Structure and function of the amphibian follicular epithelium during ovulation

Summary Low concentrations of cytochalasin B (CCB) are known to inhibit ovulation in the frog, Hyla regilla. Examination of amphibian thecal cell ultrastructure reveals filaments (average diameter 71 Å) arranged in bundles parallel to the surface of the oocyte. These filaments are often associated with hemidesmosome-like plaques on the basal plasmalemma. While individual filaments appear unaltered morphologically by CCB (1–5 g/ml), their organization into bundles, apparent relationship to the hemidesmosomes, and the highly contorted configuration of the thecal cells after oocyte expulsion, suggest that a nonmuscular contractile system residing within the follicle plays a fundamental role in ovulation.Our data suggest that the flattened epithelioid thecal cells shorten all axes that run parallel to the oocyte surface via filament bundle contractions, while they remain tightly bound together by macular attachment plaques. These cells thus increase in height to become cuboidal-low columnar in shape; the area covered by the base of each is greatly reduced. As this thecal sac decreases in size, the compression generated by the contractile mechanism forces the oocyte through the enzymatically weakened apex of the follicle and ovulation results.This investigation was supported by grant HD-07194 from the National Institutes of Health. The authors are grateful to Dr. Arthur L. Cohen, Director of the Electron Microscope Center for use of the Center's scanning electron microscope. We are also indebted to Mrs. Gail M. McDole and Mr. James D. Huber for able technical assistance     

The action of cytochalasin B during egg cleavage in Xenopus laevis: dependence on cell membrane permeability

By exposing Xenopus eggs during the first cleavage to cytochalasin B (CCB) for successive periods of 4 min, it has been shown that CCB sensitivity becomes manifest approximately 7 min after the onset of furrow formation. However, even before this time furrow regression can be induced by the injection of CCB under the membrane in the furrow. This shows that during the first 7 min of cleavage the operative contractile system is CCB sensitive. Using microelectrode techniques, electrical membrane characteristics (membrane potential and resistance) were measured continuously in normally cleaving eggs and in cleaving eggs injected with CCB. It was found that the onset of sensitivity to externally applied CCB coincides with a rapid alteration of the membrane potential and resistance. We have concluded that externally applied CCB can only enter the egg when the membrane permeability increases. No evidence has been found that CCB alters the ionic permeability of preexisting cell membrane.     

Studies on silk secretion in the trichoptera (F. Limnephilidae)

Summary The ultrastructure and amino acid composition of the secreted silk of two species of trichopteran larvae, Pycnopsyche guttifer (Walk.) and Neophylax concinnus McL., were investigated. The spinnerets of these two animals were also examined by scanning electron microscopy. The silk consists of double-stranded, flat ribbons (1–4 wide), composed of bundles of 15–25 Å filaments. There are two components of the silk: the fiber proper and a surrounding coat thought to be a silk gum. Only the outer coat is positive to the EM PATP technique of Thiery (1967), which indicated the presence of neutral sugars. Amino acid analyses of Pycnopsyche silk show that, like other silks, two predominant amino acids are glycine and serine. Arginine, unexpectedly, is the third most abundant and there are a large number of basic and long side-chain amino acids. X-ray diffraction studies of the silk indicate that it has a less crystalline, more amorphous structure than that of other silks.Submitted to the Department of Biological Sciences of the State University of New York at Albany in partial fulfillment of the requirements for the degree of Doctor of Philosophy Acknowledgements. This study was supported in part by a National Institutes of Health Graduate Student Traineeship grant # GM-02014. The author would like to express sincere gratitude to Dr. Stephen Brown for his encouragement and help during the course of this study. I would also like to thank Dr. Curtis Hemenway and Mr. Douglas Halgren of Dudley Observatory for the use of their scanning electron microscope as well as Dr. Helen Ghiradella and Mr. William Radigan for help with the scanning electron microscopy. I owe special appreciation to Dr. Y. Myer of the Chemistry Department of SUNYA for doing an amino acid analysis of the silk and to Dr. K.M. Rudall of the University of Leeds for doing the X-ray diffraction studies of the silk samples     

An argyrophil fibrillar system and amitotic nuclear division in pars intermedia cells of the rainbow trout (Salmo irideus)

Summary Argyrophil fibrils were revealed in pars intermedia chromophobe cells of Salmo irideus. For the light microscopical demonstration of the fibrils a recently developed copper sulphate-silver protein technique for Bouin-fixed hypophyses was used.The fibrils, apparently belonging to one fibrillar system, are found only in limited regions of the cytoplasm. They occur in ring-, loop-, or cracknel shapes in the close vicinity of the rounded nucleus in the chromophobe cell, and continue in linear shape into the cell base or cell apex where they may end near the adjacent basement membrane.Electron microscopically, the fibrils are composed of filaments, 70–90 Å in diameter, which are arranged in parallel. Bundles of filaments are frequently found near the nucleus. In addition to perinuclear filaments, bundles of filaments with tiny offshoots occur in different areas of the cytoplasm, extending sometimes as far as the cell membrane which is located near the basement membrane of the pars intermedia epithelium.In cells with a bi- or trilobular, apparently amitotically dividing, nucleus a fibrillar loop or ring usually surrounds the interlobular part(s) of the nucleus. This relation of the fibrils to the dividing nucleus and their consisting of regularly arranged filaments indicates the significance of the fibrillar system in the chromophobe cell. It is thus suggested that the fibrillar system is involved in the constriction of the nucleus during amitosis.The number of chromophobe cells in which fibrils are light microscopically visible varied greatly among the rainbow trout used.Miss M. C. Wentzel is thanked for her technical assistance, Mr. M. Veenhuis for his advice and excellent help with the preparation of the electron micrographs, and Mr. L. Hoekstra for preparing the final drawings. Mr. N. J. Williams' corrections to the English are gratefully acknowledged.     

The fine structure of cephalopod blood vessels III. Vessel innervation

Summary The cephalic aorta of Octopus vulgaris has a fairly complete endothelium lining the lumen, a thick complete basement membrane, a layer of circularly orientated and a layer of longitudinally orientated muscle fibres. Presumptive synaptic endings are of two types. In the circular muscle, axons containing vesicles, contact club-shaped projections of the muscle. The gap between the pre- and postsynaptic membranes is less than 100 Å and in some places apparently forms a tight junction. The second type of ending has been found in the longitudinal muscle; here axons full of vesicles end on the muscle. The ending is enclosed by a mesaxon of muscle and the synaptic gap is approximately 100 Å. In the smaller blood vessels, axons end on myofilament-containing pericytes of blood vessels (equivalent to small arterioles). The endings contain vesicles and have a synaptic gap of 100 Å. Only some of the pericytes seem to be innervated and transmission between one pericyte to another may be mediated by specialized junctions between the cells. The smaller non-myofilament containing vessels (equivalent to capillaries) are not thought to be innervated.We would like to thank Professor J. Z. Young and Dr. E. G. Gray for advice and encouragement, Mrs. Jane Astafiev for drawing Figs. 1 and 12, Mr. S. Waterman for photography, and Miss Cheryl Martin for secretarial assistance.     

The nerve fibres in the basement membrane and related structures in Saccoglossus horsti (Enteropneusta)

Summary The structure of the basement membrane of Saccoglossus horsti has been examined with the electron microscope. The membrane consists of two lamellae each of two layers. An outer amorphous layer 150 nm across and an inner fibrillar layer 1–3 m across. The fibrils of the fibrillar layer are two sizes, the majority are 5–9 nm in diameter and at least 2 m long. The thicker 30 nm fibrils occur in small patches and have striations with a 30 nm period.Within the lamellae of the basement membrane are blood spaces. The only regularly found structures in these spaces are blood particles some 12–16 nm in diameter.Nerve fibres of varying diameters traverse all the layers of basement membrane. These fibres run longitudinally and obliquely through the basement membrane, and emerge amongst the muscle cells inserted into the coelomic side of the membrane. No motor end plates have been seen. Preliminary observations suggest that many of the nerve fibres have no sheath other than the cell membrane of the fibre itself.The muscle cells are attached to the basement membrane by structures that resemble hemidesmosomes. The blood vessels of Saccoglossus have a basement membrane on the lumenal side of the endothelial cell cytoplasm.I wish to thank Professor J. Z. Young, F. R. S. for continuous encouragement and advice. To Dr. R. Newell I am indebted for the collection and identification of the specimens. I am pleased to acknowledge my debt to Dr. R. Bellairs for the use of electron microscope facilities, and to Mrs. J. Hamilton and Mr. R. Moss for skillful technical assistance.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Vinblastine-induced precipitation of phloem proteinsin vitro

Summary Purified proteins from sieve tubes ofCucurbita maxima were precipitated with vinblastine and the precipitates were analyzed with the electron microscope. Filaments (35–40 Å in diameter) and tubular structures (160 Å in width) were observed in negatively stained preparations. The predominant structures of the negatively stained and of the thin sectioned material, however, were membrane-like sheets (100–120 Å in width) which showed the typical unit membrane aspect.Dedicated to Professor Dr. W.Schumacher on his 70th birthday.The investigations were supported by the Deutsche Forschungsgemeinschaft.     

The fine structure of neuromuscular junctions and contact zones between body wall muscle cells of Ascaris lumbricoides (var. suum)

Summary Neuromuscular junctions and close membrane apposition between body wall muscle cells of Ascaris lumbricoides (var. suum) have been examined with the light and electron microscopes. It was found that the body wall muscle cells send out elongate processes from their basal, myofibril containing portion to terminate on dorsal and ventral nerves. When observed with the aid of the electron microscope the neuromuscular junctions were seen to consist of several muscle cell processes in apposition to a single axon. The intersynaptic cleft was approximately 350–500 Å wide. Both the axolemma and sarcolemma were triple layered membranes which were 75–80 Å thick. Electron dense patches were observed at intervals on the apposed membranes which were due to increased thickness of the inner membrane leaflets of axolemma and sarcolemma. Muscle cell membranes, at the level of the neuromuscular junction, were in close apposition resulting in an apparently five-layered membrane complex which was 170–210 Å thick. The sarcolemmata in these regions were separated by 10–50 Å. Presynaptic axons contained mitochondria, microtubules which were 180–270 Å in diameter, and two, morphologically distinct types and sizes of synaptic vesicles. One was 200–600 Å in diameter, with a single, triple-layered membrane bounding a center of low electron density. The other was 600–1200 Å in diameter, with a single, triple-layered membrane bounding a central, electron dense granule of 500–800 Å size.The functional significances of the close membrane appositions between body wall muscle cells and of the two types of synaptic vesicles found at the neuromuscular junctions of Ascaris lumbricoides were discussed with respect to their possible role in neuromuscular physiology.Supported by U.S.P.H.S. Grant No. NB-01528 and Research Career Development Award No. 9-K3-NB-15255. — The author wishes to express his grateful appreciation for the excellent technical assistance given by Miss Gabrielle Rouiller during the course of this investigation.     

Ultrastructure of spermatozoa of Peregrinus maidis (Homoptera,Delphacidae)

Summary The spermatozoa of Peregrinus maidis Ashm. are thread-like, approximately 650 long and 1 wide including the head (approximately 28 ).The main part of the spermatozoa consists of two mitochondria derivatives, a central body between them, the axial filament complex, and a newly found element consisting of two wing-shaped bodies. Each mitochondrion derivative shows a peripheral and an inner part. The peripheral part is formed by cristae arranged perpendicularly to the long axis of the spermatozoon. The cristae are approximately 70 Å wide. The dense layers between them measure approximately 280 Å. The inner part of the mitochondrion derivative shows a crystalline array, formed by sub-units of approximately 100 Å diameter. The wing-shaped bodies consist of tubular elements.The head has an elongated nucleus with an electron transparent space inside. At the anterior end of the nucleus lies a tapered acrosome. This appears fibrous and parts of the acrosome fibers seem to run along the nucleus. Acknowledgements. The authors wish to thank Dr. G. H. Bergold for suggestions and support, Drs. J. André, D. W. Fawcett, P. Maillet and G. F. Meyer for very helpful discussion. They are also grateful to Mr. O. Suárez for assistance in the preparation of the organs of P. maidis and to Mrs. M. de Pingarrón for technical assistance.     

A Thiococcus sp. nov. gen., its pigments and internal membrane system

Summary A Thiococcus sp. nov. gen., the first Thiorhodaceae known to contain bacteriochlorophyll b, was isolated from river mud using filtered light above 900 m as the selecting agent in enrichments. The orange-brown colour of the immotile, Gram-negative, photolithotrophic cocci is due to two new carotenoids, 3,4,3,4-tetrahydrospirilloxanthin and 3,4,3,4-tetrahydrospirilloxanthinal. The photosynthetic pigments appear to be associated with a unique internal membrane system consisting of branched membrane tubes of even diameter (approx. 450 Å) continuous with the cell membrane. The negatively stained tubes examined in the electron microscope reveal a surface fine structure of raised bodies arranged in a regular hexagonal pattern.Dedicated to Professor Dr. C. B. van Niel on the occasion of his 70th birthday.     

Elektronenmikroskopischer Nachweis spezieller cytoplasmatischer Vesikel bei Micrasterias denticulata Bréb.

Summary Electronmicroscopic studies of growing and non-growing cells of Micrasterias denticulata Bréb., fixed with glutaraldehyde-OsO₄, showed a special kind of cytoplasmic vesicle which has so far not been found in other cells. These particles (1000–1200 Å in diameter) are characterized, by an unusual, multilayered membrane and a rod-like content of high electronoptic density. The vesicles are found to be accumulated in the vicinity of the nucleus and in a positional relationship to the nuclear pores. Although no evidence could be found either for a direct passage of the vesicles through the pores or for a blebbing-process from the nuclear membrane, the rod-containing vesicles could be functional in the process of nuclearcytoplasmic exchange.

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Herrn Prof. Dr. H. Drawert zum 60. Geburtstag gewidmet.

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Diese Arbeit wurde zum Teil durch ein National Science, Foundation Senior Foreign Scientist Fellowship an Dr. Oswald Kiermayer und durch einen Training Grant 5-T1-GM-707-06 an Dr. Keith R. Porter, Harvard University, unterstützt.     

Phagocytoseverhalten vonPseudomicrothorax dubius unter Normalbedingungen und nach Applikation von Colchicin sowie Cytochalasin B

Zusammenfassung Das Phagocytoseverhalten des mit einem Reusenapparat ausgestatteten CiliatenPseudomicrothorax dubius wurde unter Normalbedingungen sowie nach Applikation von Colchicin und Cytochalasin B untersucht. Bei Konzentrationen von 0,03–0,05% Colchicin — höhere Konzentrationen zeigten letale Wirkungen auf die Organismen — konnte kein signifikanter Einfluß auf die Nahrungsaufnahme festgestellt werden. Andererseits blockierte Cytochalasin B ab Konzentrationen von 7 g/ml die Phagocytose vollständig; membranumgebene Blasen erschienen an der Buccalöffnung. Die Ergebnisse werden hinsichtlich der Mechanismen und der Krafterzeugung für die Nahrungsaufnahme diskutiert.

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Phagocytic behaviour ofPseudomicrothorax dubius under normal conditions and after application of colchicine and cytochalasin B

Summary The phagocytic behaviour ofPseudomicrothorax dubius-a ciliate with a cytopharyngeal basket-was investigated under normal conditions as well as after application of colchicine and cytochalasin B. Colchicine, applied in concentrations of 0.03–0.05%, does not show a significant influence upon normal occuring food uptake. Higher concentrations cause finally cell's death. On the other hand, cytochalasin B blocks the phagocytosis entirely when applicated at concentrations of 7 g/ml and more; membrane limited bubbles appear at the buccal opening. The findings are discussed with regard to the mechanisms and the generating forces of the process of food ingestion.

     

Anaphase progression and furrow establishment in nocodazole-arrested PtK1 cells

The relationship between progression through anaphase and furrow establishment was investigated in PtK₁ cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 m or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK₁ cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.     

Electron microscopic observations on synapse-like contacts between pituicytes and different types of nerve fibers in the anuran pars nervosa

Summary Pituicytes of Rana pipiens could be classified into two types, pale and dense, according to their relative densities of cytoplasm and the populations of free ribosomes and cell organelles. An intermediate type of pituicyte was also recognized.Lipid droplet such as are typical in the cytoplasm of mammalian pituicytes, are not in the cytoplasm of either types of frog pituicyte. Both types have long cytoplasmic processes which run among the nerve fibers, and some of them end at the pericapillary space.Nerve endings making synapse-like contacts with the cell bodies or the processes of the pituicyte are frequent. According to the structures and sizes of granules and vesicles in the nerve endings, these endings are classified into one of three types: 1) A, which appears to be a peptidergic neuronal ending containing dense granules 1,200–2,000 Å in diameter and small clear vesicles 300–400 Å in diameter; 2) B, which appear to be monoaminergic endings containing cored vesicles 600–1,000 Å in diameter and small clear vesicles 300–500 Å in diameter; 3) C, which appear to be cholinergic endings containing only small clear vesicles. Type C endings are relatively rare. In the synaptic area the axonal membranes appose those of the pituicytes across a gap of about 200 Å and numerous presynaptic vesicles are clustered or accumulated near the presynaptic membranes.The author wish to express his hearty thanks Professor Dr. A. Gorbman, Zoology Department, University of Washington, Seattle, U.S.A. and Professor Dr. H. Fujita for their helpful advices and criticisms. The frog tissues were obtained and fixed in Professor A. Gorbman's laboratory supported by U.S.P.H.S. grant NS 04887.     

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

Über schlauchförmige Ausstülpungen an der Zellmembran der Makrogametocyten von Eimeria perforans

Summary The surface of the gametocytes and gametes of Eimeria perforans reveals tube-like extrusions which have not been discovered so far in coccidia. These slender tubes are 650 Å in width and at least 1,3 in length. Probably they represent resorbing organelles. The tubes occur only at the surface of older female gametocytes and gametes and have not been observed in merozoites, schizonts, microgametocytes and male gametes. In transversal sections the tubes look like vesicles and are bordered by a membrane the layers of which are not sharply defined. The interior of the tubes seems to be empty after electron microscope observations. In longitudinal sections the membrane of the tubes is striated. The repeating unit of the dark and light bands amounts to about 165 Å. This appearance cannot be explained so far.

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Für beratende Hilfe sind wir Herrn Prof. Dr. R. Danneel und Herrn Prof. Dr. K. E. Wohlfarth-Bottermann zu Dank verpflichtet, für technische Unterstützung Fräulein cand. rer. nat. B. Volkmann. Herrn Dr. D. Spiecker von der Forschungsstelle für Jagdkunde und Wildschadenverhütung, Beuel, danken wir für die Durchführung der Infektionen. Die Mittel für die Untersuchungen stellte uns die Deutsche Forschungsgemeinschaft zur Verfügung.     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 g of cytochalasin B per ml but inhibited by 10 M oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Structural aspects of amitosis: a light and electron microscope study of the isolated macronuclei of Paramecium aurelia and Tetrahymena pyriformis

Thin sections show the macronuclei of Paramecium aurelia and Tetrahymena pyriformis to contain two types of bodies. The smaller, measuring 0.1–0.2 in diameter, have been resolved in the light microscope by first removing the macronuclei from the cells in the presence of Mg⁺⁺, then chelating that divalent cation with EDTA, resulting in expansion of the nuclear material. By staining with methyl green, Azure B, and the Feulgen procedure, the small bodies were shown to contain DNA. In whole mounts these small bodies appear to be joined to one another producing a complex network suspended in which are the larger bodies, or nucleoli. — Macronuclei from both ciliates were isolated in large quantities and purified for spreading on an air-water interface. When the nuclei burst from surface tension forces and are examined with the electron microscope, the DNA containing bodies remain attached to one another by means of 100 Å fibrils. The pattern of attachment is non-linear. Occasionally individual DNA-containing bodies loosen revealing a coil resembling both in shape and dimensions the 250 Å coil characteristic of eucaryotic chromatin. The substructure of the 250 Å coil has not been directly observed. However, the frequent association of pairs of 100 Å fibrils makes it likely that two such fibrils are tightly complexed in the 250 Å coil. The 100 Å fibril, in turn, contains two 20 Å strands, each presumably a DNA double helix. — In Paramecium each small body of the macronucleus contains approximately one chromosome-equivalent of DNA. The fact that these small bodies are joined to form large structured masses of chromatin within the macronucleus indicates that the distribution of genetic material is not random. It is possible that, similar to bacteria, entire genomes segregate as units, thus accounting for successful amitotic division.This work was supported by a Predoctoral Fellowship to the author from the National Institutes of Health, U.S. Public Health Service, and by grant GM-13882 (NIH-USPHS) to Dr. Daniel Mazia.     

Structural transformation of the phagosomal membrane in Tetrahymena cells endocytosing latex beads

A model system with a high phagosomal membrane turnover has been developed: During a 45-min period Tetrahymena cells endocytoze 186 latex beads (diameter: 2.02 m) per average cell; 166 of these beads are then exocytozed in the course of the following 145 min. During the endocytotic phase an average cell is approximated to fabricate 1200 m² phagosomal membrane. Freeze-etch electronmicroscopy reveals that both fracture faces of the nascent phagosomal membrane are associated with the typical 85 Å-particles in approximately equal numbers. Mature phagosomal membranes, however, show an unequal particle distribution. Smooth areas, smooth areas bordered with a fracture rim, and particle-associated depressions up to a diameter of 130 nm can be observed especially on fracture faces of mature phagosomes in the endocytotic phase. These are discussed with respect to membrane fusion.This paper is dedicated to Mr. W. Batz who died tragically on February 7, 1974