Occurrence of the putative microfibril-synthesizing complexes (linear terminal complexes) in the plasma membrane of the epiphytic marine red algaErythrocladia subintegra Rosenv.

Summary Cells of thalli at different developmental stages of the epiphytic marine red algaErythrocladia subintegra have been studied by freeze-etching. It was found that the plasma membrane exhibits linear microfibril-termnal synthesizing complexes (TCs), randomly distributed consisting of four rows of linearly-arranged particles (average diameter of particles 8.6 nm); each row of TCs consists of 5–33 particles (average 15). The TCs were observed on both fracture faces (PF and EF) but more clearly on the PF face. These structures appear to span both the outer and inner leaflets of the plasma membrane (transmembrane complexes)-The TCs have stable width (35 nm) and vary in length (41–311 nm, average 181 nm). The TCs subunits are highly ordered arrays forming a semicylinder. The average density of TCs on the PF face is 5.5TC/m². The microfibrils are randomly distributed and have a mean width of 39.4 nm (ranging from 16 to 70 nm). Many TCs are associated with the ends of microfibrils and microfibril imprints. The structural characteristics of linear TCs in the red algaErythrocladia are compared with those of the so far investigated Chlorophyta spp. All results favour the suggestion that TCs in the plasma membrane ofErythrocladia cells are involved in the biosynthesis, assembly and orientation of microfibrils.     

Fine structure ofPenicillium megasporum conidiospores

Summary An investigation was conducted on the fine structure ofP. megasporum conidiospores using both chemical fixation and the freeze-etching techniques. The spore cell wall was shown to consist of several layers, one of which is chiefly fibrillar in nature. The surface of the spore appears to be covered by ordered arrays of rodlets which are embedded in or resting upon a thin layer of homogenous material. At higher magnification, these rodlets appear as linear arrays of particles approximately 50 Å in diameter. This layer may be of a cutaneous nature. In addition, the plasma membrane was shown to be covered by particles, some of which appear to migrate to the inner layer of the cell wall. Numerous invaginations of the plasma membrane were also seen.On leave of absence from the Department of Botany, University Nijmegen, Holland.On leave of absence from the Department of Botany, Brigham Young University, Provo, Utah.     

Ultrastructure of the freeze-dried mouse thyroid gland

Summary Mouse thyroids were preserved for electron microscopy by means of freeze-drying. The tissue specimens were frozen in liquid isopentane, dried at a temperature of –79° C to a pressure of 4×10^-5 mm Hg, stained in vacuo with osmium tetroxide vapour at room temperature, and embedded in vacuo in Epon or Vestopal W.The ultrastructure of the freeze-dried thyroid gland was found to be fundamentally similar to that observed after ordinary chemical fixation. However, some differences were noticed. Thus the plasma membrane of the lateral cell surfaces appeared to be asymmetrical, its inner dense layer being thicker than the outer one. The mitochondria had a finely undulating contour and a matrix of rather high density. The mitochondrial outer and inner membranes appeared as five-layered structures and comprised three dense and two less dense layers. Small dense granules with an undulating outline and larger, less dense granules with a distinct surface membrane were observed. The intra-cisternal space had a lower density than the extra-cisternal space. 150 Å particles occurred very sparsely. The karyoplasm had a low density and the particulate component of the nucleus was rather scanty. By means of pores in the nuclear envelope the karyoplasm appeared to communicate with the extra-cisternal space. No basement membranes were observed. The appearance of myelin sheaths in freeze-dried specimens seemed to agree well with the picture after chemical fixation.     

The demonstration of the existence of an interlayer between the cytoplasmic membrane and the cell wall proper of staphylococci

By disintegration of the cell wall of staphylococci a definite interlayer located between the cytoplasmic membrane and the cell wall proper could be demonstrated for the first time (=MW-interlayer). This MW-interlayer contains a sort of cloddy material in which clusters of embedded ring-like disks are hexagonally arranged in a crystal-like manner. The ring-like disks, approximately 40 Å in diameter and with center-to-center spacings of approximately 75 Å, lie in direct contact either with a rhombically arranged fibrillar network of the outer parts of the cytoplasmic membrane or they themselves are part of (or interconnected by) such an apparently rhombical network. The crystal-like arranged ring-like disks of the interlayer between the cytoplasmic membrane and the cell wall shall be called MW-particles in order to differentiate them from intramembrane particles and particles on the outer surface of the cell wall. At present, nothing more than speculation on the function of the MW-particles located within the space where final processes of the cell wall polymerization are taking place is possible.Abbreviations MW membrane-wall - EF external face - PF protoplasmic face - PS protoplasmic surface - IM intramembrane     

Attachment of muscle filaments to the outer membrane of the nuclear envelope

Summary Myofilaments of striated muscles can be recognized in the electron microscope to be in structural continuity with the outer membrane of the nuclear envelope. The very site of insertion of these myofilaments at the membrane surface frequently appears characterized by a dense basal knob of 85–135 Å. It is hypothesized that this attachment of myofilaments to the nuclear membrane plays a role in mechanically transmitting the contraction of the fiber to the nucleus, thus bringing about the harmonica-like folded appearance of the nucleus which is known for the contracted states of striated, smooth and cardiac muscles.The work was supported in part by the Deutsche Forschungsgemeinschaft.The author is indebted to Miss Sigrid Krien and Miss Marianne Winter for careful technical assistance as well as to Drs. Heinz Falk and U. Scheer for valuable discussions.     

The fine structure of the polian vesicles of holothurians

Summary The Polian vesicles are tubules of variable length consisting of four layers: an external peritoneal epithelium, a connective tissue layer, a muscular layer, and an inner epithelium. The two simple epithelia produce a polysaccharide in their Golgi complexes. Their cellular junctions consist of an extensive zonula adhaerens, and an apical septate desmosome. Their surfaces possess microvilli and cilia. A basement membrane is lacking. The muscular layer is composed of paramyosin fibers. The connective tissue layer contains abundant collagen fibers that have a period of 640 Å and are embedded in an acid mucopolysaccharide matrix. Amoebocytes containing waste products and an acid mucopolysaccharide enter the Polian vesicles from the coelom, pass through them, and empty into the lumen along with material secreted by the inner epithelium. While establishing close contact with cells of the muscular layer and inner epithelium the amoebocytes seem to transfer part of their contents to these cells.Polian vesicles appear to be a very primitive excretory organ.Research performed under C.N.R. contract.     

Feinstruktur von Zellwand und Plasmamembran beiMicrasterias denticulata Bréb. nach Gefrierätzung

Zusammenfassung Gefrierätz-Untersuchungen an rasch eingefrorenen sonst aber unvorbehandelten Zellen vonMicrasterias denticulata ergaben, daß die Sekundärwand aus einer fibrillenfreien Außenschicht, die möglicherweise Lipid-Charakter aufweist, und einer Fibrillenschicht aufgebaut ist.Jede der zu Bändern zusammengefügten Mikrofibrillen (mit Durchmessern bis zu 300 Å) der Fibrillenschicht besteht aus zahlreichen Elementarfibrillen, die einen ungefähren Durchmesser von 40 bis 50 Å aufweisen. In tieferen Lagen der Fibrillenschicht laufen die Mikrofibrillen um den Porus herum, während die äußerste Lage der Fibrillenschicht vom Porus durchbrochen wird. Nur diese Lage wird sekundär perforiert; der übrige Porenkanal entsteht gleichzeitig während des Sekundärwandwachstums.Die Bruchfläche der Plasmamembran ist mit zahlreichen, statistisch verteilten Partikeln bedeckt und weist Eindellungen unter jedem Porus auf. Am Rande dieser Eindellungen sind Membranlöcher festzustellen, die als Fusionsorte von schleimenthaltenden Vesikeln und der Plasmamembran gedeutet werden.

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Fine-structure of the cell wall and plasma membrane inMicrasterias denticulata bréb. after freeze-etching

Summary Cells of the algaMicrasterias denticulata, frozen in their growth medium without further pretreatment, have been examined by means of freeze-etching. The outer surface of the secondary wall as well as the side walls of the slime pores are covered with a continuous thin, fibril free layer with cleaving properties resembling those of multiple lipid bilayers. Elementary fibrils (40 to 50 Å in diameter) are shown to make up the microfibrils (200 to 300 Å in diameter), 2 to 15 of which may aggregate laterally to form the characteristic bands of secondary wall fibrils. While the microfibrils in the inner layers of the secondary wall appear to circumvent the pores those belonging to the outermost layer seem to be frequently disrupted by the pore apparatus. These observations suggest that the main part of the pore channel is formed during secondary cell wall formation. Only the outermost fibrillar layer of the secondary wall seems to be perforated after its deposition.The cleaved face of the plasma membrane carries numerous randomly distributed particles and is marked by large depressions underlying each of the pores. In the rim areas of these depressions the membrane is frequently interrupted by small holes, which are interpreted as points of fusion between the slime secreting vesicles and the plasma membrane.

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Die elektronenmikroskopischen Untersuchungen wurden in den Biological Laboratories der Harvard University, Cambridge, U.S.A., durchgeführt. Die Arbeit wurde durch die Deutsche Forschungsgemeinschaft und durch einen Health Science Advancement Award Grant 5-SO 4-FRO 6084 an die University of Colorado unterstützt.     

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC)

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided -strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 Å resolution. In mitochondrial porins the N-terminal region forms an amphipathic -helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.     

Vergleichende Untersuchungen an Amoebozyten und Blasenzellen von Cepaea nemoralis L. (Gastropoda,Stylommatophora)

Summary The pecten oculi of the sparrow consists of capillaries, pigment cells and a superficial membrane. Because of the loose structure of the first two components broad intercellular spaces occur in the pecten. The capillary wall consists of endothelial cells and a perivascular membrane. The bodies of the endothelial cells are flattened, while the plasmalemma of both their surfaces (basal and luminal) is strongly folded and forms numerous microfolds with an average thickness of 700 Å. The height of the inner microfolds is 1.4–1.8 m, the outer microfolds measure 1.3–1.6 m. They lie densely packed side by side and are separated by recesses of the capillary lumen ca. 500 Å wide. Due to this the surface of the endothelial cell is increased by approximately 20-fold. The adjoining endothelial cells abut or overlap with margins, and are joined by the zonulae adherentes. Pigment cells form numerous processes and microvilli. Some rest on the capillary walls, while others penetrate the superficial membrane of the pecten or fill the intercellular spaces.     

Effects of taurine on the phosphorylation of specific proteins in subcellular fractions of the rat retina

The effects of 20 mM taurine on the phosphorylation of specific proteins in mitochondrial and rod outer segment subcellular fractions of the rat retina were measured. A band of protein with an apparent molecular wieght of 20K was consistently inhibited by taurine. Densitometry measurements performed on gel electrophoresis autoradiograms from the mitochondrial fraction demonstrated a 42.7±8.3% decrease due to taurine (20 mM) in the area corresponding to radioactivity from the 20K phosphoprotein. However, only a 21.2±9.0% decrease was observed due to taurine in the rod outer segment preparation. These data suggest that taurine is exerting its primary effect on the phosphorylation of the 20K molecular weight protein in the mitochondria of the retina. In addition, calmodulin and phorbol ester had no effect on the phosphorylation of the 20K molecular weight protein.     

Electron microscopical observations on the brush border of proximal tubule cells of mammalian kidney

Summary The ultrastructure of the plasma membrane and the core of microvilli of proximal tubule cells has been investigated by electron microscopy using sectioned and negatively stained material. By the technique of negative staining, a particulated coat is disclosed on the outside of the plasma membrane of microvilli of brush borders isolated from rat, rabbit and ox. This coat is composed of 30 to 60 Å particles and is 150 to 300 Å thick and appears to be a distinguishing feature for the luminal plasma membrane (brush border) of proximal tubule cells. The plasma membrane of the basal part of tubule cells is found to be smooth. By thin sectioning, an axial bundle of 50 to 70 Å diameter filaments regularly arranged in an 1+6 configuration, one axially located filament being surrounded by a ring of six, is disclosed. The distance from the ring of filaments to the inner surface of the plasma membrane is 250–300 Å, the diameter of the ring 300 Å and the center-to-center distance between filaments 120 Å. Negative staining also discloses 60 Å filaments in microvilli of isolated brush borders. Broken off, single microvilli (fingerstalls) are observed with thin filaments projecting from their broken ends. Filaments up to 1 in length are seen. At high magnification, the filaments appear beaded and show strong resemblance with actin filaments isolated from skeletal muscle. Based on present evidence, it is postulated that microvilli constituting renal brush borders possess contractile properties, which may play a role in the absorption process operating at the luminal part of the cells.The authors are indebted to Miss Kirsten Sjöberg for skilled technical assistance, and to the Danish State Research Foundation and the Tuborg Foundation for financial support.     

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by dense lamellae, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 9 nm particles m^-1 and an EF face with 150–700 11–12 nm particles m^-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles m^-1, mean size 10.9 nm, on the PF face and 560 particles m^-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.     

Possible sites of release of neurosecretory granules in the sinus gland of the crayfish,Orconectes nais

Summary The sinus gland is a neurohemal organ located in the crayfish eyestalk and represents a storage site for neurohormones prior to their release into the circulation. The sinus gland contains three classes of electron dense, membrane-limited granules. Class 3 granules are the largest and most electron dense of the granules found in the sinus gland. Granules of class 1 are the smallest, while those of class 2 are the most abundant. Generally, all granules undergo similar changes during their release.Release of neurosecretory material may be initiated by a preliminary fragmentation of the parent granules into smaller granules. Following the formation of numerous smaller granules, these move to the plasma membrane and their limiting membrane apparently fuses with it thus releasing its contents into the external lamina which is applied to the sinusoidal surface of the axon terminals. Granule release does not appear to occur along the entire plasma membrane adjacent to the blood sinus but, instead, probably occurs only at specific active sites on the membrane. The active sites are characterized in part by an accumulation of small granules and clear vesicles against the cytoplasmic side of the plasma membrane. At the site of release of the neurohormone, there is often an accumulation of dense homogeneous material beneath the axolemma.Occasionally, axon endings filled with large, electron lucent vesicles are seen. These clear granules vary from 1150–1750 Å in diameter and often exhibit broken limiting membranes. Few small vesicles are seen near the plasma membrane of these endings; however, instances of invaginations of the plasma membrane occur. The significance of endings filled with clear granules is discussed.Supported by a grant from the National Research Council of Canada (No. A-4675).     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.     

Ultrastructural aspects of cell wall synthesis inSpirogyra

Summary Filaments ofSpirogyra were fixed in 2% osmium tetroxide dehydrated in alcohol and embedded in Araldite. The fine structure of cells with regard to wall synthesis was studied. The cell wall was shown to have four layers. The inner one contains microfibrils and is considered to be the cell wall proper. The outer three layers are components of the slime layer. The innermost of these, the second layer of the wall, was shown to be between 1m to 3m and the third 0.3m to 1m. The fourth layer appears as no more than a dark black line measuring 10 nm across. In the cytoplasm two types of vesicles were seen. The largest of these has contents similar in appearance to the slime layer of the wall. This same material was also seen in the large vesicles attached to the Golgi bodies. It is suggested that the smaller vesicles are derived from the larger vesicles and later fuse with the cell membrane. The Golgi bodies were found to be fairly large measuring up to 5m across. Small electron opaque blobs and flecks on the outside of the plasmalemma and in between the microfibrils of the cell wall proper are considered to be mucilage droplets travelling to the slime layer. It cannot be excluded that some of the material of the large vesicles is released directly into the cytoplasm and is transferred without vesicles through the plasma membrane. The negative contrast appearance of the microfibrils seen in the cell wall is thought to be due to the spaces between them being filled with this electron opaque mucilage.Intercisternal rodlets measuring 2.5 nm across were seen in the Golgi bodies.Transverse microtubules were found to occur near the plasmalemma having the same orientation as some of the microfibrils.Lomasome-like structures sometimes with many 5 nm fibrils in their vicinity were seen.     

Specialised plasma membranes in the preovulatory follicle op the fowl

Summary In the hen's follicle processes from the zona granulosa cells extend into the zona radiata. The terminal plasma membrane of these processes has a total thickness of around 160 Å and consists of five layers. Small granules spaced at regular intervals are attached to the cytoplasmic aspect of the inner layer by short stalks. In the 2 mm and 7 mm follicles the plasma membrane of the ovum facing the specialised terminal membranes has a striated appearance and shows a regular arrangement of granules attached by stalks to both its inner and outer aspects. The terminal and striated membranes are separated by an interval although there are areas of closer contact. In the 15 mm and 35 mm pre-ovulatory follicles the plasma membrane round the whole surface of the ovum is now typical striated membrane with bristles and attached granules. No explanation can be given at present of the function of the terminal membranes of the granulosa processes. They may indicate some change in the permeability permitting the intercellular diffusion of particles. It is suggested that the striated ovum plasma membrane is associated with the adsorption and transport of substances into the ovum for yolk synthesis.     

Synaptic junction development in the spinal cord of an amphibian embryo: An electron microscope study

Summary An electron microscopical study has been made of the cervical spinal cord of Xenopus laevis embryos, from the time that the neural tube closes until the larvae were hatched and could swim. Sections of the whole cord were searched for intercellular junctions during this period. Two nonsynaptic types were found, the first were widely distributed puncta adherentia, the second were rare and similar to gap junctions. Membrane specializations with synaptic vesicles were first found when the neural folds had fused; membrane-vesicle clusters which looked like the presynaptic half of a synaptic junction were present, together with synaptic junctions lacking any postsynaptic membrane thickening or cytoplasm density. About four hours later, mature synaptic junctions with full thickening of the postsynaptic membrane, dense cytoplasm and striated or dense material in the synaptic cleft were present. Presynaptic mitochondria, dense-cored and flattened vesicles, fibre to fibre and fibre to cell body synapses were present from the first, as were synapses onto very fine dendrites which might be filopodia from dendritic growth cones. Synaptogenesis may start with the accumulation of vesicles in dense cytoplasm near a thickened cell membrane; the postsynaptic element becomes associated with this membrane-vesicle cluster and matures by increasing cleft and cytoplasmic density, and by membrane thickening.     

Asymmetric diffusion into the postsynaptic neuron: An extremely efficient mechanism for removing excess GABA from synaptic clefts on the Deiters' neurone plasma membrane

Microdissected Deiters' neuron plasma membranes have been used for studying the passage of GABA through the membrane both in the inward and outward direction. Working with 0.2 mM GABA in the compartment simulating the outside of the neurone and with 2.0 mM GABA in the one simulating the inside we found a net transport of GABA towards the inside. This mechanism does not require a Na⁺ ion gradient across the membrane. The nature of the transport process involved was studied by determining the rate of [³H]-GABA inward passage as a function of GABA concentration (1 nM–800 M) on the outward side of the membrane. The results have shown that until 50 M a diffusion process (v=D₁×C, where D₁=3.1×10^–11 1/m²×sec) is the sole mechanism involved. Above 50 M a second diffusion process is activated v=D₂×(C–50×10^–6), where D₂=2.8×10^–11 1/m²×sec. Taking in account both inward and outward directed diffusion, one can calculate 16 M as the equilibrium concentration of GABA on the outward side of the membrane. From a kinetic point of view, these diffusion processes are able to reduce GABA concentration in a synaptic cleft from 3 mM to 20 M within 3 sec. These diffusion systems are discussed as extremely efficient in removing the excess of released GABA in the synaptic cleft.     

Ultrastructure of cell wall and plugs of tobacco pollen tubes after chemical extraction of polysaccharides

Tobacco pollen tubes grown in vitro and from pollinated tobacco styles were treated by chemical solvents to remove one or more of the following polysaccharides from the tube walls: pectin (ethylenediamine tetraacetic acid); hemicellulose (alkali); callose (alkali; potassium hypochlorite); cellulose (cuprammonium); and all polysaccharides with exception of cellulose (H₂O₂/glacial acetic acid). Both the inner tube wall, which we had regarded as the secondary wall, and the plugs contained, in addition to callose, microfibrils of cellulose and non-cellulosic microfibrils that had pectin-like properties. When using the expressions callosic or callose layer and callose plugs in reference to pollen tubes, one should realize that they do not imply the exclusive presence of callose in the inner tube wall layer and its localized thickenings.Extended version of a contribution (poster) presented at the International Symposium Advances in Plant Cytoembryology in Lublin, Poland, in June 1980 Dedicated to Professor J. Straub (Köln-Vogelsang) on his 70th birthday in 1981