Probing the Subcellular Distribution of Phosphatidylinositol Reveals a Surprising Lack at the Plasma Membrane; Defining the Subcellular Distribution and Metabolic Channeling of Phosphatidylinositol

2019 ◽  
Author(s):  
Sruthi Balakrishnan
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Metabolic Channeling

scholarly journals Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1563-1573 ◽  
Author(s):  
MT Quinn ◽  
ML Mullen ◽  
AJ Jesaitis ◽  
JG Linner
Keyword(s):  
Plasma Membrane ◽  
Cytochrome B ◽  
Subcellular Distribution ◽  
Intact Cell ◽  
Human Neutrophils ◽  
Cytochrome B559 ◽  
Thin Sections ◽  
Specific Granule ◽  
Generating System ◽  
Peptide Antibodies

Abstract Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti- cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule- associated Rap1A was translocated to the plasma membrane in PMA- stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide- generating system.

Internalization of receptor-bound human chorionic gonadotrophin in preovulatory rat granulosa cells in vivo

1983 ◽  
Vol 103 (3) ◽  
pp. 406-412 ◽  
Author(s):  
Kalle Jääkeläinen ◽  
Seppo Markkanen ◽  
Hannu Rajaniemi
Keyword(s):  
Plasma Membrane ◽  
Granulosa Cells ◽  
Subcellular Distribution ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Female Rats ◽  
Electron Microscope Autoradiography ◽  
Silver Grains ◽  
Pregnant Mare Serum Gonadotrophin

Abstract. The subcellular distribution of 125I-labelled human chorionic gonadotrophin (hCG) in preovulatory rat granulosa cells was studied in vivo. Pregnant mare serum gonadotrophin-pretreated immature female rats received an iv injection of [125I]hCG a few hours before the endogenous preovulatory gonadotrophin surge. The animals were killed at 2 or 6 h after the [125I]hCG injections. Light microscope autoradiographs showed that the mural granulosa cells of large follicles were the most highly labelled cells in the ovaries. Electron microscope autoradiography was used to study the subcellular distribution of radioactivity in the mural granulosa cells. At 2 h 45% of the counted silver grains were associated with the plasma membrane and 10% with the lysosomes, at 6 h the values were 51% and 9%, respectively. The distribution of the observed silver grains was compared with the generated expected source to grain pairs by computerized linear multiple regression analysis. The magnitudes of the regression coefficients revealed that the plasma membrane and the lysosomes were the only specifically 125I-labelled organelles, that a few radioactive molecules were located diffusely over the cytoplasm at 2 h and that the 125I-radioactivity of the nuclei was negligible. The present results suggest that preovulatory rat granulosa cells are in vivo able to internalize into lysosomes [125I]hCG initially bound to LH/hCG receptors of the plasma membrane.

Prostaglandin E2 interaction with AVP: effects on AQP2 phosphorylation and distribution

2000 ◽  
Vol 278 (3) ◽  
pp. F388-F394 ◽  
Author(s):  
Marina Zelenina ◽  
Birgitte Mønster Christensen ◽  
Johan Palmér ◽  
Angus C. Nairn ◽  
Søren Nielsen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Collecting Duct ◽  
Water Channel ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Centrifugation Technique ◽  
Renal Inner Medulla ◽  
Intracellular Vesicles ◽  
Dose Dependent

Prostaglandin E2 (PGE2) antagonizes the action of arginine vasopressin (AVP) on collecting duct water permeability. To investigate the mechanism of this antagonism, rat renal inner medulla (IM) was incubated with the two hormones, and the phosphorylation and subcellular distribution of the water channel, aquaporin-2 (AQP2) were studied. Using a phosphorylation state-specific AQP2 antibody, we demonstrated that AVP stimulates AQP2 phosphorylation at the Ser256 protein kinase A consensus site in a time- and dose-dependent manner. In parallel studies using a differential centrifugation technique, we demonstrated that AVP induced translocation of AQP2 from an intracellular vesicle-enriched fraction to a plasma membrane-enriched fraction. PGE2(10− 7 M) added after AVP (10− 8 M) did not decrease AQP2 phosphorylation but reversed AVP-induced translocation of AQP2 to the plasma membrane. Preincubation of IM with PGE2 did not prevent the effects of AVP on AQP2 phosphorylation and trafficking. PGE2 alone did not influence AQP2 phosphorylation and subcellular distribution. Our data indicate that 1) recruitment of AQP2 to the plasma membrane and its retrieval to a pool of intracellular vesicles may be regulated independently, 2) PGE2 may counteract AVP action by activation of AQP2 retrieval, 3) dephosphorylation of AQP2 is not a prerequisite for its internalization.

Trypsin-uncoupled synthesis and secretion of yeast invertase: implications for the mechanism of secretion

1979 ◽  
Vol 25 (4) ◽  
pp. 528-534 ◽  
Author(s):  
Bruce E. Holbein ◽  
Denis K. Kidby
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
External Surface ◽  
Internal Surface ◽  
Low Molecular Weight ◽  
Synthetase Activity ◽  
Trypsin Treatment ◽  
Yeast Invertase

The subcellular distribution of invertase was examined after synthesis and secretion by sphaeroplasts had been uncoupled by the addition of 30 μg mL−1 trypsin. Sphaeroplasts secreted only the high molecular weight invertase during uncoupling by trypsin. The level of low molecular weight, 'small' invertase in the soluble internal pool was found to be elevated by over fivefold, and the membrane-associated pool was found to contain low molecular weight invertase in addition to intermediate molecular weight invertase, after 1.5 h of trypsin treatment. Purified plasma membranes from trypsin-treated sphaeroplasts had no detectable mannan synthetase activity. On the basis of these and previous findings, a working hypothesis wherein invertase is synthesized on the internal surface of the plasma membrane and glycosylated during its transit to the external surface is presented.

Subcellular distribution of the Entamoeba histolytica 140 kDa FN-binding molecule during host-parasite interaction

Parasitology ◽  
2006 ◽  
Vol 134 (2) ◽  
pp. 169-177 ◽  
Author(s):  
V. I. HERNÁNDEZ-RAMÍREZ ◽  
A. RIOS ◽  
A. ANGEL ◽  
M. A. MAGOS ◽  
L. PÉREZ-CASTILLO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Cdna Clone ◽  
Subcellular Distribution ◽  
Cultured Cells ◽  
Receptor Molecule ◽  
Specific Regulation ◽  
Host Parasite ◽  
Adhesion Process

Entamoeba histolytica trophozoites recovered from the host-parasite interface during abscess development obtain different stimuli compared with long-term cultured cells. In order to have a better understanding about the mechanisms in which the 140 kDa fibronectin (FN)-binding molecule (EhFNR) is involved during the invasive process, we decided to compare the regulation process of this molecule among long-term cultured trophozoites, FN-stimulated trophozoites, and trophozoites recently recovered from a liver abscess. A cDNA clone (5A) containing a fragment of the EhFNR that shows identity to the C-terminal region of the intermediate galactose lectin subunit Igl, was selected with a mAb (3C10). Identity of EhFNR with Igl was confirmed by immunoprecipitation with 3C10 and EH3015 (against the Gal/GalNAc intermediate subunit) mAbs. The 3C10 mAb was used as a tool to explore the modulation of the amoebic receptor (EhFNR). Our results showed specific regulation of the EhFNR in FN-interacted amoebas, as well as in trophozoites recovered at different stages of abscess development. This regulation involved mobilization of the receptor molecule from internal vesicles to the plasma membrane. Therefore, we suggest that in the host-parasite interface, the EhFNR (Igl) plays an important role in the adhesion process during abscess development.

Rab10 in insulin-stimulated GLUT4 translocation

2008 ◽  
Vol 411 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Hiroyuki Sano ◽  
William G. Roach ◽  
Grantley R. Peck ◽  
Mitsunori Fukuda ◽  
Gustav E. Lienhard
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Subcellular Distribution ◽  
Protein Kinase B ◽  
Muscle Cells ◽  
Low Density ◽  
Glut4 Translocation ◽  
Gtpase Activating Protein ◽  
Intracellular Vesicles

In fat and muscle cells, insulin stimulates the movement to and fusion of intracellular vesicles containing GLUT4 with the plasma membrane, a process referred to as GLUT4 translocation. Previous studies have indicated that Akt [also known as PKB (protein kinase B)] phosphorylation of AS160, a GAP (GTPase-activating protein) for Rabs, is required for GLUT4 translocation. The results suggest that this phosphorylation suppresses the GAP activity and leads to the elevation of the GTP form of one or more Rabs required for GLUT4 translocation. Based on their presence in GLUT4 vesicles and activity as AS160 GAP substrates, Rabs 8A, 8B, 10 and 14 are candidate Rabs. Here, we provide further evidence that Rab10 participates in GLUT4 translocation in 3T3-L1 adipocytes. Among Rabs 8A, 8B, 10 and 14, only the knockdown of Rab10 inhibited GLUT4 translocation. In addition, we describe the subcellular distribution of Rab10 and estimate the fraction of Rab10 in the active GTP form in vivo. Approx. 5% of the total Rab10 was present in GLUT4 vesicles isolated from the low-density microsomes. In both the basal and the insulin state, 90% of the total Rab10 was in the inactive GDP state. Thus, if insulin increases the GTP form of Rab10, the increase is limited to a small portion of the total Rab10. Finally, we report that the Rab10 mutant considered to be constitutively active (Rab10 Q68L) is a substrate for the AS160 GAP domain and, hence, cannot be used to deduce rigorously the function of Rab10 in its GTP form.

scholarly journals Carbonic Anhydrase II Associated with Plasma Membrane in a Human Pancreatic Duct Cell Line (CAPAN-1)

2001 ◽  
Vol 49 (8) ◽  
pp. 1045-1053 ◽  
Author(s):  
Laetitia Alvarez ◽  
Marjorie Fanjul ◽  
Nicholas Carter ◽  
Etienne Hollande
Keyword(s):  
Plasma Membrane ◽  
Carbonic Anhydrase ◽  
Pancreatic Duct ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Brefeldin A ◽  
Duct Cell ◽  
Carbonic Anhydrase Ii ◽  
Apical Plasma Membrane ◽  
Cytosolic Side

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH. (J Histochem Cytochem 49:1045–1053, 2001)

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4006-4014 ◽  
Author(s):  
Dian Feng ◽  
Katharine Crane ◽  
Nataliya Rozenvayn ◽  
Ann M. Dvorak ◽  
Robert Flaumenhaft
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Molecular Mechanisms ◽  
Protein Composition ◽  
Immunoblot Analysis ◽  
Snare Proteins ◽  
Ultrastructural Studies ◽  
Streptolysin O ◽  
Membrane Compartments ◽  
Granule Secretion

Morphologic studies have demonstrated a process by which α-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for α-granule secretion. These observations raise the possibility that morphologic features of α-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins—human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca++-induced α-granule secretion by approximately 50%. Inhibition of α-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of α-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of α-granule secretion that include homotypic α-granule fusion and the fusion of α-granules with the open canalicular system and plasma membrane.

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