scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

scholarly journals The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1α,25(OH)2-Vitamin D3in Vivo and in Vitro

2004 ◽  
Vol 18 (11) ◽  
pp. 2660-2671 ◽  
Author(s):  
Johanna A. Huhtakangas ◽  
Christopher J. Olivera ◽  
June E. Bishop ◽  
Laura P. Zanello ◽  
Anthony W. Norman
Keyword(s):  
Vitamin D ◽  
Plasma Membrane ◽  
Vitamin D Receptor ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Kidney Tissue ◽  
Mouse Lung ◽  
Nuclear Fraction

Abstract The steroid hormone 1α,25(OH)2-vitamin D3 (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). This study characterized the VDRmem present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [3H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D KD binding dissociation constant = 1–3 nm. Our data collectively support the classical VDR being the VDRmem in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [3H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r2 = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [3H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [3H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

scholarly journals Revealing the in vivo growth and division patterns of mouse gut bacteria

2020 ◽  
Vol 6 (36) ◽  
pp. eabb2531
Author(s):  
Liyuan Lin ◽  
Qiuyue Wu ◽  
Jia Song ◽  
Yahui Du ◽  
Juan Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gut Microbiota ◽  
Growth Dynamics ◽  
Gut Bacteria ◽  
Metabolic Labeling ◽  
Taxonomic Identification ◽  
In Vivo Growth

Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. We propose a method that integrates the use of sequential d-amino acid–based in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid–based probes containing different fluorophores to mice by gavage, the resulting dual-labeled peptidoglycans provide temporal information on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the in vivo growth dynamics of the bacterial gut microbiota, which will advance our understanding of bacterial cytology and facilitate elucidation of the basic microbiology of this gut “dark matter.”

scholarly journals Sphingosine kills bacteria by binding to cardiolipin

2020 ◽  
Vol 295 (22) ◽  
pp. 7686-7696 ◽  
Author(s):  
Rabea Verhaegh ◽  
Katrin Anne Becker ◽  
Michael J. Edwards ◽  
Erich Gulbins
Keyword(s):  
Plasma Membrane ◽  
Bactericidal Activity ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Central Element ◽  
Lipid Binding ◽  
Bacterial Cells ◽  
Bacterial Killing

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively–charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo. We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

scholarly journals Oms38 Is the First Identified Pore-Forming Protein in the Outer Membrane of Relapsing Fever Spirochetes

2008 ◽  
Vol 190 (21) ◽  
pp. 7035-7042 ◽  
Author(s):  
Marcus Thein ◽  
Ignas Bunikis ◽  
Katrin Denker ◽  
Christer Larsson ◽  
Sally Cutler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Outer Membrane ◽  
Signal Sequence ◽  
Amino Acid Identity ◽  
Amino Acid Sequences ◽  
Relapsing Fever ◽  
Small Water ◽  
Membrane Spanning

ABSTRACT Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia. During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii, B. hermsii, B. recurrentis, and B. turicatae. The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic β-sheets.

scholarly journals Mitochondrial clearance of Ca2+ controls insulin secretion

2019 ◽  
Author(s):  
N Vishnu ◽  
A Hamilton ◽  
A Bagge ◽  
A Wernersson ◽  
E Cowan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Selective Inhibition ◽  
Underlying Mechanism ◽  
Β Cells ◽  
Intact Cells ◽  
Membrane Hyperpolarization ◽  
Knock Out

SUMMARYTransport of Ca2+ from the cytosol to the mitochondrial matrix of insulin-secreting pancreatic β-cells facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. The establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins has allowed mitochondrial Ca2+ transport to be modified in intact cells. We examined the consequences of deficiency of the accessory protein, MICU2, in rat and human insulin-secreting cell lines as well as in mouse islets. Glucose-induced mitochondrial Ca2+ elevation and inner membrane hyperpolarization were reduced, together with cytosolic ATP/ADP-ratios and insulin secretion. Insulin secretion in Micu2 knock out mice was attenuated in vitro as well as in vivo. While KCl-evoked sub-plasmalemmal Ca2+ increases were more pronounced, the global cytosolic Ca2+ response was, surprisingly, diminished in MICU2-deficient cells. These findings were supported by selective inhibition of mitochondrial Ca2+ uptake by mitochondrial depolarization. It is concluded that mitochondrial Ca2+ transport plays an additional and hitherto unrecognized role in stimulated β-cells by regulating net Ca2+ entry across the plasma membrane. This is likely accounted for by clearing of sub-plasmalemmal Ca2+ levels by mitochondria located near the plasma membrane.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Plasma Membranes ◽  
Fertilization Rate ◽  
Apical Plasma Membrane ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fertilization Rates

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine

1995 ◽  
Vol 133 (5) ◽  
pp. 626-634 ◽  
Author(s):  
Marianne Voldstedlund ◽  
Jørgen Tranum-Jensen ◽  
Aase Handberg ◽  
Jørgen Vinten
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Insulin Stimulation ◽  
Rat Adipocytes ◽  
Adipocyte Plasma Membranes ◽  
Glucose Transporter Glut4

Voldstedlund M. Tranum-Jensen J, Handberg A, Vinten J. Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine. Eur J Endocrinol 1995:133:626–34. ISSN 0804–4643 The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 μg of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(α1- and α2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of α2 was unaffected, In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of α2 were decreased by 19% and 15% respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the α2 -isoform of Na/K-ATPase. Marianne Voldstedlund, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

scholarly journals Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium.

1994 ◽  
Vol 126 (6) ◽  
pp. 1433-1444 ◽  
Author(s):  
A L Hitt ◽  
J H Hartwig ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Cell Shape ◽  
Plasma Membranes ◽  
Axenic Culture ◽  
Actin Binding ◽  
Growth Media ◽  
Cortical Actin ◽  
Gene Encoding ◽  
High Affinity

Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.

Membrane fluidity and hemilayer temperature sensitivity in trout hepatocytes during brief in vitro cold exposure

1994 ◽  
Vol 266 (3) ◽  
pp. R773-R780
Author(s):  
E. E. Williams ◽  
J. R. Hazel
Keyword(s):  
Plasma Membrane ◽  
Cold Exposure ◽  
Plasma Membranes ◽  
Temperature Acclimation ◽  
Short Term ◽  
Fluorescence Depolarization ◽  
Intact Cells ◽  
Induced Membrane ◽  
Membrane Probes

Fluorescent membrane probes were used to assess the fluidity of hepatocyte plasma membranes (PM) from 20 degrees C-acclimated trout after exposure to 20 and 5 degrees C. PM isolated from cells after 6 h at 5 degrees C were significantly more fluid [fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH)] than control membranes at both temperatures. The increased fluidity was sufficient to offset 45-50% of the cold-induced membrane ordering. In contrast, the fluidity of PM in intact cells from 20 degrees C-acclimated fish remained constant when exposed to 5 degrees C for a similar period. In addition, the fluidity of the inner hemilayer [1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate (TMA-DPH)] was significantly less sensitive to temperature change than was the fluidity of the outer hemilayer [3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (PA-DPH)]. Because the isolated membrane preparation was most likely enriched with canalicular membranes (based on 5'-nucleotidase recovery), these results suggest that the canalicular domain of the plasma membrane is preferentially modified during short-term cold exposure and that the fluidity of the inner hemilayer of the plasma membrane of intact cells is relatively temperature insensitive, thus requiring fewer modifications than the outer hemilayer during temperature acclimation.

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