scholarly journals Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthew B Stone ◽  
Sarah A Shelby ◽  
Marcos F Núñez ◽  
Kathleen Wisser ◽  
Sarah L Veatch
Keyword(s):  
Plasma Membranes ◽  
B Lymphocyte ◽  
Membrane Lipids ◽  
Super Resolution ◽  
Cell Receptor ◽  
Biochemical Networks ◽  
Lipid Phase ◽  
Intact Cells ◽  
Signaling Function ◽  
Liquid Ordered

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

scholarly journals Modulation of 5′-nucleotidase activity in plasma membranes and intact cells by the extracellular matrix proteins laminin and fibronectin

1992 ◽  
Vol 282 (1) ◽  
pp. 181-188 ◽  
Author(s):  
N Olmo ◽  
J Turnay ◽  
G Risse ◽  
R Deutzmann ◽  
K von der Mark ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Inhibitory Activity ◽  
Plasma Membranes ◽  
Extracellular Matrix Proteins ◽  
Cell Receptor ◽  
Inhibitory Effect ◽  
Matrix Proteins ◽  
Nucleotidase Activity ◽  
Intact Cells

Modulation of 5′-nucleotidase activity by the extracellular matrix proteins fibronectin, laminin and their fragments has been studied in plasma membrane preparations as well as in intact BCS-TC2 and Rugli cells. The ectoenzyme on plasma membranes is activated by laminin; fibronectin inhibits the AMPase activity on BCS-TC2 plasma membranes but no inhibitory effect is found in plasma membrane preparations from Rugli cells. These effects are dependent on the preincubation time and protein concentration. When the effect of the extracellular matrix proteins is studied on intact cells, both BCS-TC2 and Rugli cells show similar behaviour. A decrease in the enzyme activity is observed in the presence of fibronectin. The AMPase inhibitory activity is located on its 40 kDa fragment. No inhibitory activity is found in other fibronectin fragments, including the 140 kDa fragment which contains the RGDS cell-adhesion sequence. Laminin and its E1-4 and E8 fragments are able to activate the ecto-5′-nucleotidase activity of both BCS-TC2 and Rugli cells. The effect of the E1-4 fragment on intact cells is greater than that observed for the E8 fragment and uncleaved laminin. Our results suggest a bifunctional role for 5′-nucleotidase as ectoenzyme and cell receptor for extracellular matrix proteins.

scholarly journals Temperature optimum of insulin-stimulated 2-deoxy-d-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data

1984 ◽  
Vol 218 (1) ◽  
pp. 29-36 ◽  
Author(s):  
P A Hyslop ◽  
C E Kuhn ◽  
R D Sauerheber
Keyword(s):  
Temperature Dependence ◽  
Glucose Uptake ◽  
Spin Label ◽  
Plasma Membranes ◽  
Lipid Phase ◽  
Cellular Transport ◽  
Intact Cells ◽  
Rat Adipocytes ◽  
Fluorescence Label ◽  
Adipocyte Plasma Membranes

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells.

scholarly journals Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

2016 ◽  
Vol 27 (22) ◽  
pp. 3627-3636 ◽  
Author(s):  
Sophie V. Pageon ◽  
Philip R. Nicovich ◽  
Mahdie Mollazade ◽  
Thibault Tabarin ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Cell Receptor ◽  
Intact Cells ◽  
Analysis Strategy ◽  
Signaling Activity

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

Ultrastructure of the hepatopancreas of the Pacific white shrimp, Penaeus vannamei (Crustacea: Decapoda)

1988 ◽  
Vol 68 (2) ◽  
pp. 323-337 ◽  
Author(s):  
Thomas Caceci ◽  
Kay F. Neck ◽  
Donal D H. Lewis ◽  
Raymond F. Sis
Keyword(s):  
B Cells ◽  
Plasma Membranes ◽  
Ultrastructural Level ◽  
Pacific White Shrimp ◽  
White Shrimp ◽  
Penaeus Vannamei ◽  
Intact Cells ◽  
The Pacific ◽  
Total Complement ◽  
Electron Microscopes

Fourteen specimens of the hepatopancreas of the Pacific white shrimp, Penaeus vannamei, were prepared for examination with the transmission and scanning electron microscopes and with the light microscope. The histology and ultrastructure of this organ is similar to that seen in other Decapoda. At the ultrastructural level, it was observed that B-cells rupture at approximately the level of gap junctions located on the lateral plasma membranes of the cells, and discharge the contents of their large vacuoles into the intercellular space. This efflux of enzymatic material may be the mechanism by which cells are released from the wall of the tubule at the proximal end: the rupture and collapse of a B-cell may be analagous to the removal of the keystone which supports an arch. Deprived of support, and lacking structural adaptations for cohesion (there are no desmosomes or interdigitations in the epithelium) and with the intercellular material digested, the remaining intact cells collapse into the lumen of the tubule. The lysis of individual cells of all types - R-, F-, and B-cells - may contribute to the tubules’ total complement of digestive enzymes.

Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling

1996 ◽  
Vol 109 (10) ◽  
pp. 2453-2460 ◽  
Author(s):  
K. Fujimoto ◽  
M. Umeda ◽  
T. Fujimoto
Keyword(s):  
Plasma Membranes ◽  
Membrane Lipids ◽  
Secretory Granules ◽  
General Scheme ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Electron Microscopic Observation ◽  
Erythrocyte Membranes ◽  
Electron Microscopic ◽  
Intracellular Membranes

We propose the use of membrane splitting by freeze-fracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, freeze-fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unfractured membranes and to release cytoplasm or contents. While the detergent dissolves unfractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested freeze-fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca(2+)-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.

scholarly journals Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

1982 ◽  
Vol 202 (3) ◽  
pp. 739-745 ◽  
Author(s):  
Clive J. Dix ◽  
Matthias Schumacher ◽  
Brian A. Cooke
Keyword(s):  
Cyclic Amp ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Linear Increase ◽  
Guanine Nucleotide ◽  
Production Rates ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Guanine Nucleotide Binding ◽  
Guanine Nucleotide Regulatory Protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED50 (dose that produces a response that is 50% of the maximum response) 60±5.7ng/ml and 8±1.8ng/ml (mean±s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/106 cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific 125I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4°C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5′-[β,γ-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50–60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- and the p(NH)ppG-stimulated cyclic AMP production rates were unchanged but no effect of lutropin could be demonstrated with or without added p(NH)ppG. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.

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