scholarly journals Paralemmin, a Prenyl-Palmitoyl–anchored Phosphoprotein Abundant in Neurons and Implicated in Plasma Membrane Dynamics and Cell Process Formation

1998 ◽  
Vol 143 (3) ◽  
pp. 795-813 ◽  
Author(s):  
Christian Kutzleb ◽  
Gabriele Sanders ◽  
Raina Yamamoto ◽  
Xiaolu Wang ◽  
Beate Lichte ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Species Conservation ◽  
Cell Types ◽  
Membrane Dynamics ◽  
Morphological Properties ◽  
Developmentally Regulated ◽  
Membrane Flow ◽  
Western Blots ◽  
Process Formation

We report the identification and initial characterization of paralemmin, a putative new morphoregulatory protein associated with the plasma membrane. Paralemmin is highly expressed in the brain but also less abundantly in many other tissues and cell types. cDNAs from chicken, human, and mouse predict acidic proteins of 42 kD that display a pattern of sequence cassettes with high inter-species conservation separated by poorly conserved linker sequences. Prenylation and palmitoylation of a COOH-terminal cluster of three cysteine residues confers hydrophobicity and membrane association to paralemmin. Paralemmin is also phosphorylated, and its mRNA is differentially spliced in a tissue-specific and developmentally regulated manner. Differential splicing, lipidation, and phosphorylation contribute to electrophoretic heterogeneity that results in an array of multiple bands on Western blots, most notably in brain. Paralemmin is associated with the cytoplasmic face of the plasma membranes of postsynaptic specializations, axonal and dendritic processes and perikarya, and also appears to be associated with an intracellular vesicle pool. It does not line the neuronal plasmalemma continuously but in clusters and patches. Its molecular and morphological properties are reminiscent of GAP-43, CAP-23, and MARCKS, proteins implicated in plasma membrane dynamics. Overexpression in several cell lines shows that paralemmin concentrates at sites of plasma membrane activity such as filopodia and microspikes, and induces cell expansion and process formation. The lipidation motif is essential for this morphogenic activity. We propose a function for paralemmin in the control of cell shape, e.g., through an involvement in membrane flow or in membrane–cytoskeleton interaction.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

A novel H+ permeability dominating intracellular pH in the early mouse embryo

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1353-1361
Author(s):  
J.M. Baltz ◽  
J.D. Biggers ◽  
C. Lechene
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Preimplantation Embryos ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
K Concentration ◽  
Early Mouse

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

scholarly journals Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

1987 ◽  
Vol 104 (5) ◽  
pp. 1239-1248 ◽  
Author(s):  
E S Sztul ◽  
D Biemesderfer ◽  
M J Caplan ◽  
M Kashgarian ◽  
J L Boyer
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Alpha Subunit ◽  
Gamma Glutamyl Transferase ◽  
Surface Distribution ◽  
Western Blots ◽  
Glutamyl Transferase

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

scholarly journals Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation

1995 ◽  
Vol 309 (3) ◽  
pp. 905-912 ◽  
Author(s):  
J W Kok ◽  
T Babia ◽  
K Klappe ◽  
D Hoekstra
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Differentiation ◽  
Plasma Membranes ◽  
Cell Types ◽  
Short Chain ◽  
Synthetic Activity ◽  
Intact Cells ◽  
Ht29 Cells ◽  
Extensive Degradation

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins.

1990 ◽  
Vol 111 (6) ◽  
pp. 2785-2794 ◽  
Author(s):  
E Brown ◽  
L Hooper ◽  
T Ho ◽  
H Gresham
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Synthetic Peptides ◽  
Plasma Membranes ◽  
Protein A ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Cell Biol ◽  
Leukocyte Response ◽  
Stimulation Of

Phagocytosis by monocytes or neutrophils can be enhanced by interaction with several proteins or synthetic peptides containing the Arg-Gly-Asp sequence. Recently we showed that an mAb, B6H12, specifically inhibited this enhancement of neutrophil phagocytosis by inhibiting Arg-Gly-Asp binding to the leukocyte response integrin (Gresham, H. D., J. L. Goodwin, P. M. Allen, D. C. Anderson, and E. J. Brown. 1989. J. Cell Biol. 108:1935-1943). Now, we have purified the antigen recognized by B6H12 to homogeneity. Surprisingly, it is a 50-kD molecule that is expressed on the plasma membranes of all hematopoietic cells, including erythrocytes, which express no known integrins. On platelets and placenta, but not on erythrocytes, this protein is associated with an integrin that can be recognized by an anti-beta 3 antibody. In addition, both the anti-beta 3 and several mAbs recognizing the 50-kD protein inhibit Arg-Gly-Asp stimulation of phagocytosis. These data demonstrate an association between integrins and the 50-kD protein on several cell types. For this reason, we call it Integrin-associated Protein (IAP). We hypothesize that IAP may play a role in signal transduction for enhanced phagocytosis by Arg-Gly-Asp ligands.

scholarly journals Novel Evidence of Expression and Activity of Ecto-Phospholipase C γ1 in Human T Lymphocytes

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3833-3840
Author(s):  
Sebastiano Miscia ◽  
Angela Di Baldassarre ◽  
Amelia Cataldi ◽  
Rosa Alba Rana ◽  
Valerio Di Valerio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Lymphocytes ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Cell Types ◽  
T Lymphocyte Subsets ◽  
Membrane Flow ◽  
Inositol 1 ◽  
Outer Leaflet ◽  
Expression And Activity

Abstract Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This ectoenzyme seems to play particular roles in cellular function by hydrolyzing inositol lipids located on the outer leaflet of the plasma membrane. Here, we provide the first evidence that peripheral T lymphocytes express a discrete level of a PLCγ1 at the outer leaflet of the plasma membrane. Flow cytometry showed that the PLCγ1-positive (PLCγ1+) cells (∼37%) were CD8+ and CD45RA+. Biochemical evidence indicated that (1) this ectoenzyme displays a mass similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine residues, and (3) its activity is Ca2+-dependent. In addition, this enzyme appeared to be correlated with the proliferative state of the cell, since stimulation with phytohemagglutinin (PHA) downregulated both its expression and activity, which were restored by treatment with an antiproliferative agent like natural interferon beta. Moreover, the different kinetics of formation of its hydrolytic products, inositol 1 phosphate and inositol 1:2 cyclic phosphate (Ins(1)P and Ins(1:2 cycl)P), formed upon incubation of the lymphocytes with [3H]-lyso-phosphatidylinositol (PI), allow the hypothesis of a selective involvement of the two inositol phosphates in the mechanisms regulating the metabolism of particular T-lymphocyte subsets.

Proton conductance of the plasma membrane: properties, regulation, and functional role

1993 ◽  
Vol 265 (1) ◽  
pp. C3-C14 ◽  
Author(s):  
G. L. Lukacs ◽  
A. Kapus ◽  
A. Nanda ◽  
R. Romanek ◽  
S. Grinstein
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Second Messengers ◽  
Cell Types ◽  
Membrane Properties ◽  
Transport Systems ◽  
Extracellular Acidification ◽  
Phagocyte Nadph Oxidase ◽  
Selective Permeability ◽  
Conductive Pathway

H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in the case of phagocytes. When stimulated, these cells undergo a sustained depolarization, while generating large amounts of metabolic acid. In addition, conductive H+ fluxes may also provide counterions to neutralize the activity of electrogenic enzymes, as suggested for the phagocyte NADPH oxidase.

scholarly journals Localization and quantitation of the vitamin D binding protein (Gc- globulin) in human neutrophils

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 274-283 ◽  
Author(s):  
RR Kew ◽  
MA Sibug ◽  
JP Liuzzo ◽  
RO Webster
Keyword(s):  
Vitamin D ◽  
Plasma Membrane ◽  
Western Blotting ◽  
Binding Protein ◽  
Apparent Molecular Weight ◽  
Cell Types ◽  
Human Neutrophils ◽  
Vitamin D Binding Protein ◽  
Western Blots ◽  
Extracellular Milieu

Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell- free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.

scholarly journals Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method.

1987 ◽  
Vol 105 (6) ◽  
pp. 2589-2601 ◽  
Author(s):  
R S Molday ◽  
L L Molday
Keyword(s):  
Plasma Membrane ◽  
Perturbation Method ◽  
Plasma Membranes ◽  
Prolonged Exposure ◽  
Gradient Centrifugation ◽  
Protein Composition ◽  
Major Protein ◽  
Binding Studies ◽  
Western Blots ◽  
Retinal Rod

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.

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