and photodynamic diagnosis (PDD) is an area of extensive research. One such compound that has been studied in our group is Hypericin (HY). To study the mechanism of action we have investigated uptake, intracellular localization, cell phototoxicity and morphological changes especially to ultrastructures following photodynamic treatment in poorly (CNE2) and moderately (TW0-1) differentiated human nasopharyngeal carcinoma (NPC) cells and also other tumor cells such as colon (CCL-220.1) and bladder (SD) cells in vitro. Following irradiation, phototoxicity was determined by crystal fast violet assay and apoptosis was assessed using annexin-V assay. Using spectrofluorimetry and confocal laser scanning microscopy (CLSM) we have determined cellular fluorescence localization and uptake of HY. Co-labeling with HY and fluorescent dyes specific for cell organelles revealed an intracellular localization of HY predominantly in mitochondria and lysosomes. Since many photosensitizing agents in current clinical use have mitochondrial targets, HY may be a valuable addition to current protocols. In addition, our results also indicate that leakage of lysosomal protease into cytosolic compartment might be involved in the induction of apoptosis. Electron microscopy revealed damage to plasma membrane with high drug dose (>5 microM); indicating a mechanism related to necrosis, whereas sub-lethal lower doses (<2.5 microM) resulted in induction of apoptosis indicated by typical ultrastructural signs of apoptosis. Our results based on mitochondrial and lysosomal localization support the idea that PDT can contribute to elimination of malignant cells by the induction of apoptosis, and can be of physiological significance.

Anthracenes↗
PubMed1992

Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells.

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arterial and venous thrombosis, it has been shown that it induces tissue plasminogen activator [tPA] and prostacyclin [PGI2] release from the vessel wall. We have previously shown the presence of specific binding sites with a Kd of 4.2 micrograms/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent cultures of endothelial cells from human umbilical vein were incubated with media containing 3H-acetyl-defibrotide for various intervals of time. Cells were then washed and harvested nonenzymatically. Subcellular location of 3H-defibrotide was investigated by fractionating cells on discontinuous sucrose gradient and measuring the distribution of radioactivity. 5'-nucleotidase enzyme activity was also measured to ensure the location of membrane fraction. Our results suggest that the major location of 3H-defibrotide in endothelial cells is the plasma membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.

Binding Sites↗