Brain myosin-V is a two-headed unconventional myosin with motor activity.

Chicken myosin-V is a member of a recently recognized class of myosins distinct from both the myosins-I and the myosins-II. We report here the purification, electron microscopic visualization, and motor properties of a protein of this class. Myosin-V molecules consist of two heads attached to an approximately 30 nm stalk that ends in a globular region of unknown function. Myosin-V binds to and decorates F-actin, has actin-activated magnesium-ATPase activity, and is a barbed-end-directed motor capable of moving actin filaments at rates of up to 400 nm/s. Myosin-V does not form filaments. Each myosin-V heavy chain is associated with approximately four calmodulin light chains as well as two less abundant proteins of 23 and 17 kd.

A technique for the preparation of cross-sectional TEM samples of ZnSe/GaAs heterostructures which eliminates process-induced defects.

Cross-sectional transmission electron microscopy (TEM) sample preparation of ZnSe/GaAs epitaxial films is investigated. Conventional argon ion milling is shown to produce a high density (approximately 5-8 x 10(11)/cm2) of small (diameter approximately 60-80 A) extended defects (stacking faults, microtwins, double positioning twins, etc.). In addition, transmission electron diffraction results indicate a thin ZnO layer can also occasionally form upon ion milling or electron-beam irradiation although the exact conditions for ZnO formation are not well understood. Conventional TEM (amplitude contrast) and high-resolution TEM (phase contrast) imaging in combination with transmission electron diffraction studies were performed to determine the optimum method of removing the ion milling related damage and ZnO layers during sample preparation. HF/HCl, NaOH/H2O, H2SO4/H2O2/H2O and Br2/CH3OH etching mixtures as well as low voltage argon or iodine ion milling were studied. A low energy (2 keV) iodine or argon ion milling step was shown to remove the ZnO layer and reduced the density of the extended defects associated with Ar+ ion milling, but was unsuccessful in removing all of the defects. Auger electron spectroscopy results indicate residual iodine was either left on the surface or implanted beneath the surface during iodine ion milling. Etching the XTEM samples in HF/HCl was shown to be effective in removing the ZnO layer but had little or no effect on the ion milling induced defects. Etching the samples in a 0.5% Br2/CH3OH solution resulted in complete elimination of the ion milling induced extended defects including the residual defects associated with iodine ion milling. In addition the Br2/CH3OH etch produced the best surface morphology. Thus a brief (1-2 seconds) Br2/CH3OH etch after conventional preparation (argon ion milling) of cross-sectional ZnSe/GaAs TEM samples appears to be an inexpensive and superior alternative to iodine ion milling.

Proliferation of microbodies in Saccharomyces cerevisiae.

The development of microbodies in the yeast Saccharomyces cerevisiae was studied in response to different conditions of growth. Various strains of S. cerevisiae were investigated, using cells from the exponential growth phase on glucose as an inoculum in all transfer experiments. Electron microscopy, including serial sectioning, revealed that these cells generally contained one to four small microbodies which were localized in the vicinity of the cell wall and characterized by the presence of catalase. Transfer of these glucose-grown cells into media supplemented with various compounds known to induce microbody proliferation in other yeasts--i.e. uric acid, alkylated amines, amino acids, C2-compounds such as ethanol or acetate, in the presence or absence of compounds that induce oxygen radical formation--did not result in a significant change in the number of microbody profiles observed. Marked microbody proliferation was, however, observed after a shift of cells into media containing oleic acid and was associated with the induction of activities of beta-oxidation enzymes. In addition, catalase and isocitrate lyase were present in enhanced levels. Kinetic experiments suggested that these microbodies developed from those originally present in the inoculum cells. In thin sections up to 14 microbody profiles were occasionally observed, often present in small clusters. Their ultimate volume fraction amounted to 8-10% of the cytoplasmic volume.

A procedure for obtaining complementary replicas of ultra-rapidly frozen sandwiched samples.

Complementary replicas of samples prepared for electron microscopy by the freeze-fracture/etch technique are extremely valuable in the interpretation of the exposed surfaces, the nature and location of the membrane fracture plane, and as an aid in the recognition of the potential artefacts of this technique. This paper describes a procedure for the preparation of complementary replicas of thin samples sandwiched between copper foil strips and frozen ultra-rapidly in the absence of chemical pretreatments. In this procedure, the copper foil support bearing the replica is floated on the surface of a chromic acid solution, resulting in the controlled dissolution of the copper metal. The replica which remains at the surface of the chromic acid is then stabilized against fragmentation during subsequent cleaning and rinsing steps by placing a 50 mesh gold grid on top of the replica. To minimize agitation of the replica/grid, all cleaning steps are performed in a single depression plate well. The clean replica/grid is picked up from below on a thin Formvar film, dried, and then separated from the extra film. Careful placement of the gold grid on the replicas and low magnification electron micrograph montages of the complementary grids facilitate the location of complementary regions and simplify examination of complementary specimen areas at higher magnification.

Crystallization of intramembrane particles in rabbit sarcoplasmic reticulum vesicles by vanadate.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle appear in freeze-fracture as 0.15-0.2 micron vesicles. The concave fracture surface (P-face) contains a dense population of 8.5 nm particles that were previously identified as the Ca2+-transport ATPase. The convex surface (E-face) is mostly smooth, displaying an occasional particle but no complementary arrays of pits. Incubation of the vesicles at 4 degrees C in calcium-free solutions containing 5 mM Na3VO4 induces the formation of two-dimensional crystalline arrays of the Ca2+, Mg2+-ATPase, accompanied by structural changes visible by freeze-etch electron microscopy. Most vesicles elongate into tubules 60-80 nm in diameter and the 8.5 nm intramembrane particles of the P-face become regularly organized into parallel ridges. The ridges are coiled around the tubules in right-handed helices, oriented at 50-60 degrees angle to the long axis of the tubules. The particles repeat along the rows at about 5.5 nm and the rows repeat at 10.5-11.0. Occasionally the ridges seem to break up into 8.5 nm particles. Parallel furrows are visible on the (convex) E-face of the tubules. In high resolution replicas, the furrows are resolved into rows of pits that are complementary images of the ridges. Deep etching and rotary shadowing reveal oblique crests on the protoplasmic surface, consisting of dimeric particles close to 8.5 X 5.5 nm in size, in which each monomer can frequently be resolved into two structural domains. These data suggest that vanadate induces a conformational change in the Ca2+-transport ATPase, with crystallization of the intramembrane particles.

Actin-myosin interactions visualized by the quick-freeze, deep-etch replica technique.

A new method of preparing biological samples for electron microscopy has been used to re-examine the structure of actin filaments, actin filaments decorated by myosin subfragment-1 (S1), and insect flight muscles. Samples were quick-frozen by contact with a block of copper cooled to approximately 4 K; then were freeze-fractured, deep-etched, rotary-replicated with platinum, and viewed in a transmission electron microscope. By this approach, actin filaments display prominent transverse bands whose repeat (approximately 5.5 nm) and pitch (approximately 15 to 20 degrees) fit with the expected left-handed "genetic" helix. Freeze-etched actin filaments do not, however, display the usual two-start helix as prominently as is seen after negative staining, and they also appear substantially thicker than after negative staining (9 to 10 nm versus 8 nm). The latter two-start helix appears very clearly after S1 decoration. Nevertheless, freeze-etched acto-S1 does not display the "arrowheads" that are seen after negative staining. Instead it displays the outer envelope of the helically deployed S1, and as would be expected from current models derived from optical reconstruction of negatively stained samples, this surface view looks only slightly polarized. Finally, the quick-freeze, deep-etch approach provides particularly distinct images of the crossbridges in insect flight muscles. These are plentiful and regularly arranged in rigor muscles, but rare in muscles relaxed with ATP before freezing. In rigor muscles fixed with aldehydes, these crossbridges assume a broad distribution of inclination, ranging from 45 degrees to 90 degrees with a mean of approximately 80 degrees, which is less tilt than has been seen before in thin-sectioned muscles. However, when aldehyde fixation is followed by exposure to tannic acid with or without uranyl acetate block-staining, crossbridges assume a more acute angle with respect to the fiber axis, centering around 45 degrees. This is associated with a commensurate reduction in interfilament spacing within the muscle fibers, such that tilted crossbridges are not any longer than untilted ones (both measuring approximately 15 nm). At the opposite extreme, crossbridges often become stretched in unfixed muscles, owing to an unnatural increase in interfilament spacing that occurs during sample preparation; in such regions, crossbridges display narrow "stalks", which invariably emerge from the thick filaments at close to 90 degrees. We conclude that crossbridge shape and orientation is strongly affected by different methods of sample preparation, and this will make it difficult to visualize natural crossbridge movements by electron microscopy.

Thin sectioning and freeze-etching studies of isolated islets of Langerhans of rats in gestation and after hypothalamic lesion.

Morphometrical studies of B-cell granules have not demonstrated marked morphological changes in various states of hyperinsulinemia.

Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum).

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.

Euglena plasma membrane during normal and vitamin B12 starvation growth.

Freeze-fracture and optical diffraction techniques were used to study the organization of the Euglena pellicle during the normal and replicative stages of the cell cycle and during vitamin B12 starvation. It was shown that the diffuse layer underlying the tripartite structure has a fibrillar structure. Despite the absence of homology in the 2 fracture faces of the pellicle, the EF striated and the PF particulate ones appear complementary as shown by optical diffraction studies; it must therefore be considered as a true membrane. The grooves are free from such particles and striations. They appear as a specific pattern of the cortex, different from the ridges in their structural organization and their replicative capacity as observed during vitamin B12 starvation. This notion is confirmed by the mode of pellicular growth which is characterized by 2 steps. The first occurs during the early replicative stage (pre-mitotic phase of the cell cycle) when the formation of a new ridge is correlated with the appearance of the 'minor' orientation of a 2-dimensional lattice on the EF and the PF faces and the spread of the particles over the PF face of the space between the old ridges. The second takes place during the lengthening of the ridges from the initiating posterior side (non-replicative stage). During this second step, the 'major' orientation of the lattice is preferentially observed in control cells and exclusively in starved cells. The striking differences between the grooves and the ridges is discussed, as well as the 2 modes of growth and their significance in morphogenesis.

Ultrastructure and partial characterization of a regular array in the cell wall of Lactobacillus brevis.

The cell wall of Lactobacillus brevis was revealed by electron microscopy to have an outer layer composed of a regular array. The morphological unit of the regular array appeared to consist of four spherical subunits, each about 2 nm in diameter, which were arranged in a tetragonal pattern about 4.5 by 7.0 nm in dimension. The regular array was composed of the tetragonal units in rows in two directions at an angle of about 75 degrees to each other. The average spacing between the rows was about 10 nm in one direction and about 7 nm in the other. The tetragonally arranged subunits were removed from the cell wall by treatment with guanidine hydrochloride, urea, or sodium dodecyl sulfate (SDS) but not by the action of ethylenediaminetetraacetate, nonionic detergents, or proteolytic enzymes except pepsin. The regular subunits were shown to be composed of a protein with a molecular weight of about 51,000 by SDS-polyacrylamide gel electrophoresis.

A freeze-etching and replication study of wall deposition in elongating plant cells.

The architecture of the expanding wall of mung bean hypocotyl (Phaseolus aureus) and collenchyma of celery (Apium graveolens) was examined using freeze-etching without any cryoprotectant, and surface-replication of frozen-ground and air-dried specimens. The polylamellated organization of the wall was seen. Freeze-etching clearly visualized, within one single fracture plans, the intermediate strata in which the microfibril orientation gradually changes between the main transverse and longitudinal directions. They corresponded to the bow-shaped arcs seen with surface replication and conventional microtomy. The organization of newly-formed microfibrils (periplasmic microfibrils) was seen by their imprints on the plasmalemma. When they were being deposited the microfibrils were loose and sinuous though tightly packed, rigid and parallel on the further layers. Therefore it seems that the fibrillogenesis and the spatial orientation of the microfibrils are two subsequent steps. The role of the periplasm in controlling the three-dimensional arrangement of the wall is emphasized.

Qualitative x-ray spectrometric study of E. histolytica trophozoite nuclei.

Qualitative X-ray energy spectroscopical analysis was performed in two strains of Entamoeba histolytica trophozoites: HK-9; NIH strain, and HM-2; IMSS strain, cultured in axenic and monoxenic conditions, respectively. The trophozoites were fixed in buffered-glutaraldehyde, embedded in Epon, thick sectioned )50-200 nm), and mounted on nickel grids. X-ray emissions were analyzed with an Ortec X-ray energy detector and multichannel and analyzer adapted to a transmission electron microscope. The study was concentrated on the refractive intranuclear bodies, characteristically numerous in axenically grown E. histolytica trophozoites. Nuclei of trophozoites with those intranuclear bodies contained phosphorus, calcium, and magnesium in greater amounts than nuclei not containing those structures. This was true for all axenically grown trophozoites and two monoxenically grown trophozoites which contained four intranuclear bodies. It is concluded that the composition of the refractive intranuclear bodies in E. histolytica trophozites does not appear to change with the culture conditions or with the composition of the culture media.

Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter species HO1-N.

The ultrastructure of Acinetobacter sp. strain HO1-N grown on hydrocarbon and nonhydrocarbon substrates was compared using thin sections and freeze-etching. Hydrocarbon-grown cells were characterized by the presence of intracytoplasmic membrane-bound hexadecane inclusions. This membrane did not exhibit a typical unit membrane structure but appeared as a monolayer. The freeze-etch technique revealed the internal structure of the hexadecane inclusions and provided evidence for the presence of a smooth-surfaced limiting membrane. Freeze-etching also revealed intracytoplasmic membranes in the hexadecane-grown cells. These ultrastructural modifications were not present in nonhydrocarbon-grown cells. The hexadecane inclusions were isolated from Acinetobacter. Negative-staining of the inclusions revealed electron-transparent vesicles approximating the size of the inclusions seen in whole cells. Freeze-etching of the purified inclusions revealed membrane-bound vesicles. The purified inclusions exhibited a relatively high value of lipid phosphorus to protein. The lipid composition and the electrophoretic banding pattern of the inclusions on sodium dodecyl sulfate-polyacrylamide gels were determined and compared with other membrane fractions (outer membrane and cytoplasmic membrane) previously isolated from this organism.

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.

Ultrastructural and chemical alteration of the cell envelope of Pseudomonas aeruginosa, associated with resistance to ethylenediaminetetraacetate resulting from growth in a Mg2+-deficient medium.

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.