Rough surfaced smooth endoplasmic reticulum in rat and mouse cerebellar Purkinje cells visualized by quick-freezing techniques.
The in vivo structure of the smooth endoplasmic reticulum (ER) was visualized in rat and mouse cerebellar Purkinje cells by using quick-freezing techniques followed by freeze-substitution for ultrathin-sectioning or freeze-fracturing and deep-etching for replicas. High magnification electron microscopy of the ultrathin sections revealed a surprising finding that all the smooth ER are apparently rough surfaced, and heavily studded with a large number of small dense projections. In the soma the smooth ER appears to be similar to its rough counterpart, except that the projections are slightly smaller, less electron dense and less protrusive on the ER membranes than the ribosomes. The projections were short rectangles, 20 x 20 x 6 nm3 in size, covering the cytoplasmic surface of the smooth ER in a checker-board manner where closely packed. After freeze-etching and replication, they appeared to be composed of four subparticles, surrounding a central channel. Thus the projections are very similar to the foot structure (ryanodine receptor) of the sarcoplasmic reticulum. Furthermore, they were distributed exclusively in the ER compartment and were highly concentrated especially in the smooth ER. This localization of the projections coindides with the intracellular distribution of the inositol 1,4,5-trisphosphate (IP3) receptor determined by quantitative immunogold electron microscopy. These findings would suggest that the projections are tetramers of IP3 receptor molecules and could be used as a morphological marker for the smooth ER in Purkinje cells, which spreads from the soma to the axon and dendrite, up to the tips including the spines. In Purkinje cells tubular smooth ER runs freely in a serpentine fashion or are intertwined to make large membraneous tangles without forming cisternal stacks. It is highly probable that the ER cisternal stacks do not exist naturally in Purkinje cells but are formed artificially during the various procedures for chemical fixation. (+info
Modulation of nanotube formation by structural modifications of sphingolipids.
Galactosylceramides (GalCers) containing nervonoyl (24:1(Delta15(cis))) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (. Biophys. J. 69:1976-1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1((X-9)). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1(Delta15(cis)) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25-30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1(Delta15)GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1(Delta15)Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1(Delta15)GalCer. 22:1(Delta13)GalCer dispersions consisted of long, smooth tubules (35-40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1(Delta11)GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30-35 nm, with helical pitches of 80-90 nm. 18:1(Delta9)GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation. (+info
Apo A-I inhibits foam cell formation in Apo E-deficient mice after monocyte adherence to endothelium.
We have previously shown that expression of the human apo A-I transgene on the apo E-deficient background increases HDL cholesterol and greatly diminishes fatty streak lesion formation. To examine the mechanism, prelesional events in atherosclerotic plaque development were examined in 6- to 8-week-old apo E-deficient and apo E-deficient/human apo A-I transgenic mice. A quantitative assessment of subendothelial lipid deposition by freeze-fracture and deep-etch electron microscopy indicated that elevated apo A-I did not affect the distribution or amount of aortic arch subendothelial lipid deposits. Immunohistochemical staining for VCAM-1 demonstrated similar expression on endothelial cells at prelesional aortic branch sites from both apo E-deficient and apo E-deficient/human apo A-I transgenic mice. Transmission electron microscopy revealed monocytes bound to the aortic arch in mice of both genotypes, and immunohistochemical staining demonstrated that the area occupied by bound mononuclear cells was unchanged. Serum paraoxonase and aryl esterase activity did not differ between apo E-deficient and apo E-deficient/human apo A-I transgenic mice. These data suggest that increases in apo A-I and HDL cholesterol inhibit foam cell formation in apo E-deficient/human apo A-I transgenic mice at a stage following lipid deposition, endothelial activation, and monocyte adherence, without increases in HDL-associated paraoxonase. (+info
Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study.
We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others. (+info
Cellulose microfibrils: visualization of biosynthetic and orienting complexes in association with the plasma membrane.
Cellulose microfibril biosynthesis, assembly, and orientation in the unicellular green alga, Oocystis, is visualized in association with a linear enzyme complex embedded in the B face of the plasma membrane. Granule bands of the A face and complementary ridges of the B face are postulated to assist in the orientation of recently synthesized microfibrils. A model for microfibril synthesis and orientation is proposed and correlated with current hypotheses regarding cellulose biosynthesis in higher plants. (+info
Solenoidal model for superstructure in chromatin.
Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei. (+info
Outer-membrane penetration barriers as components of intrinsic resistance to beta-lactam and other antibiotics in Escherichia coli K-12.
A new technique has been devised to investigate the penetration of antibiotics through the gram-negative outer membrane; the application here was to study intrinsic resistance of Escherichia coli K-12. Exponential cells in broth were briefly treated with 2.5 mM ethylenediaminetetraacetic acid at 5 degrees C to disrupt the outer membrane penetration barrier, and the response of treated and untreated cells to antibiotics was compared by turbidimetry. A barrier index was derived to describe the ability of 7 beta-lactam and 10 other antibiotics to penetrate the outer membrane of strain Y10. There was correlation between the molecular weight and log(10) barrier index (r = 0.59, P congruent with 0.01). The envelope mutant D22 (envA) had low barrier indexes for erythromycin, rifampin, ampicillin, and cloxacillin. For the beta-lactams, outer membrane penetration and affinity for inner membrane target site(s) triggering cell lysis were measured as independent components of the overall activity; although penetration and overall activity varied greatly, the affinities of most were within a narrow range. (+info
Role of cell shape in determination of the division plane in Schizosaccharomyces pombe: random orientation of septa in spherical cells.
The establishment of growth polarity in Schizosaccharomyces pombe cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle. (+info