Freeze-fracture: a personal history.

The sequence of events leading to the development of freeze-fracture replication is described. Subsequent developments discussed include complementary replicas, replica interpretation with stereo micrograph and reversal negatives, replica reinforcement, and control of resistance evaporation.

Plasmodium falciparum: freeze-fracture of the gametocyte pellicular complex.

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.

Vitrification of human monocytes.

Human monocytes purified from peripheral blood by counterflow centrifugal elutriation were cryopreserved in a vitreous state at 1 atm pressure. The vitrification solution was Hanks' balanced salt solution (HBSS) containing (w/v) 20.5% Me2SO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol. Fifteen milliliters of this solution was added dropwise to 1 ml of a concentrated monocyte suspension at 0 degrees C. Of this, 0.8 ml was drawn into silicone tubing and rapidly cooled to liquid nitrogen temperature, stored for various periods, and rapidly warmed in an ice bath. The vitrification solution was removed by slow addition of HBSS containing 20% fetal calf serum. The numerical cell recovery was about 92% and most of these retained normal phagocytic and chemotactic ability. Differential scanning calorimeter records of the solution show a glass transition at -115 degrees C during cooling and warming, but no evidence of ice formation during cooling. Devitrification occurs at about -70 degrees C during warming at rates as rapid as 80 degrees C/min. The amount of devitrification is dependent upon the warming rate. Freeze-fracture freeze-etch electron microscope observations revealed no ice either intra- or extracellularly in samples rapidly cooled to liquid nitrogen temperatures except for small amounts in some cellular organelles. However, if these cell suspensions were warmed rapidly to -70 degrees C and then held for 5 min, allowing devitrification to occur, the preparation contained significant amounts of both intra- and extracellular ice. Biological data showed that this devitrification was associated with severe loss of cell function.

Cold shock hemolysis in human erythrocytes studied by spin probe method and freeze-fracture electron microscopy.

When human erythrocytes are osmotically stressed or chemically treated, they hemolyze on cooling below 10 degrees C (called cold shock). We have studied the effects of osmotic stress and cooling on the state of membrane by the spin-probe method and freeze-fracture electron microscopy. At room temperature, the membrane fluidity detected by 12-doxyl stearate spin probe showed a steady decrease with osmolality in hypertonic NaCl solutions up to 900 mOsm/kg, above which it remained unchanged. In hypertonic sucrose solutions, the electron paramagnetic resonance spectra showed an additional pair of absorptions, indicating development of regions, in the membrane, further immobilized than in NaCl solutions. Mobility of a cholesterol analogue probe, androstane, did not show change by hypertonicity, but the spectral intensity dropped at 1,200 mOsm/kg, probably due to formation of loose aggregates in the cholesterol phase. On cooling the osmotically stressed cells in NaCl solution, the isotropic rotational correlation time vs. inverse temperature plot of 12-doxyl stearate probe exhibited a step-wise discontinuity at approximately 10 degrees C, suggestive of a drastic transition in the state of the membrane. At about the same temperature, the freeze-fracture pattern of osmotically stressed cells revealed the development of large wrinkles and aggregation of membrane particles, in contrast to the case of the cells in isotonicity. Significance of these findings in understanding cold shock hemolysis is discussed.

Reliable use of resistance evaporation of Pt and C for high resolution freeze-fracturing and a crystal surface image complementary to the E-face of yeast plasma membranes.

Freeze-fracture specimens of bakers yeast plasma membranes faces were prepared in both a modified Denton DFE-2-freeze-etch module and a modified Balzers BAF-301 freeze-etch unit. Each unit was equipped with a liquid nitrogen cooled shroud, resistance evaporators with PT-C and C sources 7 cm from the specimens and with a resistance monitor to control PT-C shadow film thickness. Optical diffraction patterns of specimens prepared in these units have fourth, fifth or sixth order spots. Therefore, on the basis of optical diffraction patterns, resolution of yeast plasma membrane specimens prepared in these units is equivalent to or better than that obtained by others with an ultrahigh vacuum system equipped with specially redesigned electron guns. A new image with tube-like particles in hexagonal arrays, each surrounded by six substructure particles, nearly perfect high-resolution complement to the hexagonal array of ring-like depressions and the six surrounding subunit depressions of the E-face, has been revealed on the surfaces of cubic crystals (presumably ice) which formed in the gap between the P- and E-faces within fissures that occurred when the samples were frozen in liquid Freon 22. When the samples were subsequently freeze-fractured at 77 K at a chamber vacuum of 13 microPa in which the specimens were protected from surface contamination by a liquid nitrogen cooled shroud, these crystals remained attached to the P-face but pulled away from the E-face against which they had apparently made molecular contact.

Prefracture and cold-fracture images of yeast plasma membranes.

Fracture-temperature related differences in the ultrastructure of plasmalemma P faces of freeze-fractured baker's yeast (Saccharomyces cerevisiae) have been observed in high-resolution replicas prepared in freeze-etch systems pumped to 2 X 10(-7) torr in which the specimens were protected from contamination by use of liquid nitrogen-cooled shrouds. Two major P-face images were observed regardless of the source of the yeast, the age of the culture, the growth temperature, the physiological condition, or the suspending medium used: (a) a "cold-fracture image" with many strands closely associuated with tubelike particles (essentially the same image as those previously published for yeast freeze-fractured at 77 degrees K), and (b) a "prefracture image" characterized by the presence of more distinct tubelike particles with few or no associated strands (for aging cultures, the image recently referred to as "paracrystalline arrays" of "craterlike particles"). Both types of P-face image can be found in separate areas of single replicas and occasionally even within a single plasma membrane. Whereas portions of replicas known to be fractured at any temperature colder than 218 degrees K reveal only the cold-fracture image, prefracture images are found in cells intentionally fractured at 243 degrees K and in cracks or fissures which develop during the freezing of other specimens. These findings demonstrate that the prefracture image results from the fracturing of specimens at some temperature above 230 degrees K, no t from fracturing specimens at some temperature between 173 degrees and 77 degrees K, and not from the use of "starved" yeast cells.

A resistance monitor with power cut-off for automatic regulation of shadow and support film thickness in freeze-etching and related techniques.

A resistance monitor with sensors and automatic power cut-off has been developed to control the thickness of Pt-C shadow and C replica films in freeze-etching and related techniques. The monitor and sensors, in conjunction with newly modified evaporators, should considerably reduce the amount of C and Pt required and should prove useful in other applications employing vacuum evaporation of thin films of Pt, C, or other conducting materials.

Separation and infectivity of circular and linear forms of potato spindle tuber viroid.

Potato spindle tuber viroid can be separated into two fractions by polyacrylamide gelelectrophoresis in the presence of formamide and urea. One fraction contains predominantly circular molecules; the second fraction contains almost exclusively linear molecules. The purity of the fractions was estimated by electron microscopy of formaldehyde-denatured molecules. The length distributions of denatured circular and linear molecules were determined. Both circular and linear molecules were found to be infectious.

Helical filaments produced by a Mycoplasma-like organism associated with corn stunt disease.

Mycoplasma-like bodies with helical filaments were seen by phase contrast microscopy in juice expressed from tissues of plants infected with corn stunt agent. Each filament is bounded by a "unit membrane" and no cell wall, sheath, envelope, or second membrane has yet been discerned by electron microscopy. The association of these filaments with development of disease, their occurrence in phloem cells as seen by both freeze-etching and thin-section electron microscopy, the diagnosis of infection based on their presence in plants without symptoms, and their absence in noninfected corn are consistent with the hypothesis that these unusual filaments are formed by the mycoplasma-like organism presumed to be the corn stunt agent.

Remission of aster yellows disease by antibiotics.

Suppression of symptoms of aster yellows by antibiotics supports the tentative hypothesis that the etiologic agent is a mycoplasma-or bedsonia-like organism rather than a virus. Development of symptoms was supressed by chlortetracycline, tetracycline, or chloramphenicol, but not by penicillin. When plants were treated with chlortetracycline at 1000 parts per million before symptoms appeared, symptoms developed only after cessation of the treatment. Assay of the agent of aster yellows, extracted from plants, indicated inhibition of growth of the pathogen by treatment with chlortetracycline. Plants severely affected before treatment began developed new symptomless axillary growth, including flowers; previously yellowed leaves often became green. Acquisition of the agent of aster yellows by leafhoppers was drastically reduced when infected plants were treated with chlortetracycline continuously for 1 week before exposure to the vectors. Our data, and preliminary evidence from purification studies, are consistent with a possible mycoplasma-or bedsonia-like etiology of the aster yellows disease.