Faculty Opinions recommendation of Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

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.

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B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Blood ◽

10.1182/blood.v73.7.1814.1814 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1814-1820 ◽

Cited By ~ 15

Author(s):

L Feldman ◽

N Dainiak

Keyword(s):

Growth Factors ◽

T Lymphocytes ◽

B Lymphocytes ◽

Plasma Membranes ◽

B Lymphocyte ◽

Temperature Stability ◽

Hematopoietic Growth Factors ◽

Serum Free ◽

Gm Csf ◽

Lineage Specificity

Abstract Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

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Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase

Biochemical Journal ◽

10.1042/bj2100437 ◽

1983 ◽

Vol 210 (2) ◽

pp. 437-449 ◽

Cited By ~ 61

Author(s):

A D Whetton ◽

L M Gordon ◽

M D Houslay

Keyword(s):

Fatty Acid ◽

Adenylate Cyclase ◽

Spin Probe ◽

Plasma Membranes ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Cholesterol Concentration ◽

Lipid Phase ◽

Lipid Order ◽

Liver Plasma Membranes

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.

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Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes

Biochemical Journal ◽

10.1042/bj1970675 ◽

1981 ◽

Vol 197 (3) ◽

pp. 675-681 ◽

Cited By ~ 48

Author(s):

M D Houslay ◽

I Dipple ◽

L M Gordon

Keyword(s):

Adenylate Cyclase ◽

Spin Probe ◽

Plasma Membranes ◽

Chemical Constituents ◽

Lipid Phase ◽

Lipid Phase Transition ◽

Arrhenius Plots ◽

Lipid Environment ◽

Lipid Phase Separation ◽

Outer Half

The glucagon-stimulated (coupled) activity of rat liver plasma-membrane adenylate cyclase could be selectively modulated by the anionic drug phenobarbital, whereas the fluoride-stimulated (uncoupled) activity remained unaffected. It is suggested that the cationic drug phenobarbital preferentially interacts with the external half of the bilayer, as the negatively charged phospholipids are found at the cytosol-facing side. This results in a selective fluidization of the external half of the bilayer, leading to a depression in the high-temperature onset of the lipid phase transition (from 28 degree to 16 degree C) occurring there. This was detected both by e.s.r. analysis, using a fatty acid spin probe, and also by Arrhenius plots of glucagon-stimulated activity, where the enzyme forms a transmembrane complex with the receptor and is sensitive to the lipid environment of both halves of the bilayer. However, in the absence of hormone, adenylate cyclase only senses the lipid environment of the inner (cytosol) half of the bilayer. Thus its fluoride stimulated activity and Arrhenius plots of this activity remained unaffected by the presence of phenobarbital (less than 12 mM) in the assay. These results support the view that independent modulation of the fluidity or chemical constituents of each half of the bilayer can selectively affect the receptor-coupled and uncoupled activities of adenylate cyclase.

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The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment

Biochemical Journal ◽

10.1042/bj1740179 ◽

1978 ◽

Vol 174 (1) ◽

pp. 179-190 ◽

Cited By ~ 114

Author(s):

I Dipple ◽

M D Houslay

Keyword(s):

Adenylate Cyclase ◽

Benzyl Alcohol ◽

Plasma Membranes ◽

Break Point ◽

Alcohol Concentration ◽

Cyclase Activity ◽

Adenylate Cyclase Activity ◽

Lipid Phase ◽

Glucagon Receptor ◽

Arrhenius Plots

1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The ‘break’ points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5′-Nucleotidase activity was stimulated by benzyl alcohol, and the ‘break’ point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.

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B-lymphocyte-derived erythroid burst-promoting activity is distinct from other known lymphokines

Blood ◽

10.1182/blood.v73.7.1814.bloodjournal7371814 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1814-1820

Author(s):

L Feldman ◽

N Dainiak

Keyword(s):

Growth Factors ◽

T Lymphocytes ◽

B Lymphocytes ◽

Plasma Membranes ◽

B Lymphocyte ◽

Temperature Stability ◽

Hematopoietic Growth Factors ◽

Serum Free ◽

Gm Csf ◽

Lineage Specificity

Previously we have demonstrated that, in contrast to various panspecific or multilineage hematopoietic growth factors, lymphocyte- derived erythroid burst-promoting activity (BPA) is lineage specific, stimulating BFU-E proliferation in serum-free culture by up to 600% of control values while failing to enhance nonerythroid colony formation. To further determine the cellular source(s) of this important erythropoietic growth regulator, we have separated normal nonadherent peripheral blood and splenic lymphocytes by nylon wool fractionation, SRBC rosetting, and panning with monoclonal antibodies (MoAbs). These unstimulated T- and B-lymphocyte-enriched populations were used as cell sources to produce conditioned media (CM) and to prepare plasma membranes (PM). When CM fractions or purified PM were assayed in serum- free human bone marrow culture, BPA was localized entirely to the B- lymphocyte-derived fractions. While CM or PM from unstimulated T lymphocytes failed to stimulate BFU-E proliferation, activation of T cells by either phytohemagglutinin-M (1%) or concanavalin A (Con A; 5 micrograms/mL) induced the expression of a factor on the PM and in the resultant CM that stimulated the formation of erythroid bursts. In addition to enhancing BFU-E proliferation, this T-cell factor stimulated the proliferation of CFU-GM and CFU-GEMM in serum-free culture. When compared biochemically (in terms of temperature stability, localization by ammonium sulfate fractionation, and sensitivity to dithiothreitol) or immunochemically (using antibodies specific for lymphocyte-derived BPA, GM-CSF, or interleukin-3 [IL-3]), as well as by lineage specificity, B- and activated T-lymphocyte- derived growth factors appeared to be distinct. The burst stimulatory activities expressed by recombinant human GM-CSF and IL-3 were immunologically distinct from that associated with octylglucoside extracts of plasma membranes from resting B lymphocytes. Our results suggest that the BFU-E-directed growth-promoting activity released from activated T lymphocytes is apparently due to GM-CSF, while both resting and mitogen-stimulated normal B lymphocytes express erythroid-specific BPA and neither GM-CSF nor IL-3.

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Aberrant Protein Sorting to the Nucleus during Erythroblast Enucleation: Mechanistic Basis for Membrane Protein Loss in Hereditary Elliptocytosis and Spherocytosis.

Blood ◽

10.1182/blood.v108.11.1559.1559 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1559-1559

Author(s):

Marcela A. Salomao ◽

Sarah Short ◽

Gloria Lee ◽

Xiuli An ◽

Mohandas Narla ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Plasma Membranes ◽

Molecular Mechanisms ◽

Protein Sorting ◽

Transmembrane Proteins ◽

Hereditary Elliptocytosis ◽

Skeletal Protein ◽

Aberrant Protein ◽

Protein 4.1R

Abstract During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of membrane components to plasma membranes surrounding expelled nuclei and young reticulocytes. This protein partitioning performs a crucial role in regulating the protein content of reticulocyte plasma membranes. Although it is known that cytoskeletal actin, spectrin and protein 4.1R distribute to reticulocytes, little is known about the sorting patterns of erythroblast transmembrane proteins. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), erythrocytes contain well-described deficiencies of various transmembrane proteins, in addition to those encoded by the mutant genes. For example, elliptocytic human and murine erythrocytes resulting from mutations in the 4.1R gene lack not only protein 4.1R but also transmembrane protein glycophorin C (GPC), known to be a 4.1R binding partner with a key role in linking cytoskeleton to bilayer. Similarly, in HS resulting from mutations in the ankyrin gene, deficiencies of band 3, Rh and GPA have been documented. Various molecular mechanisms could explain deficiencies of membrane proteins in HS and HE erythrocytes including: perturbed trafficking to the erythroblast membrane; aberrant protein sorting during erythroblast enucleation; and selective loss during reticulocyte membrane remodeling. We explored whether aberrant protein sorting during enucleation might be responsible for GPC deficiency in HE. First we performed immunochemical analysis of the sorting pattern of GPC using highly purified extruded nuclei and immature reticulocytes derived from terminally differentiated murine erythroblast cultures. Proteins from equivalent numbers of expelled nuclei and reticulocytes were analyzed by Western blotting. Using antibodies specific for GPC we determined that 90% of GPC sorted to reticulocyte plasma membranes. To validate these results we used live cell, three-color immunofluorescent microscopy and analyzed enucleating erythroblasts, reticulocytes and extruded nuclei from freshly harvested murine wild type bone marrow. Independently confirming the Western blot data, we found that GPC sorted almost exclusively to reticulocytes with little or no GPC in association with nuclear plasma membrane. Strikingly, in 4.1R null erythroblasts GPC was distributed exclusively to expelled nuclei. These findings unequivocally establish that skeletal protein 4.1R is critical for normal sorting of GPC to young reticulocytes and provide clear evidence that specific skeletal protein associations can regulate protein sorting during enucleation. Moreover, our data provide a molecular explanation for the underlying basis of GPC deficiency observed in 4.1R-deficient individuals with HE. We speculate that aberrant protein sorting may be a prevalent mechanism for the deficiencies of various membrane proteins in HS and HE and that their differential loss could contribute to the variable phenotypic expression of these hemolytic disorders.

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Plasma membranes from experimental granulation tissue

Biochemical Journal ◽

10.1042/bj1460565 ◽

1975 ◽

Vol 146 (3) ◽

pp. 565-573 ◽

Cited By ~ 13

Author(s):

P Lehtinen ◽

E Vuorio ◽

E Kulonen

Keyword(s):

Granulation Tissue ◽

Adenosine Triphosphatase ◽

Plasma Membranes ◽

Peritoneal Macrophages ◽

Lipid Phase ◽

Embryonic Chick ◽

Electrophoretic Patterns ◽

Tendon Cells ◽

Developmental Phases ◽

Better Than

1. A procedure was developed for the preparation of plasma membranes from experimental granulation tissue of the rat without the addition of enzymes. The yield is better than 20% and the purification at least tenfold. 2. Values are given for the activities of 5′-nucleotidase, Na-+, k-+-activated Mg-2+dependent adenosine triphosphatase and leucine β-naphthylamidase, for lipid composition, and for the gel-electrophoretic patterns of proteins and glycoporteins in the membrane preparations. 3. The plasma membranes from the mature granulation tissue contain proportionally more protein in the lipid phase, but the specific activities of 5′-nucleotidase and Na-+,K-+-activated Mg-2+-dependent adenosine triphosphatase are smaller than in the proliferating tissue. Certain differences were repeatedly observed in the gel-electrophoretic patterns of the developmental phases. 4. The plasma membranes from the granulation tissue were compared with those from rat peritoneal macrophages and from embryonic-chick tendon cells.

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