Plasma Membrane Structure as Revealed with Freeze-Fracture Methods

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The Journal of Cell Biology ◽

10.1083/jcb.76.1.158 ◽

1978 ◽

Vol 76 (1) ◽

pp. 158-174 ◽

Cited By ~ 18

Author(s):

PL Moore ◽

HL Bank ◽

NT Brissie ◽

SS Spicer

Keyword(s):

Plasma Membrane ◽

Electron Microscope ◽

Membrane Fusion ◽

Thin Section ◽

Membrane Structure ◽

Freeze Fracture ◽

Vacuole Membrane ◽

Attachment Sites ◽

Pmn Leukocytes ◽

Scanning Electron

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.

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Effect of Hyperthermia on the Plasma Membrane Structure of Chinese Hamster V79 Fibroblasts: A Quantitative Freeze-Fracture Study

Radiation Research ◽

10.2307/3576560 ◽

1986 ◽

Vol 106 (1) ◽

pp. 47 ◽

Cited By ~ 13

Author(s):

G. Arancia ◽

W. Malorni ◽

G. Mariutti ◽

P. Trovalusci

Keyword(s):

Plasma Membrane ◽

Membrane Structure ◽

Freeze Fracture ◽

Chinese Hamster ◽

Fracture Study

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The polar development of membrane structure during spermiogenesis by freeze fracture and double replica technique

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157796 ◽

1990 ◽

Vol 48 (3) ◽

pp. 50-51

Author(s):

Dang Liankai

Keyword(s):

Plasma Membrane ◽

Nuclear Membrane ◽

Membrane Structure ◽

Freeze Fracture ◽

Glycerol Solution ◽

Nuclear Pores ◽

Replica Technique ◽

Polar Development ◽

Protein Particles ◽

Different Characteristics

With the developing process from the spermatid to the sperm, the great changes of membrane structure are token place both in plasma membrane and nuclear membrane. This note will report the development of polarization in membrane and the distribution of nuclear pores and protein particles during spermiogenesis, and also compare the number of protein in P-face with that in E-face.Mice, Rats, Rabbits and Dogs were operated by taking out the seminiferous tube. It was fixed in 2.5% glutaradehyde for 2 hrs 5 washed in cacodylated buffer, and then the sample was put in to 30% glycerol solution for 6-12hrs. Samples were put into liquid nitrogen rapidly and afterwards moved to BALZAR BAF-400D instrument to get Freeze-Fracture replica and double replica for observation with TEM.The expression and the development of the polarization in membrane structure of the spermatid. The spermatid in different stepshas different characteristics and there is more polarization for membrane structure following spermiogenesis.

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Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100080407 ◽

1977 ◽

Vol 35 ◽

pp. 596-597

Author(s):

E. Keyhani

Keyword(s):

Plasma Membrane ◽

Candida Utilis ◽

Synthetic Medium ◽

Freeze Fracture ◽

Translational Diffusion ◽

Yeast Cells ◽

Distilled Water ◽

Intramembrane Particles ◽

The Matrix ◽

Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

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Deformation of Yeast Plasma Membrane by Ethidium Bromide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103334 ◽

1988 ◽

Vol 46 ◽

pp. 254-255

Author(s):

E. Keyhani

Keyword(s):

Plasma Membrane ◽

Thin Section ◽

Ethidium Bromide ◽

Freeze Fracture ◽

Mutagenic Effect ◽

Yeast Cells ◽

Hydrophobic Region ◽

Thin Section Electron Microscopy ◽

Section Electron ◽

Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

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Warm and freeze-fracture of cotton seed radicles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129206 ◽

1987 ◽

Vol 45 ◽

pp. 984-985

Author(s):

E. L. Vigil ◽

E. F. Erbe

Keyword(s):

Thin Film ◽

Water Vapor ◽

Fracture Surface ◽

Membrane Structure ◽

Room Temperature ◽

Freeze Fracture ◽

Longitudinal Axis ◽

Fracture Plane ◽

Cotton Seeds ◽

Condensed Water

In cotton seeds the radicle has 12% moisture content which makes it possible to prepare freeze-fracture replicas without fixation or cryoprotection. For this study we have examined replicas of unfixed radicle tissue fractured at room temperature to obtain data on organelle and membrane structure.Excised radicles from seeds of cotton (Gossyplum hirsutum L. M-8) were fractured at room temperature along the longitudinal axis. The fracture was initiated by spliting the basal end of the excised radicle with a razor. This procedure produced a fracture through the tissue along an unknown fracture plane. The warm fractured radicle halves were placed on a thin film of 100% glycerol on a flat brass cap with fracture surface up. The cap was rapidly plunged into liquid nitrogen and transferred to a freeze- etch unit. The sample was etched for 3 min at -95°C to remove any condensed water vapor and then cooled to -150°C for platinum/carbon evaporation.

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Freeze-fracture observations on changes in the distribution of Filipin-sterol complexes during epididymal maturation and capacitation of boar spermatozoa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157991 ◽

1990 ◽

Vol 48 (3) ◽

pp. 90-91

Author(s):

N. Seki ◽

Y. Toyama ◽

T. Nagano

Keyword(s):

Plasma Membrane ◽

Essential Role ◽

Freeze Fracture ◽

Final Concentration ◽

Freeze Fracturing ◽

Physiological Conditions ◽

Epididymal Maturation ◽

Cacodylate Buffer ◽

Sperm Membrane ◽

Boar Spermatozoa

It is believed that i ntramembra.nous sterols play an essential role in membrane stability and permeability. To investigate the distribution changes of sterols in sperm membrane during epididymal maturation and capacitation, filipin has been used as a cytochemical probe for the detection for membrane sterols. Using this technique in combination with freeze fracturing, we examined the boar spermatozoa under various physiological conditions.The spermatozoa were collected from: 1) caput, corpus and cauda epididymides, 2) sperm rich fraction of ejaculates, and 3)the uterus 2hr after natural coition. They were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4), and treated with the filipin solution (final concentration : 0.02.0.05%) for 24hr at 4°C with constant agitation. After the filipin treatment, replicas were made by conventional freeze-fracture technique. The density of filipin-sterol complexes (FSCs) was determined in the E face of the plasma membrane of head regions.

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A freeze-fracture-etched study of the physarum polycephalum plasma membrane during early formation of the macroplasmodial stage

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147570 ◽

1993 ◽

Vol 51 ◽

pp. 346-347

Author(s):

Randolph W. Taylor ◽

Henrie Treadwell

Keyword(s):

Plasma Membrane ◽

Life Cycle ◽

Growth Phase ◽

Filter Paper ◽

Physarum Polycephalum ◽

Freeze Fracture ◽

Petri Dish ◽

Wire Screen ◽

Wide Mouth ◽

Sterile Filter

The plasma membrane of the Slime Mold, Physarum polycephalum, process unique morphological distinctions at different stages of the life cycle. Investigations of the plasma membrane of P. polycephalum, particularly, the arrangements of the intramembranous particles has provided useful information concerning possible changes occurring in higher organisms. In this report Freeze-fracture-etched techniques were used to investigate 3 hours post-fusion of the macroplasmodia stage of the P. polycephalum plasma membrane.Microplasmodia of Physarum polycephalum (M3C), axenically maintained, were collected in mid-expotential growth phase by centrifugation. Aliquots of microplasmodia were spread in 3 cm circles with a wide mouth pipette onto sterile filter paper which was supported on a wire screen contained in a petri dish. The cells were starved for 2 hrs at 24°C. After starvation, the cells were feed semidefined medium supplemented with hemin and incubated at 24°C. Three hours after incubation, samples were collected randomly from the petri plates, placed in plancettes and frozen with a propane-nitrogen jet freezer.

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