The mechanics of biological membrane fusion Merger of aspects from electron microscopy and patch-clamp analysis

1992 ◽  
Vol 103 (3) ◽  
pp. 613-618
Author(s):  
HELMUT PLATTNER ◽  
GERD KNOLL ◽  
CHRISTIAN ERXLEBEN
Keyword(s):  
Electron Microscopy ◽  
Patch Clamp ◽  
Membrane Fusion ◽  
Biological Membrane

High-voltage electron microscopy of patch-clamped membranes

1987 ◽  
Vol 45 ◽  
pp. 582-583
Author(s):  
F. Sachs ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
Cell Membrane ◽  
Patch Clamp ◽  
High Voltage Electron Microscopy ◽  
Small Patch ◽  
Cellular Electrophysiology ◽  
Voltage Electron ◽  
Electron Microscopes ◽  
Patch Technique ◽  
Membrane Patch

Cellular electrophysiology has been revolutionized by the introduction of patch clamp techniques. The patch clamp records current from a small patch of the cell membrane which has been sucked into a glass pipette. The membrane patch, a few micons in diameter, is attached to the glass by a seal which is electrically, diffusionally and mechanically tight. Because of the tight electrical seal, the noise level is low enough to record the activity of single ion channels over a time scale extending from 10μs to days. However, although the patch technique is over ten years old, the patch structure is unknown. The patch is inside a glass pipette where it has been impossible to see with standard electron microscopes. We show here that at 1 Mev the glass pipette is transparent and the membrane within can be seen with a resolution of about 30 A.

Lipid Polymorphism and Virus-Membrane Fusion as Observed in Vitrified Thin Films by Cryo-Electron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 492-493
Author(s):  
P.M. Frederik ◽  
K.N.J. Burger ◽  
M.C.A. Stuart ◽  
A.J. Verkleij
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Membrane Fusion ◽  
Film Formation ◽  
Molar Ratio ◽  
Influenza B ◽  
Complex Structures ◽  
Type Ii ◽  
Suspended Material ◽  
Cryo Electron Microscopy

Cellular membranes are often composed of phospholipid mixtures in which one or more components have a tendency to adopt a type II non-bilayer lipid structure such as the inverted hexagonal (H||) phase. The formation of a type II non-bilayer intermediate, the inverted lipid micel is proposed as the initial step in membrane fusion (Verkleij 1984, Siegel, 1986). In the various forms of cellular transport mediated by carrier vesicles (e.g. exocytosis, endocytosis) the regulation of membrane fusion, and hence of inverted lipid micel formation, is of vital importance.We studied the phase behaviour of simple and complex lipid mixtures by cryo-electron microscopy to gain more insight in the ultrastructure of different lipid phases (e.g. Pβ’, Lα, H||) and in the complex membrane structures arising after Lα < - > H|| phase changes (e.g. isotropic, cubic). To prepare hydrated thin films a 700 mesh hexagonal grid (without supporting film) was dipped into and withdrawn from a liposome suspension. The excess fluid was blotted against filter paper and the thin films that form between the bars of the specimen grid were immediately (within 1 second) vitrified by plunging of the carrier grids into ethane cooled to its melting point by liquid nitrogen (Dubochet et al., 1982). Surface active molecules such as phospholipids play an important role in the formation and thinning of these aqueous thin films (Frederik et al., 1989). The formation of two interfacial layers at the air-water interfaces requires transport of surface molecules from the suspension as well as the orientation of these molecules at the interfaces. During the spontaneous thinning of the film the interfaces approach each other, initially driven by capillary forces later by Van der Waals attraction. The process of thinning results in the sorting by size of the suspended material and is also accompanied by a loss of water from the thinner parts of the film. This loss of water may result in the concentration and eventually in partial dehydration of suspended material even if thin films are vitrified within 1 sec after their formation. Film formation and vitrification were initiated at temperatures between 20-60°C by placing die equipment in an incubator provided widi port holes for the necessary manipulations. Unilamellar vesicles were made from dipalmitoyl phosphatidyl choline (DPPC) by an extrusion method and showed a smooth (Lα) or a rippled (PB’.) structure depending on the temperature of the suspensions and the temperature of film formation (50°C resp. 39°C) prior to vitrification. The thermotropic phases of hydrated phospholipids are thus faithfully preserved in vitrified thin films (fig. a,b). Complex structures arose when mixtures of dioleoylphosphatidylethanol-amine (DOPE), dioleoylphosphatidylcholine (DOPC) and cholesterol (molar ratio 3/1/2) are heated and used for thin film formation. The tendency of DOPE to adopt the H|| phase is responsible for the formation of complex structures in this lipid mixture. Isotropic and cubic areas (fig. c,d) having a bilayer structure are found in coexistence with H|| cylinders (fig. e). The formation of interlamellar attachments (ILA’s) as observed in isotropic and cubic structures is also thought to be of importance in biological fusion events. Therefore the study of the fusion activity of influenza B virus with liposomes (DOPE/DOPC/cholesterol/ganglioside in a molar ratio 1/1/2/0.2) was initiated. At neutral pH only adsorption of virus to liposomes was observed whereas 2 minutes after a drop in pH (7.4 - > 5.4) fusion between virus and liposome membranes was demonstrated (fig. f). The micrographs illustrate the exciting potential of cryo-electron microscopy to study lipid-lipid and lipid-protein interactions in hydrated specimens.

scholarly journals Antibody-induced membrane fusion in Paramecium

1984 ◽  
Vol 65 (1) ◽  
pp. 153-162
Author(s):  
A. Barnett ◽  
E. Steers
Keyword(s):  
Electron Microscopy ◽  
Membrane Fusion ◽  
Phase Contrast ◽  
Immobilized Cells ◽  
Living Cells ◽  
Immune Serum ◽  
Induced Membrane ◽  
Transmission Electron ◽  
Membrane Changes ◽  
Antigen Antibody

Immobilization of cells by specific immune serum involves crosslinking between immunoglobulin G (IgG) and the i-antigen in the cell membrane. Globular material is seen to accumulate at the ciliary tips by phase-contrast and fluorescence microscopy in a manner analogous to ‘capping’ in more typical eukaryotes. When immobilized cells of Paramecium are examined by scanning electron microscopy, the fused ciliary tips are seen to be distended, discoidal membranes. Transmission electron microscopy often reveals several ciliary axonemes enclosed within a single, enlarged membrane that is oriented with the ferritin-labelled second antibody directed against the i-antigen antibody on the outer surface only. Fixed cells or living cells treated with immune Fab do not show membrane changes, but do bind antibody. Membrane fusion occurs only if cells are alive and the i-antigen is directly or indirectly cross-linked by intact immune IgG.

scholarly journals Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

2008 ◽  
Vol 183 (2) ◽  
pp. 181-186 ◽  
Author(s):  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Membrane Fusion ◽  
Biological Membrane ◽  
Membrane Integrity ◽  
Fusion Pore ◽  
Mechanistic Models ◽  
Collateral Damage ◽  
Vacuole Fusion ◽  
Membrane Lysis

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.

Lipids in biological membrane fusion

1995 ◽  
Vol 146 (1) ◽  
Author(s):  
L. Chernomordik ◽  
M.M. Kozlov ◽  
J. Zimmerberg
Keyword(s):  
Membrane Fusion ◽  
Biological Membrane

scholarly journals Expansion of the fusion stalk and its implication for biological membrane fusion

2014 ◽  
Vol 111 (30) ◽  
pp. 11043-11048 ◽  
Author(s):  
H. J. Risselada ◽  
G. Bubnis ◽  
H. Grubmuller
Keyword(s):  
Membrane Fusion ◽  
Biological Membrane
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