Freeze-fracture study of phagocytosis in Dictyostelium discoideum

1981 ◽  
Vol 51 (1) ◽  
pp. 63-84
Author(s):  
C. Favard-Sereno ◽  
M.A. Ludosky ◽  
A. Ryter
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Freeze Fracture ◽  
Thin Sections ◽  
Latex Beads ◽  
Fracture Study ◽  
Lateral Phase Separation ◽  
Membrane Changes ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

The plasma membrane and its derivative, the phagosome membrane, were studied during and after ingestion of yeast of latex beads in Dictyostelium discoideum. Freeze-fracture electron microscopy, which provides information on the internal architecture of the membranes, and observation of thin sections of cells treated by cytochemical methods were used in parallel. For visualization of membrane sterols in the replicas, the cells were fixed in the presence of digitonin or the antibiotic filipin. No lateral phase separation occurred during yeast engulfment: the intramembranous particles (IMPs), phospholipids and sterols remained distributed at random in the forming phagosome membrane. In contrast architectural modifications of the membrane were observed upon phagosome internalization. Compared to the plasma membrane, the phagosome membrane displayed 2–3 times more IMPs a shift in the IMP size distribution and a higher sterol content. These changes were completed soon after phagosome closure; they were not related either to the nature of the ingested particles (yeast, latex beads) or to the pH in the membrane environment. The membrane changes too place when the phagosomes began to fuse with pre-existing digestive or autophagic vacuoles and lysosomes. Some of the experimental evidence suggests that the restructuring of the membrane may be related to the presence of hydrolases.

Myocardial sarcolemma in ischemia: a quantitative freeze-fracture study

1988 ◽  
Vol 255 (3) ◽  
pp. H467-H475 ◽  
Author(s):  
J. S. Frank ◽  
S. Beydler ◽  
N. Wheeler ◽  
K. I. Shine
Keyword(s):  
Electron Microscopy ◽  
Analysis Of Variance ◽  
Freeze Fracture ◽  
Mixed Effects ◽  
Fracture Face ◽  
Intramembrane Particles ◽  
Fracture Study ◽  
K Concentration ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Freeze-fracture electron microscopy permits the visualization of the intramembrane particles (IMP). These IMPs are presumably proteins responsible for the main functions of the membrane. Quantitative techniques (Clark-Evan statistics) were applied to determine in a critical manner whether IMP pattern shifts (random, clustered, or ordered) occur under the ischemic conditions (5-45 min with and without reperfusion) and whether this change is related to the experimental condition. In each case three hearts, eight replicas/heart, one area of 0.25 micron 2 of membrane fracture face/replica was measured to give a total of 6 micron 2 of membrane counted for each condition (control vs. ischemic). A mixed effects nested model analysis of variance was performed in each variable. We found that IMP aggregation can be present in some control membranes, but the degree of aggregation was greater and more consistent in membranes made ischemic and followed by reperfusion. Most striking was the significant clustering of IMPs in membranes from hearts ischemic for only 5 min. Reperfusion after only 5 min of ischemia reversed IMP clustering. Functionally at this time there is an increase in K+ concentration in the interstitial space that reaches approximately 15 mM within 10 min and reverses on reperfusion. The structural alteration in IMPs appears to parallel the function in ischemic hearts.

Phytosphingosine kills Candida albicans by disrupting its cell membrane

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Enno C.I. Veerman ◽  
Marianne Valentijn-Benz ◽  
Wim van't Hof ◽  
Kamran Nazmi ◽  
Jan van Marle ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Plasma Membrane ◽  
Adenine Nucleotides ◽  
Freeze Fracture ◽  
Direct Interaction ◽  
Histatin 5 ◽  
Membrane Probe ◽  
Ultrastructural Damage ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Abstract The mechanism of action of phytosphingosine (PHS), a member of the sphingosine family which has candidacidal activity when added externally, was investigated. Previously, it has been reported that the fungicidal activity of PHS is based on the induction of caspase-independent apoptosis. In contrast, we found that addition of PHS causes a direct permeabilization of the plasma membrane of yeast, highlighted by the influx of the membrane probe propidium iodide, and the efflux of small molecules (i.e., adenine nucleotides) as well as large cellular constituents such as proteins. Freeze-fracture electron microscopy revealed that PHS treatment causes severe damage of the plasma membrane of the cell, which seems to have lost its integrity completely. We also found that PHS reverts the azide-induced insensitivity to histatin 5 (Hst5) of Candida albicans. In a previous study, we had found that the decreased sensitivity to Hst5 of energy-depleted cells is due to rigidification of the plasma membrane, which could be reverted by the membrane fluidizer benzyl alcohol. In line with the increased membrane permeabilization and ultrastructural damage, this reversal of the azide-induced insensitivity by PHS also points to a direct interaction between PHS and the cytoplasmic membrane of C. albicans.

scholarly journals Membrane differentiations at sites specialized for cell fusion.

1977 ◽  
Vol 72 (1) ◽  
pp. 144-160 ◽  
Author(s):  
R L Weiss ◽  
D A Goodenough ◽  
U W Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Active Role ◽  
Central Dome ◽  
Thin Sections ◽  
Mating Structure ◽  
Putative Site ◽  
Freeze Fracture Electron Microscopy

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.

Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin

2002 ◽  
Vol 282 (6) ◽  
pp. F998-F1011 ◽  
Author(s):  
Tian-Xiao Sun ◽  
Alfred Van Hoek ◽  
Yan Huang ◽  
Richard Bouley ◽  
Margaret McLaughlin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Direct Evidence ◽  
Collecting Duct ◽  
Freeze Fracture ◽  
Water Channels ◽  
Dominant Negative ◽  
Coated Pits ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Before the identification of aquaporin (AQP) proteins, vasopressin-regulated “water channels” were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin-coated pits on the apical surface of collecting duct principal cells. Furthermore, by using a fracture-label technique applied to LLC-PK1cells expressing an AQP2- c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin-coated pits and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated before internalization.

scholarly journals A freeze-fracture study of early membrane events during mast cell secretion.

1977 ◽  
Vol 73 (3) ◽  
pp. 660-671 ◽  
Author(s):  
S J Burwen ◽  
B H Satir
Keyword(s):  
Mast Cell ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Ionic Composition ◽  
Freeze Fracture ◽  
Polymyxin B ◽  
Cell Secretion ◽  
Granule Membrane ◽  
Fracture Study ◽  
Freeze Fracture Electron Microscopy

The early membrane events taking place during mast cell secretion were followed in transmission and freeze-fracture electron microscopy. In order to slow down exocytosis and capture intermediate stages of membrane fusion, special conditions of incubation and stimulation were used. These were as follows: (a) the use of incubation media with altered ionic composition, and (b) stimulation with a low dosage of polymyxin B sulfate (4 microgram/ml) at low temperature (18 degrees C) for very short incubation times (30-60 s), with or without the presence of formaldehyde (0.8%). Under these conditions, unetchable circular impressions are found on the E face of the plasma membrane, 80-100 nm in diameter, with particles associated with their perimeters. In granule-to-granule fusion, the zone involved is demarcated by one or two rows of particles on the E face. In addition, raised circular areas of varying diameters (43-87 nm) surrounded by similar particles, also found on the E face, may represent potential sites before completion of fusion. Neither the circular impressions on the plasma membrane nor the sites on the granule membrane are permanent, but their appearance coincides with initiation of membrane fusion.

scholarly journals Ultrastructure of Na,K-transport vesicles reconstituted with purified renal Na,K-ATPase.

1980 ◽  
Vol 86 (3) ◽  
pp. 746-754 ◽  
Author(s):  
E Skriver ◽  
A B Maunsbach ◽  
P L Jørgensen
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Cation Transport ◽  
Coupled Transport ◽  
Vesicle Membrane ◽  
Thin Sections ◽  
Intramembrane Particles ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
K Transport

To study the size and structure of the Na,K-pump molecule, the ultrastructure of phospholipid vesicles was examined after incorporation of purified Na,K-ATPase which catalyzes active coupled transport of Na+ and K+ in a ratio close to 3Na/2K. The vesicles were analyzed by thin sectioning and freeze-fracture electron microscopy after reconstitution with different ratios of Na,K-ATPase protein to lipid, and the ultrastructural observations were correlated to the cation transport capacity. The purified Na,K-ATPase reconstituted with phospholipids to form a very uniform population of vesicles. Thin sections of preparations fixed with glutaraldehyde and osmium tetroxide showed vesicles limited by a single membrane which in samples stained with tannic acid appeared triple-layered with a thickness of 70 A. Also, freeze-fracture electron microscopy demonstrated uniform vesicles with diameters in the range of 700-1,100 A and an average value close to 900 A. The vesicle diameter was independent of the amount of protein used for reconstitution. Intramembrane particles appeared only in the vesicle membrane after introduction of Na,K-ATPase and the frequency of intramembrane particles was proportional to the amount of Na,K-ATPase protein used in the reconstitution. The particles were evenly distributed on the inner and the outer leaflet of the vesicle membrane. The diameter of the particles was 90 A and similar to our previous values for the diameter of intramembrane particles in the purified Na,K-ATPase. The capacity for active cation transport in the reconstituted vesicles was proportional to the frequency of intramembrane particles over a range of 0.2-16 particles per vesicle. The data therefore show that active coupled Na,K transport can be carried out by units of Na,K-ATPase which appear as single intramembrane particles with diameters close fo 90 A in the freeze-fracture micrographs.

scholarly journals Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study.

1979 ◽  
Vol 80 (1) ◽  
pp. 141-149 ◽  
Author(s):  
S L Tamm
Keyword(s):  
Electron Microscopy ◽  
Shear Zone ◽  
Cell Shape ◽  
Freeze Fracture ◽  
Clockwise Rotation ◽  
Fracture Study ◽  
Freeze Fracture Electron Microscopy ◽  
Specific Membrane ◽  
Fracture Electron

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.

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