Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

Localization of l-fucose in the root cap and slime of Zea mays L. utilizing fluorescent lectin conjugates and colloidal gold-lectin techniques

Ulex europaeus lectin (UEA) labelled with fluorescein isothiocyanate (FITC), rhodamine or colloidal gold, localized l-fucose in maize root cap cells and secreted root cap slime. Free-hand sections of maize root apices stained with FITC-UEA or rhodamine-UEA and examined by fluorescence microscopy yielded satisfactory results as long as the stains were freed of unconjugated dye, the sections treated with osmium tetroxide vapour to quench autofluorescence, and the samples incubated at 37°C. This resulted in successful labelling with a lower concentration of fluorochrome-lectin conjugate than reported by previous workers. Rhodamine-UEA was superior to FITC due to the lower primary fluorescence of the root tip observed under green light.Thin sections from glutaraldehyde fixed and Spurr's resin embedded maize root tips were treated with UEA bound to colloidal gold. Gold particles were found within sloughed cells and root cap cells, particularly concentrated over the Golgi complex, Golgi-derived vesicles and within the secretory slime products.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Development and cellular organization ofPinus sylvesfris pollen tubes

The organization ofPinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g.Nicotiana tabacum, was not present inP. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.     

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca²⁺ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca²⁺ gradient.Abbreviations ER endoplasmic reticulum - MES 2-[N-morpholino] ethanesulfonic acid - MFs microfilaments     

The ultrastructure of Candida krusei

Various methods of chemical fixation and freeze-drying of Candida krusei were compared to determine the most appropriate method for the ultrastructural investigation of the thick walled organisms of this genus. Freeze-drying without chemical fixation was of little value because of insufficient variation in electron density. Potassium permanganate was able to penetrate the intact cell but failed to show cytoplasmic glycogen and lipid and some details of the cell wall. While normal glutaraldehyde, formaldehyde and osmium tetroxide treatment failed to permeate and preserve intracellular structures, several cycles of rapid freezing (–155°C) and thawing followed by glutaraldehyde fixation and osmium tetroxide post-fixation demonstrated the intracellular details of the majority of cells so treated.     

Effects of myrmicacin (β-hydroxydecanoic acid) on protoplasmic movement and ultrastructure ofCamellia japonica pollen

Summary While tube elongation of growing pollen ofCamellia japonica was stopped by treatment with 50–100 ppm of myrmicacin, protoplasmic movement in the pollen tube still continued. However, higher concentration (200 ppm) of the inhibitor arrested the movement. The vesicles containing membrane substances disappeared at the tip of the tube of the growth-inhibited pollen. Removal of the inhibitor resulted in the reappearance of the vesicles at the tip region and tube elongation was restored.     

SEC1A and SEC6 synergistically regulate pollen tube polar growth

Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles. The exocyst and Sec1/Munc18 (SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through co-immunoprecipitation-mass spectrometry screening, protein interaction analysis and in vivo microscopy, we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

SYSTEMATIC SIGNIFICANCE OF THE ABSENCE OF PIT-PLUG CAP MEMBRANES IN THE BATRACHOSPERMALES (RHODOPHYTA)1

Four species of the Batrachospermales were examined by transmission electron microscopy to determine whether or not cap membranes, a typical structural component of pit plugs in several orders of red algae, were present. Routine specimen preservation methods used in past studies led to contradictory reports, so Batrachospermum keratophytum Bory, B. sirodotii Skuja ex Reis, Sirodotia suecica Kylin, and S. tenuissima (Collins) Skuja ex Flint were prepared by secondary fixation in potassium permanganate or a combination of potassium ferrocyanide-osmium tetroxide to enhance membrane contrast. These fixation procedures produced clear, well-contrasted images in which cap membranes were absent. The absence of cap membranes in S. suecica and S. tenuissima and the presence of cap membranes in two members of the Nemaliales was confirmed by freeze-substitution methods. Absence of cap membranes in representatives of the Batrachospermales further distinguishes the Batrachospermales from the Nemaliales, the order in which they long resided, and demonstrates the value of this character in elucidating ordinal alliances among the Rhodophyta.     

Cytoplasmic vesicles in germinating spores ofGilbertella persicaria

Summary Germ tube apices ofGilbertella persicaria contain cytoplasmic vesicles, similar to the secretory vesicles found at the tips of vegetative hyphae. The vesicles are present at all stages of development, from the time of germ tube initiation to the establishment of branched hyphae. In contrast to the abundant vesicles at tips of established hyphae, the germ tubes have only a few apical vesicles in a layer next to the plasma membrane. When germinated spores are treated by washing and centrifuging prior to fixation, the cytoplasm is often disrupted near the apex, and the clusters of apical vesicles disappear. The findings indicate the delicate nature of hyphal tips and the necessity of avoiding prefixation stresses in order to preserve the apical apparatus of growing hyphae.     

Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.Li Wang and Yi-Min Liu are considered joint first authors     

FINE STRUCTURE AND CYTOCHEMISTRY OF LILIUM POLLEN TUBES

Growth of the Lilium longiflorum pollen tube in vitro is restricted to a zone extending back 3–5 μ from the tip. Electron micrographs of cross and longitudinal thin sections of L. longiflorum and L. regale pollen tubes reveal that the cytoplasm of the nongrowing region of the tube contains an abundance of mitochondria, amyloplasts, Golgi bodies, endoplasmic reticulum, lipid bodies, and vesicles. In contrast, the growing tip is characterized by an abundance of vesicles and an absence of other cytoplasmic elements. The vesicles appear to be of 2 types. One is spherical, about 0.1 μ in diameter, stains strongly with phosphotungstic acid, apparently arises from the Golgi apparatus and appears to contribute to tube wall and plasmalemma formation. The other type is irregular in shape, 0.01-0.05 μ in diameter, stains strongly with lead hydroxide, and is of unknown origin and function. Cytochemical analysis indicates that the tips of L. longiflorum pollen tubes are singularly rich in ribonucleic acid, protein, and carbohydrate. These findings are discussed in relation to tube growth.     

Cytochemical localization of NADH-ferricyanide oxido-reductase in hypocotyl segments and isolated membrane vesicles of soybean

Summary NADH-ferricyanide oxido-reductase activity was demonstrated at the inner (cytoplasmic) aspect of plasma membranes and plasma membrane vesicles from hypocotyls of etiolated soybean (Glycine max L.) seedlings by cytochemical procedures. The plasma membrane-associated activity, observed in both tissue and vesicle preparations, resisted fixation in 0.1 % glutaraldehyde, required the presence of exogenous pyridine nucleotide and was inhibited by adriamycin. With tissue, the activity could be demonstrated only with broken cells where reactants could penetrate freely. With vesicles of plasma membrane origin, activity was seen only with cytoplasmic side out vesicles (fraction E) prepared by free-flow electrophoresis. Activity was observed also on the cytoplasmic surface of the tonoplast and on putative tonoplast vesicles oriented cytoplasmic side out.Recipient of a NSF/CNRS post doctoral fellowship.     

Investigations into the structure and fixation properties of cytoplasmic lamellae in the hamster oocyte

Summary Lamellar structures have been revealed in the cytoplasm of rapidly growing hamster oocytes by glutaraldehyde fixation and by fixation in 30% ethanol followed by osmication. The structures are not preserved after osmium tetroxide either used alone or followed by glutaraldehyde; nor are they preserved by absolute ethanol, formaldehyde, glyceraldehyde, glyoxal, 2-hydroxy-adipaldehyde or potassium permanganate. Immersion in 30% ethanol followed by extraction in distilled water and fixation in glutaraldehyde and osmium tetroxide exposes the lattice-like skeletal structure of the lamellae. The lamellae are present but slightly altered after short digestion in pepsin. Longer digestion results in complete dissolution of the structures.Supported by U.S.P.H.S. Post-doctoral Fellowship 5 F2 HD-25, 190–02.I wish to thank Prof. R. E. Coupland for his continued interest in this work and for his helpful criticisms.     

Cytochemical localization of myrosinase (β-thioglucosidase) in root tips ofSinapis alba

Summary A method for electron microscopic cytochemical localization of a-thioglucosidase (myrosinase) has been developed. Since sulphate is one of the products of the hydrolysis of sinigrin by myrosinase, it was felt that if the incubation was carried out in the presence of Pb⁺⁺-ions an insoluble precipitate of electron-dense PbSO₄ would be formed at the reaction sites. Following formaldehyde fixation a few different cell organelles in the extreme root tip ofSinapis alba showed reaction specificity for myrosinase but following glutaraldehyde fixation the enzymatic activity was inhibited. Biochemical tests of the isolated enzyme showed complete inhibition of the myrosinase by glutaraldehyde. Variations in the substrate concentration and incubation time indicated that the enzyme was confined to the dilated cisternae of the endoplasmic reticulum and in a limited extent to the mitochondria.     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

Improved Ultrastructural Detail in Tissues Fixed with Potassium Permanganate

An improved method has been developed for fixation with potassium permanganate. Although this is one of the methods widely used to preserve the dense cores of adrenergic storage vesicles, fixation of other tissue components is usually poor. The main differences from previously reported methods using potassium permanganate are the use of a physiological saline as the vehicle for all solutions, and, following this, very rapid dehydration before infiltration with plastic. Cellular and intercellular details of tissue ultrastructure may, in general, be evaluated as satisfactorily as with conventional fixatives, with the exception of certain protein elements associated with ribosome, microtubule, and myofilament organization. Nerve endings with agranular or clear vesicles may be distinguished from adrenergic endings since the dense cores of the vesicles of the latter are preserved by this method.