Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

Calorimetric and fluorescence characterization of interactions between enkephalins and liposomal and synaptic plasma membranes containing gangliosides

The interactions of the opioid peptide [D-Ala2]methionine-enkephalinamide with dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing gangliosides GM1, Gd1a, and Gt1b and synaptic plasma membranes selectively enriched with dimyristoylphosphatidylcholine (DMPC) and ganglioside Gd1a have been investigated by using high-sensitivity differential scanning calorimetry. In the absence of gangliosides, the addition of enkephalinamide in concentrations of up to 10(-3) M does not induce any appreciable change in the heat capacity function of DPPC. In the presence of gangliosides, however, changes in the heat capacity function were observed with as little as micromolar concentrations of the enkephalinamide; the same is true for DMPC-Gd1a-enriched synaptic membranes. The magnitude and the nature of the enkephalinamide effect depend on the type of ganglioside studied. For DPPC vesicles containing ganglioside GM1 only a slight broadening in the heat capacity function and a small upward shift in the transition temperature were observed. For DPPC vesicles containing ganglioside Gd1a the effect was more dramatic; enkephalinamide concentrations as low as 10(-5) M caused the appearance of two well-defined peaks in the heat capacity function in contrast to the one peak observed in the absence of enkephalinamide. In the case of DPPC vesicles containing ganglioside Gt1b the enkephalinamide effect was seen at concentrations of 10(-4) M or higher. Synaptic plasma membranes were isolated from bovine brain, selectively enriched with exogenous lipid, and their thermotropic behavior was characterized by steady-state fluorescence spectroscopy and differential scanning calorimetry. This lipid enrichment results in the appearance of a membrane phase transition otherwise absent in the intact membrane preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intranuclear annulate lamellae in oocytes of the tunicate,Styela partita

Summary Intranuclear annulate lamellae have been observed with the electron microscope in oocytes of the tunicate, Styela partita. Morphological evidence suggests that the annulate lamellae may arise by a specialized fusion process of individual vesicles. Intranuclear vesicles appear to be formed, in time, before differentiated annulate lamellae. It is also suggested that the position and structure of an annulus is in large part determined by the fusion of the vesicles. An annulus may be present as soon as two vesicles have completed their fusion process. Finally, it is again suggested on the basis of morphological evidence that the intranuclear vesicles are derived by the blebbing activity of the inner layer of the nuclear envelope.This investigation was supported by grants (RG-9229, 9230) from the National Institutes of Health, Public Health Service. The electron microscope facilities used were also supported by a grant (GM-05479) from the National Institutes of Health to Professor H. W. Beams.     

The Golgi complex

Summary The ultrastructural arrangement of membranes of the Golgi complex has been characterized in Golgi fractions isolated from rat liver. Procedures for isolation of these fractions have been modified to provide a good yield of Golgi membranes (60 to 70%) with greater than 50-fold purification of sialyl transferase, an enzyme specific for the Golgi complex. The isolated membranes appear well preserved and both the dimensions and appearance of the Golgi complex observed by negative staining and in sections of the isolated membranes correlate well with that in liver sections.The Golgi complex consists of a series of platelike structures, each consisting of a central sac or cisterna from which a network of fine tubules arises. The tubules increase in diameter towards the periphery of the plate and are associated with the formation of vacuoles or secretory vesicles. The structure of the Golgi complex has been related to its role in glycoprotein biosynthesis.     

ELECTRON MICROSCOPY OF CENTRIFUGED HYPHAE OF NEUROSPORA

Normal and centrifuged hyphae of Neurospora were studied with the electron microscope. The following cell structures could be identified: nuclei with nucleoli, mitochondria, endoplasmic reticulum, ribosomes, glycogen, fat bodies, vacuoles, and vesicles with an inner canalicular system, of unknown nature. In centrifuged hyphae, the glycogen layer appeared as a light area, with a slight indication of granular structure. The ribosome layer consisted of densely packed ribosomes without any membranes. The mitochondrial layer contained spaces filled with ribosomes. The nuclei were loosely packed, with endoplasmic reticulum between them. The "enchylema" layer was composed of vesicles belonging to the endoplasmic reticulum. The vacuolar layer was poorly preserved and consisted of double-walled vesicles. Fat appeared as stellate osmiophilic droplets. These observations were compared with previous observations under the optical microscope and their meaning for cell physiology was discussed.     

EFFECTS OF PHOSPHOTUNGSTATE NEGATIVE STAINING ON THE MORPHOLOGY OF THE ISOLATED GOLGI APPARATUS

Isolated Golgi complexes can be recognized in phosphotungstate (PTA) negative stain as stacks of membranous plates surrounded by a complex anastomosing network of tubules and vesicles. The extent of this tubular network is, however, much greater than can be observed in thin sections of whole cells. To determine which of the steps leading to the final negatively stained image may produce the observed changes, we have monitored each of the steps by other electron microscope and biochemical methods. The first damage to the membranes seems to occur during the initial isolation procedure as judged by the appearance of smooth patches on the freeze-fractured membrane faces that are normally covered with particles. Subsequent suspension of the Golgi fraction in water, to dilute the sucrose for negative staining, leads to the disappearnce of the stacking, to some tubulation and some vesiculation of the membranes as judged by thin section and freeze-cleave microscopy. The latter technique also reveals an increase in smooth-cleaving membrane faces. Application of the negative stain to the water-washed Golgi fraction, finally, produces extensive tubular arrays and a simultaneous decrease in the remaining large membranous vesicles. The freeze-cleaved tubular membranes appear essentially smooth except for small patches of aggregated particles. Parallel gel electrophoresis studies of the membranes and of the water and negative stain wash extracts indicate that protein extraction is involved in these morphological changes. PTA seems to be a particularly effective solvent for certain membrane proteins that are not removed by the water wash. These observations suggest that removal of membrane proteins alters structural restraints on the membrane lipids so that they behave semiautonomously like myelinics and form new artificial structures. This does not eliminate the possibility, however, that some tubules also exist in the Golgi apparatus in vivo.     

Structural changes of chloroplasts and galactolipid contents in Chlorella cells during clinorotation

Research of chloroplast ultrastructure in Chlorella cells grown during long-term period under clinorotation has been carried out. Different changes of the chloroplast structure, concerning both the amount of starch grains and stroma electron density as well as membrane system have been revealed. Occurrence of more significant bends of the thylakoids compared to the control and more loose arrangement of the thylakoids in a bunch were noted. The most significant changes were observed in a membrane system, in particular, appearance of the non-uniform expansions of the inter- and intrathylakoid spaces in Chlorella chloroplasts. Taking into account the role of galactolipids as important components of the photosynthetic membranes, the content of general galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglicerol in Chlorella cells was determined. It was assumed that statistically significant increase of the galactolipid content, especially of MDG, can probably be one of the reasons of membrane system reorganizations in Chlorella cells under altered gravity.     

Water movement from intracristal spaces in isolated liver mitochondria

When analyzing mitochondria isolated in a sucrose medium that had been embedded for thin sectioning according to one low denaturation embedding technique, large intracristal spaces were present in close to 90% of the mitochondria. The two crista membranes were closely apposed in only 40% of all cristae. When the mitochondria were transferred to an incubation medium, the percentage of mitochondria with intracristal spaces was reduced to 40%. About 90% of all cristae were lacking any space separating the two crista membranes. The presence of inorganic phosphate in the medium was required for the closing of the intracristal spaces. The percentage of cristae lacking an intracristal space remained the same after addition of substrate for respiration (state 4) and of ADP (state 3). Inhibition or uncoupling of respiration led to an increase in the percentage of intracristal spaces, showing that oxidative phosphorylation is required to maintain the crista membranes closely apposed. The appearance and disappearance of the intracristal spaces was an indication of water movements across the crista membranes. The mean volume of the mitochondria increased 33% when they were transferred from the sucrose medium to the incubation medium, showing that the removal of water from the cristae was not caused by a passive osmotic effect. Addition of substrate made the volume decrease by 28%. After further addition of ADP, the volume decreased another 23%. No change in volume was associated with inhibition or uncoupling of respiration. The observations revealed that water can move into or out of the cristae independently of water movement out from the entire mitochondrion. Therefore, the water moving out from or into the cristae is translocated across the cristae membrane. The observations are interpreted to reveal the presence of a mechanism that actively prevents water from accumulating in the crista membrane. This mechanism allows for a low water activity to be maintained within the membrane. The variations in the frequency of intracristal spaces occurred without any simultaneous changes in the width of the space appearing between the two surface membranes after isolation of the mitochondria. The observations, therefore, do not agree with the concept that there is an outer compartment that communicates freely with intracristal spaces.     

Histological and cytological studies on the ovary of Nauphoeta cinerea (Blattaria,Oxyhaloidea) during the first reproductive cycle

Histological studies were performed on the ovary of the ovoviviparous cockroach Nauphoeta cinerea during the first reproductive cycle by means of optical microscopy and histoautoradiography, and electron microscopy. The oöcyte chamber is composed of follicle cells, the oöcyte and a layer of symbiotic bacteria at the level of the microvillous border of the oöcyte. The first reproductive cycle begins with a short inactive period preceding the appearance of vitellogenin. During this period, the follicular epithelium achieves its development by a mitotic flare. From the 3rd day on, vitellogenin is synthesized by the fat body and large intercellular spaces appear between the follicular cells, in conjunction with a rapid growth of the oöcyte, which takes up selectively the vitellogenin by means of pinocytotic vesicles. These coalesce to give the yolk globules. Along with these phenomena, the proteosynthetic apparatus and its activity in the follicular cells increase slowly. After about the 12th day, the intercellular spaces disappear and the follicular epithelium which has now a very well developed proteosynthetic apparatus, begins to synthesize and lay down the egg membranes. After ovulation, the empty oöcyte chamber collapses and the follicular epithelium shows rapid degeneration processes (large cytolysosomes) that destroy the chamber completely during the gestation period. At the beginning of the 2nd cycle, there only remain a cell or two of the previous follicular epithelium and a very large annular piece of basement membrane.     

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali- stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.     

Electron microscopy of isolated cell walls of Bacillus subtilis var. niger

Isolated cell walls of Bacillus subtilis have a striated appearance in the electron microscope. The structure persists when teichoic acids are removed. It is inferred that the structure bears on the arrangement of the peptidoglycan chains.     

Penetration of Host Cell Membranes by Adenovirus 2

Highly purified human adenovirus type 2 directly penetrated the plasma membranes of KB cells. The process of membrane penetration resulted in the appearance of large numbers of adenovirions free in the cytoplasm of the infected cells. The virions underwent a morphological change as they penetrated the cell surface. Penetration of the plasma membranes and the accompanying alteration in virion morphology was dependent on a function associated with the intact cells, because neither event occurred when purified virions were added to isolated cell membranes. Inactivation of the adenovirions with heat or antibodies before inoculation of the cells reduced the infectivity of the virus population and prevented the appearance of virions free in the cytoplasm. The inactivation of the virions did not significantly reduce the number of virus particles which were found in cell vacuoles and pinocytotic vesicles.     

Innervation of the rabbit cornea. A histochemical and electron-microscopic study

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluorescence methods revealed adrenergic nerves in the stroma; a few fibres were also observed between the basal epithelial cells near the limbus. Acetylcholinesterase- (AChE-) positive nerves were found both in the stroma and in the epithelium, whereas nonspecific cholinesterase (NsChE) activity appeared only in the stromal nerves. Under the electron microscope, both AChE and NsChE activities were observed to be located in the axon membranes. A weak NsChE reaction also appeared in the Schwann cells. When the specimens fixed with KMnO4 were examined under the electron microscope, most nerve fibres did not contain any special axoplasmic structures, although several axons contained mitochondria. Moreover, two vesicle-containing axon types were found in the stromal nerves; axons with small granular vesicles and axons containing small agranular vesicles. In the epithelium, two types of fibres were observed; one type containing only mitochondria while the other showed both agranular vesicles and mitochondria.     

FINE STRUCTURE AND MORPHOGENIC MOVEMENTS IN THE GASTRULA OF THE TREEFROG, HYLA REGILLA

The blastoporal groove of the early gastrula of the treefrog, Hyla regilla, was examined with the electron microscope. The innermost extension of the groove is lined with invaginating flask- and wedge-shaped cells of entoderm and mesoderm. The distal surfaces of these cells bear microvilli which are underlain with an electron-opaque layer composed of fine granular material and fibrils. The dense layer and masses of vesicles proximal to it fill the necks of the cells. In flask cells bordering the forming archenteron the vesicles are replaced by large vacuoles surrounded by layers of membranes. The cells lining the groove are tightly joined at their distal ends in the region of the dense layer. Proximally, the cell bodies are separated by wide intercellular spaces. The cell body, which is migrating toward the interior of the gastrula, contains the nucleus plus other organalles and inclusions common to amphibian gastrular cells. A dense layer of granular material, vesicles, and membranes lies beneath the surface of the cell body and extends into pseudopodium-like processes and surface undulations which cross the intercellular spaces. A special mesodermal cell observed in the dorsal lining of the groove is smaller and denser than the surrounding presumptive chordamesodermal cells. A long finger of cytoplasm, filled with a dense layer, vesicles and membranes, extends from its distal surface along the edge of the groove, ending in a tight interlocking with another mesodermal cell. Some correlations between fine structure and the mechanics of gastrulation are discussed, and a theory of invagination is proposed, based on contraction and expansion of the dense layer and the tight junctions at distal cell surfaces.     

REJUVENATION OF MELOSIRA GRANULATA (BACILLARIOPHYCEAE) RESTING CELLS FROM THE ANOXIC SEDIMENTS OF DOUGLAS LAKE,MICHIGAN, II. ELECTRON MICROSCOPY 1

Detailed cytological changes that accompany the rejuvenation of resting cells of Melosira granulata were studied with the electron microscope. Dormant and viable cells that we previously classified as the condensed state generally contain definable chloroplasts, mitochondria, a nucleus and other cytoplasmic remnants. However, there appears to be a continuous cytoplasmic degradation spectrum and some cells which appear intensely colored with the light microscope have discontinuous chloroplast membranes and few other cytoplasmic remnants. Rejuvenation of viable dormant cells is initially accompanied by the accumulation of both lipids and polyphosphates. In the earliest stages of expansion, these storage products are dispersed throughout the cell. In later stages of expansion, the lipids appear to be coalesced into larger droplets which are easily identified at the light microscope level. The fully expanded stage is characterized by the normal complement of organelles and their arrangement at the periphery of the cells and central cytoplasmic bridge. These cells appear both anabolically and catabolically active as evidenced by the abundance of endoplasmic reticulum, ribosomes and secretory and lytic vesicles. Prior to cell division, both lipids and polyphosphates a re reduced or absent in the cells. The ultrastructural features of the dormant, condensed state in resting cells of M, granulata are similar to those described for hypnospores. A rejuvenation sequence that produces cytological features common to resting state formation could provide a population of cells which could easily revert should environmental conditions become adverse.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary The fine structure and pigmentation of an apochlorotic diatom isolated from decaying Macrocystis pyrifera is described. The morphology of acid cleaned shells suggests that the isolate is Nitzschia alba Lewin and Lewin. Light microscope observations indicated a centrally located nucleus and numerous highly refractile bodies which stained differentially with Nile blue and Sudan black B. The stained globules could be correlated in thin-sectioned profiles with either electron dense or lucent areas depending on the fixation technique. In the electron microscope the nucleus, Golgi complex, and mitochondria were similar in appearance to those described for other diatoms. Proplastid-like organelles, delimited by a double membrane, and containing small vesicles were also observed. Neither carotenoids nor chlorophylls could be detected by spectroscopic or spectrofluorometric analysis in vivo or in organic solvent extracts. Deposition of new walls was initiated by formation of silicon deposition-vesicles in the central region of dividing cells. The acentric raphes were deposited last. The genesis and interrelationship of the old plasmalemma, silicalemma, and newly formed plasmalemma are discussed.     

The purification and characterization of Dictyostelium discoideum plasma membranes

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5′-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome $\text{c}$ reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.     

Fine structure studies on phragmoplast and cell plate formation

Formation and development of phragmoplast and cell plate were studied in endosperm of Haemanthus katherinae Bak. The same cells were studied with the light and electron microscope. Several cells were studied with time-lapse microcinematography before fixation. This permitted comparison of structures during their development on both the light and electron microscope level. Movements of fibrillar components of the phragmoplast and spindle were analyzed and their transport properties were correlated with formation of the cell plate. Change of arrangement of microtubules, transport of vesicles which form the cell plate, and formation of vesicles and microtubules has also been discussed.     

Ultrastructure of intracytoplasmic membranes of Methanomonas margaritae cells grown under different conditions

The development of intracytoplasmic membranes of Methanomonas margaritae cells grown under different culture conditions was studied. Growth on methane was strongly accelerated by the addition of copper ions. Acceleration by copper, however, was not observed in the case of growth on methanol. Cells grown on methane with copper possessed intracytoplasmic membranes along the cell periphery. When the organism was grown in a medium lacking copper, intracytoplasmic membranes appeared as large vesicles surrounded by a unit membrane at the periphery of the cell. The vesicles originated from paired membranes due to the absence of copper in the medium. Cells grown on methanol with or without copper possessed a number of vesicles of different sizes arranged in a chain along the cell periphery. The possible relationship between membrane arrangement and methane oxidation is discussed.     

The purification and characterization of Dictyostelium discoideum plasma membranes.

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5'-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome c reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.