Temperature-induced physical changes in fungal plasma membranes

1980 ◽  
Vol 58 (10) ◽  
pp. 1138-1143 ◽  
Author(s):  
R. W. Miller
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Protein Complexes ◽  
Divalent Cations ◽  
Freeze Fracture ◽  
Ordered Structures ◽  
Membrane Protein Complexes ◽  
Hexagonally Ordered ◽  
Physical Changes

Ordered structures suggesting phase separation of lipids were observed in freeze-fracture replicas of plasma membranes of Fusarium sulphureum after free protoplasts had been fixed at 0 °C. Areas representing up to 50% of the protoplast plasma membrane inner fractured face assumed a lateral, hexagonally ordered structure from which the normally randomly distributed 25-nm membrane protein complexes had been excluded. Intact macroconidia only rarely showed small regions of lateral ordering of this type when fixed at the same, nonlethal temperature.The normal impermeability of the macroconidial plasma membrane to divalent cations such as Ni2+ declined rapidly when the cells were incubated at temperatures 5 °C or more above the physiological growth temperature range of the organism. Admission of divalent cations and death of the cells was associated with an irreversible thermotropic change in the fluidity in the membrane lipids as indicated by lipophilic spin probes.Partitioning spin probe and freeze-fracture techniques were best suited to the observation of thermotropic physical changes in the fungal plasma membrane which were induced at supra- and sub-physiological temperatures, respectively.

scholarly journals Cytochemical evidence for the presence of phospholipids in epithelial tight junction strands.

1993 ◽  
Vol 41 (5) ◽  
pp. 649-656 ◽  
Author(s):  
F W Kan
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Phospholipase A2 ◽  
Plasma Membranes ◽  
Membrane Lipids ◽  
Freeze Fracture ◽  
Fracture Face ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Specific Association

Previous freeze-fracture experiments using either glutaraldehyde-fixed and cryoprotected specimens or unfixed rapid-frozen samples led to the proposal that cylindrical strands of the tight junction (TJ) observed in freeze-fracture preparations are inverted cylindrical micelles made up of membrane lipids and, possibly, membrane proteins. However, no one has yet been able to directly label the structural fibrils of the TJ. To test the hypothesis that TJ strands observed on freeze-fracture preparations are composed at least partially of lipids, we have combined the phospholipase A2-gold and the fracture-label techniques for localization of phospholipids. Phospholipase A2, purified from bee venom, was adsorbed on gold particles and used for specific labeling of its substrate. Phospholipase A2-colloidal gold (PLA2-CG) complex was applied to freeze-fractured preparations of rat exocrine pancreatic cells and testicular Sertoli cells, both of which are known to have extensive TJ complexes on their plasma membranes. Fracture-label replicas of exocrine pancreatic cells revealed specific association of gold particles with TJ fibrils on the protoplasmic fracture-face of the plasma membrane. The majority of these gold particles were observed either directly on the top of the TJ fibrils or adjacent to these cylindrical structures. A high density of PLA2-CG labeling was also observed over the complementary exoplasmic fracture-face of the TJ complex. This intimate association of PLA2-CG labeling with the TJ is particularly evident in the Sertoli cell plasma membrane, where rows of gold particles were observed to be superimposed on parallel arrays of cylindrical strands of the TJ complex. The present findings provide direct cytochemical evidence to support the hypothesis that cylindrical TJ strands observed in freeze-fracture preparations contain phospholipids.

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 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 Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin.

1988 ◽  
Vol 106 (3) ◽  
pp. 649-656 ◽  
Author(s):  
L Morris ◽  
P R Crocker ◽  
S Gordon
Keyword(s):  
Plasma Membrane ◽  
Divalent Cation ◽  
Plasma Membranes ◽  
Fetal Liver ◽  
Divalent Cations ◽  
Membrane Receptors ◽  
Erythroid Cells ◽  
Mammalian Development ◽  
Trypsin Treatment ◽  
Plasma Membrane Receptors

During mammalian development the fetal liver plays an important role in hematopoiesis. Studies with the macrophage (M phi)-specific mAb F4/80 have revealed an extensive network of M phi plasma membranes interspersed between developing erythroid cells in fetal liver. To investigate the interactions between erythroid cells and stromal M phi, we isolated hematopoietic cell clusters from embryonic day-14 murine fetal liver by collagenase digestion and adherence. Clusters of erythroid cells adhered to glass mainly via M phi, 94% of which bound 19 +/- 11 erythroblasts (Eb) per cell. Bound Eb proliferated vigorously on the surface of fetal liver M phi, with little evidence of ingestion. The M phi could be stripped of their associated Eb and the clusters then reconstituted by incubation with Eb in the presence of divalent cations. The interaction required less Ca++ than Mg++, 100 vs. 250 microM for half-maximal binding, and was mediated by a trypsin-sensitive hemagglutinin on the M phi surface. After trypsin treatment fetal liver M phi recovered the ability to bind Eb and this process could be selectively inhibited by cycloheximide. Inhibition tests showed that the Eb receptor differs from known M phi plasma membrane receptors and fetal liver M phi did not bind sheep erythrocytes, a ligand for a distinct M phi hemagglutinin. We propose that fetal liver M phi interact with developing erythroid cells by a novel nonphagocytic surface hemagglutinin which is specific for a ligand found on Eb and not on mature red cells.

scholarly journals A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes

2019 ◽  
Vol 32 (5) ◽  
pp. 219-230
Author(s):  
Jason M Lajoie ◽  
Yong Ku Cho ◽  
Dustin Frost ◽  
Samantha Bremner ◽  
Lingjun Li ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Blood Brain Barrier ◽  
Membrane Trafficking ◽  
Plasma Membranes ◽  
Protein Complexes ◽  
Brain Barrier ◽  
Yeast Display ◽  
Functional Protein ◽  
Blood Brain ◽  
Membrane Protein Complexes

Abstract Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema

2002 ◽  
Vol 282 (6) ◽  
pp. L1382-L1390 ◽  
Author(s):  
Paola Palestini ◽  
Chiara Calvi ◽  
Elena Conforti ◽  
Laura Botto ◽  
Carla Fenoglio ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Activation ◽  
Membrane Lipids ◽  
Gradient Centrifugation ◽  
Mechanical Stimuli ◽  
Biophysical Properties ◽  
Interstitial Edema ◽  
Vascular Perfusion

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml · kg−1 · min−1 saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (∼4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.

scholarly journals Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

1990 ◽  
Vol 38 (10) ◽  
pp. 1421-1426 ◽  
Author(s):  
M R Torrisi ◽  
A Pavan ◽  
L V Lotti ◽  
G Migliaccio ◽  
M C Pascale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Viral Proteins ◽  
Low Ph ◽  
Surface Distribution ◽  
Transfected Cells ◽  
Cytosolic Side ◽  
Infected Cells

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

scholarly journals Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.

1984 ◽  
Vol 99 (2) ◽  
pp. 390-402 ◽  
Author(s):  
D Zucker-Franklin ◽  
S Petursson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Precursor Cell ◽  
Membrane Domains ◽  
Thin Sections ◽  
Energy Dependent ◽  
First Time ◽  
Later Phase ◽  
Peripheral Cytoplasm

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.

scholarly journals Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

1977 ◽  
Vol 74 (2) ◽  
pp. 561-577 ◽  
Author(s):  
DS Friend ◽  
L Orci ◽  
A Perrelet ◽  
R Yanagimachi
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Phosphatidylcholine Liposomes ◽  
Acrosomal Membrane ◽  
Large Particles ◽  
Fracture Appearance ◽  
Preparatory Stage

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.

A comparison of membrane fracture faces of fixed and unfixed glycerinated tissue

1976 ◽  
Vol 21 (3) ◽  
pp. 437-448
Author(s):  
A.S. Breathnach ◽  
M. Gross ◽  
B. Martin ◽  
C. Stolinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Striated Muscle ◽  
Freeze Fracture ◽  
Particle Aggregation ◽  
Buccal Epithelium ◽  
Mitochondrial Membranes ◽  
Nuclear Membranes ◽  
General Plasma

Fixed (glutaraldehyde, 3%) and unfixed specimens of rat buccal epithelium, striated muscle, and liver, were cryoprotected with glycerol, freeze-fractured, and replicated without sublimation. A comparison of fracture faces of general plasma membranes, nuclear membranes, mitochondrial membranes, and membranes of rough endoplasmic reticulum revealed no significant differences as between fixed and unfixed material. Apart from some membranes of liver endoplasmic reticulum, there was no evidence of aggregation or redistribution of intramembranous particles in the unfixed material. The results demonstrate that chemical prefixation of tissues for freeze-fracture is not always necessary, or even desirable, and that glycerol may not be as deeply or directly implicated in particle aggregation as previously thought. Fixation with glutaraldehyde alters the cleaving behaviour of plasma membrane at desmosomes and tight junctions, but not at gap junctions.

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