AN ULTRASTRUCTURAL STUDY OF CELL DIVISION IN THE CAMBIUM

The fine structure of dividing cambial cells of Ulmus americana and Tilia americana has been studied in material fixed in glutaraldehyde followed by osmium tetroxide. The cambia examined consisted of 7–9 rows of unexpanded fusiform cells, all of which had similar ultrastructural components. The fine structure and sequence of events of mitosis and cytokinesis in the dividing cambial cells apparently are similar to those of dividing cells in root tips and leaves. Of special interest was the observation that during cytokinesis, a broad cytoplasmic plate or phragmosome precedes the developing phragmoplast and cell plate through the dividing cambial cell. Smooth and coated vesicles derived from dictyosomes are associated with cell plate formation in these cells, smooth vesicles primarily with earlier stages of plate formation, and coated vesicles in later stages.     

ONTOGENY AND CYTOCHEMISTRY OF ALKALOIDAL VESICLES IN LATICIFERS OF PAPAVER SOMNIFERUM L. (PAPAVERACEAE)

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide-osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron-dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle.     

The effects of 6-hydroxydopamine on the appearance of granulated vesicles in glomus cells of the rat carotid body

The glomus cells of the rat carotid body reveal an intense fluorescence after exposure to paraformaldehyde vapor and contain catecholamines. After initial fixation in glutaraldehyde, many granulated vesicles are seen in the glomus cells. After initial fixation in osmium tetroxide, most of the vesicles are depleted of their dense interiors and granulated vesicles occur infrequently. Administration of 6-hydroxydopamine followed by initial fixation in osmium tetroxide leads to the reappearance of dense interiors in virtually all vesicles. 6-Hydroxydopamine apparently is taken up by the membrane pump of the glomus cell and is incorporated into the amine storage granules, thereby displacing the endogenous monoamines. Osmium tetroxide does not dissolve the 6-hydroxydopamine from the vesicles, as it apparently does for the normal vesicular contents. The 6-hydroxydopamine does not fluoresce, hence 6-hydroxydopamine administration results in a decreased intensity of formaldehyde induced fluorescence in the glomus cells. Administration of reserpine after 6-hydroxydopamine treatment (and subsequent initial fixation in osmium tetroxide) depletes the previously restored dense material from the vesicles of the glomus cells. 6-Hydroxydopamine acts like a monoamine in that it is taken up by the glomus cell, incorporated into the vesicles, and can be depleted from the vesicles by reserpine.     

Alkaline Bismuth Stain as A Tracer for Golgi Vesicles of Plant Cells

An alkaline solution of bismuth subnitrate reacts well with carbohydrate-rich components of Golgi bodies in sections prepared from plant leaves fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. The metal deposits formed are so fine that the stain is appropriate to ultrastructural observation at high magnification. The Golgi vesicles show polarity with respect to the localization of the reactive deposits. Golgi vesicles that had migrated farther from the Golgi cisternae showed greater reactive deposits and higher membrane contrast than those close to the Golgi cisternae. These results indicate that the alkaline bismuth stain is an excellent tracer for Golgi bodies of plant cells.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

OBSERVATIONS ON THE FINE STRUCTURE OF PHARYNGEAL MUSCLE IN THE PLANARIAN DUGESIA TIGRINA

Pharyngeal muscle of the planarian Dugesia tigrina was studied by electron microscopy after osmium tetroxide fixation. The muscle cell was observed to contain one myofibril or bundle of myofilaments parallel to its longitudinal axis. The myofilaments were of two types, different in size and distribution. No Z lines or myofilament organization into cross or helical striations were seen. Dense bodies were seen as projections from an invagination of the plasma membrane and as dense lines parallel to the myofilaments. The muscle cells are surrounded by a plasma membrane which is structurally associated with dense body projections, with vesicles and cisternae of sarcoplasmic reticulum, and with synaptic nerve endings. The cell has sarcoplasmic projections perpendicular to its long axis; these projections are seen to contain the nucleus or mitochondria and granules. Mitochondria and granules are also seen in a sarcoplasm rim around the fibril. The dense bodies may serve as attachment for thin myofilaments and function in transmission of stimuli from plasma membrane to the interior of the fibril.     

ELECTRON MICROSCOPE OBSERVATIONS ON TINGIBLE BODY MACROPHAGES IN MOUSE SPLEEN

The structure of the ciliary epithelium of the adult albino rabbit has been studied by electron microscopy. Material was fixed in osmium tetroxide and embedded in epoxy resins. Two hitherto unappreciated features of the non-pigmented epithelial layer are described. First, the "infolded plasma membranes" described by previous workers are shown by serial sections to be projections or interdigitations from adjacent cells. Second, the "rows of vesicles" described by previous workers are shown by serial sections to be part of an unusual form of smooth-surfaced tubular endoplasmic reticulum. The tubules are highly convoluted and extensively interconnected. They are arranged in sheets, so that a cross-section through a sheet gives the appearance of a row of vesicles. The other structural features of the ciliary epithelium are also described. Previous workers have reported that Diamox, which inhibits the secretory activity of the epithelium, causes profound structural changes. An effort has been made to confirm these reports under carefully controlled experimental conditions. It was found that secretion could be inhibited by a maximally effective dose of Diamox without the occurrence of any detectable structural changes. The physiological significance of these findings is discussed.     

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.     

YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L

Yolk proteins are thought to enter certain eggs by a process akin to micropinocytosis but the detailed mechanism has not been previously depicted. In this study the formation of protein yolk was investigated in the mosquito Aedes aegypti L. Ovaries were fixed in phosphate-buffered osmium tetroxide, for electron microscopy, before and at intervals after a meal of blood. The deposition of protein yolk in the oocyte was correlated with a 15-fold increase in 140 mµ pit-like depressions on the oocyte surface. These pits form by invagination of the oocyte cell membrane. They have a 20 mµ bristle coat on their convex cytoplasmic side. They also show a layer of protein on their concave extracellular side which we propose accumulates by selective adsorption from the extraoocyte space. The pits, by pinching off from the cell membrane become bristle-coated vesicles which carry the adsorbed protein into the oocyte. These vesicles lose the coat and then fuse to form small crystalline yolk droplets, which subsequently coalesce to form the large proteid yolk bodies of the mature oocyte. Preliminary radioautographs, and certain morphological features of the fat body, ovary, and midgut, suggest that the midgut is the principal site of yolk protein synthesis in the mosquito.     

AN ELECTRON MICROSCOPIC CHARACTERIZATION OF CLASSES OF SYNAPTIC VESICLES BY MEANS OF CONTROLLED ALDEHYDE FIXATION

Examination of variables of aldehyde fixation that may affect the shape of agranular synaptic vesicles has revealed that even brief storage of aldehyde-perfused nervous tissue pieces in cacodylate buffer, prior to hardening in osmium tetroxide, has an unusually severe flattening effect on agranular vesicles of a particular type. These are the vesicles of peripheral cholinergic axon endings, and of certain central synaptic bulbs. Types of synaptic bulbs can now be further defined on the basis of shape of agranular synaptic vesicles under controlled conditions of aldehyde fixation. Previously described "S" bulbs in the spinal cord contain uniformly spheroid vesicles, which are wholly resistant to flattening. Previously described "F" bulbs contain somewhat smaller agranular vesicles that are flattened after aldehyde fixation, even when this is followed by prompt hardening in osmium tetroxide solution. A third type, previously characterized as having irregularly round agranular vesicles after the above treatment, contains only severely flattened vesicles when the osmium tetroxide hardening is preceded by even a brief wash with sodium cacodylate buffer containing sucrose. Moreover, the "third" type is characteristic of all cholinergic peripheral axon endings examined, as well as the large axosomatic ("L") synaptic bulbs of the spinal cord.     

THE FINE STRUCTURE OF DIFFERENTIATING XYLEM ELEMENTS

Differentiating xylem elements of Avena coleoptiles have been examined by light and electron microscopy. Fixation in 2 per cent phosphate-buffered osmium tetroxide and in 6 per cent glutaraldehyde, followed by 2 per cent osmium tetroxide, revealed details of the cell wall and cytoplasmic fine structure. The localized secondary wall thickening identified the xylem elements and indicated their state of differentiation. These differentiating xylem elements have dense cytoplasmic contents in which the dictyosomes and elements of rough endoplasmic reticulum are especially numerous. Vesicles are associated with the dictyosomes and are found throughout the cytoplasm. In many cases, these vesicles have electron-opaque contents. "Microtubules" are abundant in the peripheral cytoplasm and are always associated with the secondary wall thickenings. These microtubules are oriented in a direction parallel to the microfibrillar direction of the thickenings. Other tubules are frequently found between the cell wall and the plasma membrane. Our results support the view that the morphological association of the "microtubules" with developing cell wall thickenings may have a functional significance, especially with respect to the orientation of the microfibrils. Dictyosomes and endoplasmic reticulum may have a function in some way connected with the synthetic mechanism of cell wall deposition.     

Pigment containing lipid vesicles. I. Preparation and characterization of chlorophyll a-lecithin vesicles.

Vesicles obtained by sonication of chlorophyll a-lecithin mixtures dispersed in anaqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophyll a. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophyll a molecule per 55 lecithins can be incorporated into membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102 +/- 5 A and independent of the chloropyll a content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

THE FINE STRUCTURE OF THE GALL BLADDER EPITHELIUM OF THE MOUSE

Sections of mouse gall bladder epithelium fixed by perfusion with buffered osmium tetroxide have been studied in the electron microscope as an example of simple columnar epithelium. The free surface presents many microvilli, each presenting a dense tip, the capitulum, and displaying a radiating corona of delicate filaments, the antennulae microvillares. Very small pit-like depressions, representing caveolae intracellulares, are encountered along the cell membrane of the microvilli. The free cell surface between microvilli shows larger cave-like depressions, likewise representing caveolae intracellulares, containing a dense material. The lateral cell borders are extensively folded into pleats, which do not interdigitate extensively with corresponding folds of the adjacent cell membrane. The terminal bars are shown to consist of thickened densities of the cell membrane itself in the region of insertion of the lateral cell wall with the free cell surface. This thickening is associated with an accumulation of dense cytoplasmic material in the immediate vicinity. The terminal bar is thus largely a cytoplasmic and cell membrane structure, rather than being primarily intercellular in nature. The basal cell membrane is relatively straight except for a conical eminence near the center of the cell, projecting slightly into the underlying tunica propria. The basal cell membrane itself is overlain by a delicate limiting membrane, which does not follow the lateral contours of the cell. Unmyelinated intercellular nerve terminals with synaptic vesicles have been encountered between the lateral walls of epithelial cells. A division of the gall bladder epithelial cell into five zones according to Ferner has been found to be convenient for this study. The following cytoplasmic components have been noted, and their distribution and appearance described: dense absorption granules, mitochondria, Golgi or agranular membranes, endoplasmic reticulum or ergastoplasm, ring figures, and irregular dense bodies, perhaps lipoid in nature. The nucleus of these cells is also described.     

ELECTRON MICROSCOPE STUDIES ON THE ORIGIN OF ANNULATE LAMELLAE IN OOCYTES OF NECTURUS

Developing oocytes, ranging from approximately 0.1 to 1.0 mm in diameter, in Necturus were studied with the electron microscope. The outer layer of the nuclear envelope is actively engaged in the formation of vesicular elements along most of its surface, especially in smaller oocytes. Groups of vesicles appear to be released into the ooplasm at about the same time, resulting in long chains of individual vesicles immediately adjacent to the nuclear membrane. This process is repeated so that chains of vesicles grouped in rather ordered ranks extend progressively into the surrounding cytoplasm. Eventually, the cytoplasm becomes more concentrated with chains of vesicles and the distance between the individual rows becomes less. Very soon after a chain of vesicles has been budded off from the nuclear membrane, fine intervesicular connections appear between certain of the vesicles comprising the rows. Several of the vesicles in a row may then fuse, forming short, flattened cisternae. Fusion of vesicles continues, individual rows of vesicles become more closely packed and, finally, regions appear in the cytoplasm which have the appearance of annulate lamellae. Further growth of the lamellae appears to occur by the progressive fusion of vesicles at the ends of those lamellae already present, as well as by the addition of other fusing rows of vesicles.     

CYTOLOGY OF DIFFERENTIATING TRACHEARY ELEMENTS I. ORGANELLES AND MEMBRANE SYSTEMS

The distribution of organelles, membrane systems, and ribosomes is not at any time obviously related to the pattern of secondary wall in helically thickened tracheary elements in leaves of Beta vulgaris L. (sugar beet) and Cucurbita maxima Duchesne, fixed with potassium permanganate and osmium tetroxide. During the differentiation of the secondary wall, cisternae of the endoplasmic reticulum and dictyosomes are particularly conspicuous, and the dictyosomes are associated with numerous vesicles. Similar vesicles appear to be in various stages of fusion with the secondary wall thickenings. The tracheary elements contain plastids which may include starch granules. Ribosomes occur free in the cytoplasm and in association with endoplasmic membranes.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

The effect of glutaraldehyde fixation on the elucidation of the morphogenesis of annulate lamellae in oocytes ofRana pipiens

Summary Primary fixation of the frog oocyte with glutaraldehyde, compared to osmium tetroxide, alters the appearance of components involved in the morphogenesis of annulate lamellae. With glutaraldehyde, the outer layer of the nuclear envelope is connected with membranous laminae of variable length. Rounded blebs of the outer layer of the nuclear envelope are infrequently observed. Further, rather than clusters or rows of cytoplasmic vesicles as are observed in osmium tetroxide-fixed cells; numerous and long, smooth-surfaced lamellae are present in the ooplasm after glutaraldehyde fixation. The long membranous laminae then become concentrated in several ooplasmic packets. This is followed by the progressive alignment or orientation of the laminae within the packet. Eventually, those aligned and formerly smooth-surfaced lamellae are converted into annulate lamellae.This study was supported by research grants (HD-00699, GM-09229) and a Career Development Award from the National Institutes of Health, U.S. Public Health Service.