Fine Structure of Selected Marine Pseudomonads and Achromobacters

The fine structure of more than 20 marine pseudomonads and more than 15 achromobacters was examined. Under the conditions extant, clear differences between members of these two groups were seen. The pseudomonads displayed the characteristic gram-negative morphology: the cell wall was irregularly undulant and the cytoplasmic membrane more nearly planar, ribonucleoprotein (RNP) particles were loosely packed throughout the periphery of the cytoplasm, and the deoxyribonucleic acid (DNA) was axially disposed. Cell division appeared to be by constriction. Some strains characteristically produced evaginations or blebs of the cell wall. Occasionally, thick, densely stained ring structures were seen which are possibly analogous to mesosomes. In contrast, the achromobacters demonstrated a regularly undulant outer cell wall element and a planar inner wall. The cytoplasmic membrane was thin and not readily observed. RNP particles were densely stained and tightly packed in the cytoplasm; the DNA was most often lobate in disposition. Cellular division was mediated by the formation of a septum which consisted of the cytoplasmic membrane and the inner element of the cell wall. Mesosomes were observed in all of the strains examined. Dense inclusion bodies were also seen in many strains.     

Effects of Treatment with Sodium Dodecyl Sulfate on the Ultrastructure of Escherichia coli

An electron microscopy study has been made of the effects of dissolution of the plasma membrane of Escherichia coli with sodium dodecyl sulfate (SDS) on the organization of the nucleoplasm and the cytoplasm. The alterations observed in time course experiments were related to absorbance changes and to release of macromolecules from the cells. As the cells became plasmolyzed, under the conditions used, the first visible effect of SDS was a collapse of the plasmolysis spaces. This was accompanied by a displacement of the nuclear material which then appeared in broad contact with the redeployed plasma membrane. This initial displacement of nuclear material to the cell border may indicate an association between the nucleoplasm and the plasma membrane. Upon further dissolution of the plasma membrane, the nuclear material receded from the cell margin and contracted into an axial filament. Meanwhile, the cytoplasm dissociated into an amorphous, Pronase-sensitive component and an electron-opaque, granular one sensitive to ribonuclease. The latter represented one continuous area of ribosomal structures surrounding the nucleoplasm, an organization which did not occur when the cells were inhibited with rifamycin before SDS treatment. During prolonged SDS interaction, approximately 65% of the cellular protein, 25% of the ribonucleic acid and 40% of the deoxyribonucleic acid were released from the cells concomitant with the disappearance of the amorphous cytoplasmic part, expansion of the ribosomal aggregate, and rearrangement of the nuclear material at the cell periphery. The observations support the contention that all ribosomal structures bear a direct relationship with the nucleoplasm.     

Solitary bodies (S-bodies) in the parasitic red algaHarveyella mirabilis (Choreocolaceae,Cryptonemiales)

Summary Unusual spherical cytoplasmic inclusions identical to S-bodies described previously in three angiosperms were found in all cells of the parasitic red algaHarveyella mirabilis collected from several locations in the northeast Pacific. The inclusions are ca. 60–80 nm and consist of an outer double membrane bounding a granular mantle and a DNase sensitive central core. S-bodies are dispersed throughout the cytoplasm and are associated occasionally with nuclei, plastids, mitochondria, ER, and vacuoles. They have not been observed in any other alga except in host algal cells, connected to parasite cells by cellular pit connections. The possible function of these inclusions is considered with respect to the parasitic nature ofHarveyella mirabilis.     

Ultrastructural Study of Pierce's Disease Bacterium in Grape Xylem Tissue

The rod-shaped rickettsia-like bacteria of Pierce's disease measure about 0.25 to 0.50 μm in diameter and 1.0 to 4.0 μm long. The bacteria have a cell wall consisting of a trilaminar outer membrane and two intermediate low-density layers separated by a dense intermediate layer. A trilaminar cytoplasmic membrane is also present, resulting in a total wall complex thickness of 25 to 40 nm. A periodic infolding of the outer membrane and intermediate layers of the wall give the wall surface a ridged apperance. The ridges appear to go around the long axis of the cell, possibly in the form of spirals. Ribosomes and nuclear regions with easily visible deoxyribonucleic acid strands and clumps are distributed throughout the cytoplasm. Binary fission, during which the cell wall and cytoplasmic membrane folded inward to partition the cell, was observed. In the xylem of infected grapes, the bacteria are either distributed evenly throughout the lumen of the xylem vessel or appressed along the inner surface of the vessel walls in an electron-lucent matrix.     

Regeneration of sialic acid on the surface of Chinese hamster cells in culture. I. General characteristics of the replacement process

Electropotential differences between the cell interior and the external medium have been studied with intracellular microelectrodes in ovarian oocytes, ovulated unfertilized eggs and fertilized eggs of R. pipiens. In ovarian oocytes the cytoplasm was 50 to 80 mV negative, relative to isotonic Ringer's solution. In contrast, electrode penetration of the oocyte nucleus in situ indicated that the nucleoplasm was about 25 mV positive, relative to the cytoplasm. After ovulation, the cortical cytoplasm became 20 to 50 mV positive with regard to an external solution of 0.1 strength Ringer's solution (ca. pond water). Penetration of the cytoplasm at levels from 0.3 to 0.6 mm below the egg surface revealed an inner zone with a potential which was about 15 mV negative, relative to the cortical cytoplasm. A slow hyperpolarization of the cortical membrane occurred at activation, with the potential returning to that of the ovulated unfertilized egg within ten minutes. After fertilization, the egg cytoplasm remained positive until the first cleavage. As division proceeded, the cytoplasm slowly depolarized and became 50 to 60 mV negative, relative to 0.1 strength Ringer's solution.     

Nuclear and cytoplasmic changes during early stages of cell differentiation in roots of the water-fern,Azolla pinnata

Summary Selected nuclear and cytoplasmic changes associated with early differentiation of four cell-types—dermatogen, inner and outer cortex, and endodermis—have been analysed using montages of electron micrographs of median longitudinal sections of young roots ofAzolla pinnata. The area fraction of nucleoplasm occupied by chromocentres (CAF) is smaller in the apical cell than in the nuclei of its most recently formed daughter cells. The CAF also differs between the four cell-types: dermatogen nuclei have a lesser mean CAF and smaller chromocentres than nuclei of the endodermis; cortical cell nuclei have intermediate values. These differences may reflect changes in nuclear activity during cell differentiation. The area fraction occupied by the vacuome (VAF) differs between the apical cell and its daughters: the apical cell seems to retain most of the vacuome at division, while the daughter cells receive less vacuolate cytoplasm. Of the four cell-types analysed, the cortical cells develop a large VAF the quickest; the dermatogen is slower to become vacuolate. Cells in the dermatogen and outer cortex derive from common mother cells, as do cells in the endodermis and inner cortex, and even the most recently-formed cells in the files of inner and outer cortex are more vacuolated than their sister cells in the other two celltypes. The onset of vacuolation may be triggered by an inductive influence emanating from older vacuolated cells in the same file. The rate of vacuolation in each of the cell-types examined may also be negatively correlated to the intensity of synthesis of protein used to construct cytoplasmic materials.     

Localization of the membrane-bound hydrogenase in Alcaligenes eutrophus by electron microscopic immunocytochemistry

Abstract The membrane-bound hydrogenase was localized in cells of Alcaligenes eutrophus by electron microscopic immunocytochemistry. Post-embedding labeling performed on ultrathin sections revealed that the enzyme was located predominantly (80%) at the cell periphery in autotrophically and heterotrophically grown bacteria harvested from the exponential phase of growth. In the stationary growth phase, however, only 50% of the enzyme was found at the cell periphery; the remaining 50% was distributed over the cytoplasm. The relative amount of electron microscopic label per cell as seen by application of the protein A—gold technique was higher in cells grown autotrophically as compared to cells grown heterotrophically on fructose. Derepression of the enzyme was followed electron microscopically in a substrate-shift experiment (growth on fructose, followed by a shift to glycerol). Major amounts of the enzyme appeared to undergo a reattachment to the cytoplasmic membrane under these conditions, starting with a reduced location of the enzyme in the cytoplasm and an accumulation in cell areas close to the cytoplasmic membrane. These findings indicate that the 'membrane-bound' hydrogenase (i.e., that material enriched as membrane-bound enzyme according to the appropriate activity test) is not, in fact, membrane bound or membrane integrated but membrane associated. It may or may not interact with the cytoplasmic face of the cytoplasmic membrane, depending on the growth phase and conditions.     

Fine Structure of Encephalitozoon cuniculi from Rabbits,Mice and Hamsters*

SYNOPSIS. Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is ∼ 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.     

ELECTRON MICROSCOPY OF THE VOLVOCACEAE AND ASTREPHOMENACEAE

Lang, Norma J. (U. Texas, Austin.) Electron microscopy of the Volvocaceae and Astrephomenaceae. Amer. Jour. Bot. 50(3): 280-300. Illus. 1963.—Clonal cultures of Gonium sociale, G. pectorale, Pandorina morum, Eudorina elegans, Eudorina sp., Volvulina steinii, V. pringsheimii, Platydorina caudata, Pleodorina illinoisensis, P. californica, Volvox aureus, V. tertius, V. globator, V. barberi, and Astrephomene gubernaculifera representing the Volvocaceae and Astrephomenaceae in the Volvocales were examined with the electron microscope and their ultrastructure compared. The ultrastructure of the various organelles is basically similar in the species studied and no increase in cellular complexity is found to accompany the evolutionary trends evidenced in the Volvocaceae. The ultrastructure of a colonial cell is basically that of Chlamydotnonas. A cytoplasmic membrane having a unit membrane structure encompasses a cell and is continuous with the double-membraned flagellar sheaths. The flagella contain the typical 9 + 2 fibril arrangement with the 2 axial fibrils terminating in a cylinder at the flagellar base and the 9 peripheral pairs continuing into the cytoplasm as a basal body. The organelles comprising the cytoplasm are: mitochondria with plate-like cristae; dictyosomes composed of stacks of agranular cisternae; small, rough or smooth-surfaced vesicles; an endoplasmic reticulum of granule-bearing and agranular tubules, lamellae and broad cisternae; vacuoles which are either contractile, contain fine granular and fibrillar material, or have dense contents probably representing polyphosphate; lipid bodies; and dense granules 100–150 A which have been called ribosomes. The finely granular nucleoplasm is surrounded by a porous, double-membraned nuclear envelope and contains a centric nucleolus composed of dense, spherical granules. The outer membrane of the nuclear envelope bears granules and may have granular extensions into the perinuclear cytoplasm. Each extension appears to encompass one or several dictyosomes and has been termed an “amplexus.” The amplexi are agranular on the surface contiguous to a dictyosome. A double-membraned chloroplast envelope is continuous around the single, cup-shaped chloroplast. The basic chloroplast units are discs closed at each end, occurring in stacks of varying number parallel to the envelope. The presumed proteinaceous matrix of the basal pyrenoid is penetrated by elongated, tubular elements which connect with the lamellar discs. Multiple rows of granules, associated with individual discs, form the anterior stigma within the chloroplast envelope. The colonial matrix is not a structureless, mucilaginous material uniting the cells in colonies, but it has rather a highly complex structure especially around the periphery of the colony and the flagellar channels. The apparent substitution of a fibrillar layer of the colonial matrix for the discrete compact cell wall, such as is found in Chlamydomonas, implies a greater degree of complexity in the evolution of these colonial genera than is generally assumed.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE : III. Continuity between Plasma Membrane and Nuclear Envelope in Androgonial Cells of Blasia

An ultrastructural study of late-stage androgonial cells of Blasia pusilla, a thallose liverwort, showed the nearly spherical nuclei often lying close or appressed to the cell walls. In some cells the two membranes comprising the nuclear envelope separated, the inner membrane continuing intact as a limiting boundary of the nucleus and the membrane on the outer, cytoplasmic side recurving away from the nucleus to continue without evident interruption around the periphery of the cell as the plasma membrane. It is believed that Blasia offers the first completely convincing demonstration of the heretofore problematic continuity of cytoplasmic membranes. A possible sequence of events leading to this unusual relationship between nucleus and cytoplasm is suggested. The sequence includes blebbing of the outer membrane of the nuclear envelope and subsequent membrane proliferation, apparent isolation of cytoplasmic ground substance, fusion of internal membrane with the ectoplast, and migration that finally brings the nucleus into flat contact with the wall. While this manifestation of membrane continuity may be anomalous, it is not presently considered the result of cell injury.     

Continuous periplasm in a filamentous, heterocyst-forming cyanobacterium

The cyanobacteria bear a Gram-negative type of cell wall that includes a peptidoglycan layer and an outer membrane outside of the cytoplasmic membrane. In filamentous cyanobacteria, the outer membrane appears to be continuous along the filament of cells. In the heterocyst-forming cyanobacteria, two cell types contribute specialized functions for growth: vegetative cells provide reduced carbon to heterocysts, which provide N2-derived fixed nitrogen to vegetative cells. The promoter of the patS gene, which is active specifically in developing proheterocysts and heterocysts of Anabaena sp. PCC 7120, was used to direct the expression of altered versions of the gfp gene. An engineered green fluorescent protein (GFP) that was exported to the periplasm of the proheterocysts through the twin-arginine translocation system was observed also in the periphery of neighbouring vegetative cells. However, if the GFP was anchored to the cytoplasmic membrane, it was observed in the periphery of the producing proheterocysts or heterocysts but not in adjacent vegetative cells. These results show that there is no cytoplasmic membrane continuity between heterocysts and vegetative cells and that the GFP protein can move along the filament in the periplasm, which is functionally continuous and so provides a conduit that can be used for chemical communication between cells.     

Immunoelectron microscopy of myosin associated with the generative cells in pollen tubes ofNicotiana tabacum L.

In this study, polyclonal anti-myosin antibodies were used for immunogold labeling of ultrathin sections of pollen tubes ofNicotiana tabacum L. to unravel the ultrastructural localization of myosin associated with the generative cells. Clusters of immunogold particles were consistently found in association with the area of the outer surface of the vegetative cell plasma membrane present around the generative cell. Compared to the generative cell cytoplasm, the nucleoplasm showed higher numbers of gold particles. This is the first direct evidence demonstrating the presence of myosin in the nuclei of the generative cell of flowering plants. The possible implications of these findings are discussed in relation to movement of the generative cell in the pollen tube cytoplasm.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Freeze-fracture features of epithelioid cells,multinucleated giant cells,and phagocytic macrophages

The freeze-fracture morphology of epithelioid cells, multinucleated giant cells (Langhans' type), and phagocytic macrophages was investigated. The intensely folded and interdigitating surface membranes of epithelioid cells and multinucleated giant cells displayed no specialized areas of cell contact. The size of the intramembranous particles (IMP) and the fact that the area density of IMPs was higher in the cytoplasmic (P) faces than in the external (E) faces of the cell membranes agreed with observations in other eukaryotic cells. The area densities of the IMPs suggest lower transport rates of molecules across the cell membranes of granuloma cells than of certain epithelial cells. Small pits were detected in the surface membranes of the granuloma cells but an extrusion of granules was not observed. The cytoplasmic granules displayed very different sizes and shapes ranging from spherical to rod-shaped. The latter type of granules (probably primary lysosomes) dominated in multinucleated giant cells. The granule membranes were studded with IMPs whose area densities increased with the granule size. Multilamellar bodies with smooth (lipid) fracture faces were found only in phagocytic macrophages. The nuclear pores of the granuloma cells were distributed over the entire surfaces of the nuclei and displayed moderate clustering. The values of the area densities of the nuclear pores were in keeping with the values observed in mammalian and human epithelial or mesenchymal cells, indicating similar exchange rates of molecules between the nucleoplasm and the cytoplasm in these different cell types. In a single phagocytic macrophage the E-face of the inner membrane of the nuclear envelope displayed a network of fine filaments whose nature is at present unknown.     

Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.     

The ultrastructure of smooth cardiac muscle in the clam,Venus mercenaria

A fine structural study of the ventricular muscle of Venus mercenaria has revealed that it is an invertebrate smooth muscle. In the relaxed state induced by acetylcholine, both thick (350 Å) and thin (80 Å) myofilaments are observed. These are loosely distributed in bundles in the periphery of the mononucleated myocytes. The central core of the cell contains an ovoid nucleus, α-glycogen rosettes, round mitochondria and numerous smooth surfaced vesicles of the endoplasmic reticulum. After exposure to serotonin, all myofilaments are compacted in the peripheral cytoplasm and become oriented parallel to the longitudinal cellular axis. This produces contraction bands visible in phase contrast microscopy. Because these myofilaments attach to the cell membrane at sites of attachment plaques, contraction of the cell results in the serial evagination or blebbing of the cell surface. The above features are clearly demonstrable in this invertebrate smooth muscle and strongly suggest a sliding filament model as the contractile mechanism in this tissue. Moreover, the cell surface is thought to play an active and major role in that process.     

Purification of cytoplasmic membranes and outer membranes from Proteus mirabilis

Summary A crude cell envelope suspension has been prepared from Proteus mirabilis after osmotic shock of penicillin-induced spheroplasts. Employing discontinuous sucrose gradients this cell envelope suspension can be fractionated into four fractions. Besides a pellet of remaining spheroplasts and an intermediate fraction with mixed composition a highly purified cytoplasmic membrane fraction and an outer membrane fraction have been obtained. The cytoplasmic membrane fraction is not contaminated with mucopeptide or outer membrane material. It has a buoyant density of 1.13 g/ml and a protein content of 38%. The specific activities of formate dehydrogenase and nitrate reductase and the content of cytochrome b₁ have increased sixfold in comparison with the crude cell envelope suspension. The outer membrane fraction contains only few contaminations with cytoplasmic membrane components and with mucopeptide.The gradient fractions have been characterized by electron microscopy and by polyacrylamide gel electrophoresis.     

Studies on the Fine Structure of Microorganisms : V. Morphogenesis of Nuclear and Membrane Structures during Ascospore Formation in Yeast

The fine structure of cells of Saccharomyces cerevisiae engaged in the formation of ascospores was studied in electron micrographs of ultrathin sections. Although the mode of the first reduction division could not be clearly determined, the second nuclear division appeared to proceed in a manner similar to that observed previously during vegetative division. That is, division by constriction of the existing nucleus occurs without dissolution of the nuclear membrane and without involvement of discrete chromosomes. Variously shaped areas of low electron density were discerned within the nucleoplasm; these had not been previously seen in the vegetative nucleus. The significance of this nuclear differentiation and its possible similarity to nuclear structures reported in bacteria and an imperfect fungus are discussed. The cytoplasmic membrane appears first in the developing ascospore. The formation of an outer coat and an inner coat then follows. The cytoplasmic vacuole was observed not to be incorporated into the spore. An unusual intracytoplasmic membrane was observed in the spore and appeared to be at least temporarily continuous with the nuclear membrane.     

Visualization of actin in situ by rhodamine-conjugated phalloin in the fungus Uromyces phaseoli

Rhodamine-conjugated phalloin, a derivative of phalloidin which binds to F-actin, was associated with three types of structures in uredospore germlings of the bean rust fungus, Uromyces phaseoli. The structures were filaments, peripheral plaques, and intranuclear inclusions. The filaments, located throughout the germ tube but especially in the more basipetal regions, were observed as either barely perceivable, fine elements or as easily detectable, coarser structures. The plaques, which we suggest to be equivalent to filasomes, occurred near the periphery of the cell's cytoplasm. They were most numerous in the hyphal tip regions. Nuclear inclusions occurred within the nucleoplasm subjacent to the spindle pole body. Treatments with KI and phalloidin substantiated that the fluorescently labelled sites were F-actin. Treatment of the germlings with cytochalasin E caused the intranuclear inclusions to become extended often with branched, fine filaments. Similar treatments led to a disappearance of cytoplasmic filaments, but had no perceivable effect on the peripheral plaques.