The membrane systems of the cardiac muscle cell of Cirolana borealis Lilljeborg (Crustacea,Isopoda)

The membrane systems of the cardiac muscle cell of the isopod Cirolana borealis Lilljeborg are described. The sarcolemma invaginates at the level of the Z band, forming transverse tubules. Narrow tubules branch off in a longitudinal direction from these transverse and radially arranged Tz-tubules forming a transverse collar at each A-I level, where dyadic and triadic junctions are formed with the sarcoplasmic reticulum. Two different orientations of the coupling discs have been detected in the supercontracted sarcomere, and this observation has been discussed. Adjacent myofibrils are separated by a double layer of sarcoplasmic reticulum.     

STRUCTURE AND DEVELOPMENT OF THE CHLOROPLAST IN CHLAMYDOMONAS : I. THE NORMAL GREEN CELL

The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO₄ fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.     

Removal of insect basal laminae using elastase

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginai discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginai discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginai discs, mechanical stirring in Ringer low in Ca²⁺ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.     

UV Micrographs and Photomicrographs from the Palynological Laboratory,Stockholm-Solna

In Equisetum both the spore and the rhizoidal and first-formed prothallial cells contain sac-like cytoplasmic particles limited by a unit membrane. After KMnO₄ fixation these bodies resemble the spherosomes described from earlier studies (e.g. Frey-Wyssling et al., 1964). They are bounded by a unit membrane, have a diameter of 0.8–1.7 μ and a fine granular content. After double fixation with buffered glutaraldehyde combined with an osmium post-fixative, or triple fixation with buffered formaldehyde added, the bodies resemble microbodies in fine structure. They have a thin unit membrane, a more coarsely granular matrix than after KMnO₄ fixation and very often a core like a 0.5 μ cluster of tubules or discs of around 200 Å in diameter. When spores are fixed in glutaraldehyde the bodies often show cytoplasmic invaginations or inclusions, which is never the case when they are fixed with KMnO₄     

Structure and development of the chloroplast in Chlamydomonas. I. The normal green cell

The cytoplasmic organization of a normal green strain of the alga Chlamydomonas reinhardi has been investigated with the electron microscope using thin sections of OsO(4) fixed material. The detailed organization of the chloroplast has been of special interest. The chloroplast, a cup-shaped organelle, surrounded by a double membrane, consists of: (1) discs about 1 micron in diameter, considered to represent the basic structural unit of the chloroplast, and each composed of a pair of membranes joined at their ends to form a flat closed vesicle; the discs are grouped into stacks resembling the grana of higher plants; (2) matrix material of low density in which the discs are embedded; (3) starch grains; (4) the pyrenoid, a non-lamellar region associated with starch synthesis, and containing tubules which connect with the lamellae; (5) the eyespot, a differentiated region containing two or three plates of hexagonally packed, carotenoid-containing granules, located between discs, and associated with phototaxis. In addition to the chloroplast, the cytoplasm contains various membranous and granular components, including mitochondria, endoplasmic reticulum, and dictyosomes, identified on the basis of morphological comparability with structures seen in animal cells. The nucleus, not investigated in detail in this study, contains a large, granular nucleolus and is surrounded by a nuclear envelope which is provided with pores and exhibits instances of continuity with the endoplasmic reticulum of the cytoplasm.     

MEMBRANES OF MESOPHYLL CELLS OF NICOTIANA RUSTICA AND PHASEOLUS VULGARIS WITH PARTICULAR REFERENCE TO THE CHLOROPLAST

Weier , T. E., and W. W. Thomson . (U. California, Davis.) Membranes of mesophyll cells of Nicotiana rustica and Phaseolus vulgaris with particular reference to the chloroplast. Amer. Jour. Bot. 49(8): 807–820. Illus. 1962.—The endoplasmic reticulum in mesophyll cells is represented by short lengths of irregularly disposed, paired membranes. It is occasionally associated with a typically double nuclear envelope. Groups of irregularly parallel, paired membranes suggesting disorganized dictyosomes occur infrequently. Mitochondria are unevenly distributed in mesophyll; they are large and have sparse tubular cristae around their periphery. In the great majority of instances the bounding membrane is diffusely stained with KMnO₄. When it is sharp and distinct, it may be double as usually pictured, or it may have well-delineated stretches of a single membrane bounding 25–50% of its circumference. The tonoplast and ectoplast are very fragile, the former appearing as a single dark line. In young leaves the ectoplast is visualized as a continuous single membrane adjacent to the cell wall, but in our micrographs of mature leaves it is always discontinuous. The plastid membrane sometimes is distinctly double, having 2 dark components bounding a light component. In the great majority of cases, however, this membrane is either a solid dark line, or the clear component of the double membrane is crossed by delicate dark lines giving the membrane a braided, or scalariform appearance. The various appearances of the membrane may intergrade with each other. The width of the plastid membrane is variable, ranging from 200 to 400 A. The inner component may invaginate into the stroma, and bodies may form in the clear space between the 2 outer membrane components. Micrographs suggest that these bodies, and others formed by small masses of stroma, may be expelled into the hyaloplasm, where they exist as spherical single-membraned particulates. The reality of the variable structure of the plastid membrane is discussed in light of concepts of membrane activity, molecular structure, and the relation of these factors to possible artifacts.     

THE STRIATED MUSCULATURE OF BLOOD VESSELS : II. Cell Interconnections and Cell Surface

The interconnections and the surfaces of the striated muscle cells which occur in thoracic and in lung veins of the mouse were studied with the electron microscope. The osmium-fixed tissues were embedded in methacrylate or in araldite and sectioned with a Porter-Blum microtome. Many preparations were stained before embedding with phosphotungstic acid or after sectioning with uranyl acetate. Typical intercalated discs are observed in this muscle. They are similar to the discs found in heart muscle. These intercalated discs represent boundaries between separate muscle cells. Along the discs, cells are joined in planes normal to their myofilaments. The same cells are also joined in planes parallel to the myofilaments by means of lateral interconnections. These lateral cell boundaries are in continuity with the intercalated discs. Three morphologically distinct parts occur within the lateral cell interconnections: One is characterized by small vesicles along the plasma membrane, the second part has the structure of desmosomes, and a third part represents an external compound membrane (formed by the two plasma membranes of the adjoining cells) and is termed "quintuple-layered cell interconnection." Small vesicles and plasma membrane enfoldings along the free surface of muscle cells are interpreted as products of a pinocytosis (phagocytosis) process. Some of them are seen to contain small membrane-bounded bodies or granules. The free cell surface shows a characteristic outer dense layer ("basement membrane") which accompanies the plasma membrane. The topographic relation of this dense layer with the plasma membrane seems to vary in different preparations. The significance of this variation is not well understood. On two occasions a typical arrangement o vesicles and tubules was observed at Z band levels, just beneath the plasma membrane. These structures are believed to represent endoplasmic reticulum. Their possible significance for the conduction of excitation is discussed.     

OBSERVATIONS ON SPERMIOGENESIS IN THE FUNGUS GNAT SCIARA COPROPHILA

Although 9-membered centrioles are found in somatic tissues of Sciara, the centriole which lies at the spindle pole of the second meiotic division in male Sciara is composed of a row of approximately 70 short tubules in an oval array. Shortly after telophase of this unequal division, in the daughter cell destined to undergo spermiogenesis, microtubules become confluent with the tubules of the centriole. These tubules have the same density as other cytoplasmic microtubules after glutaraldehyde-OsO₄ fixation and, like them, are not preserved with OsO₄ fixation. As the centriole, now with approximately 70 attached, posteriorly directed, doublet tubules, migrates from the polar to the apolar end of the nucleus to take a final position in an oval groove which forms in the nuclear envelope, the tubules lengthen and become demonstrable after OsO₄ fixation and more electron opaque than other cytoplasmic microtubules following glutaraldehyde-OsO₄ fixation. Later, a singlet tubule appears peripherally to each doublet of the oval and 4 "arms" develop at specific sites on the tubules. Posteriorly, where the oval of tubules becomes discontinuous and forms a spiral, the arrangement of arms is different and the singlet tubules are lacking. Dense solid bodies develop inside this odd flagellum and become enclosed by a smooth double membrane. A single mitochondrial derivative has three components: a central area of homogeneous, moderately electron-opaque, proteinaceous material; a peripheral ring of cristae; and a crystalloid which is specifically oriented with respect to the flagellar tubules.     

Structures located at the levels of the Z bands in mouse ventricular myocardial cells

Within ventricular myocardial cells of the mouse, the myoplasmic regions located immediately adjacent to the Z lines of the sarcomeres contain a variety of structures. These include: (1) transversely oriented 10 nm (‘intermediate’) filaments that apparently contribute to the cytoskeleton of the myocardial cell; (2) the majority of the transverse elements of the T-axial tubular system; (3) specialized segments of the sarcoplasmic reticulum (SR) that are closely apposed to the sarcolemma or T-axial tubules (junctional SR); (4) ‘extended junctional SR’ (‘corbular SR’) that exists free of association with the cell membrane; (5) ‘Z tubules’ of SR that are intimately apposed to the Z line substance; and (6) leptofibrils. In addition, fasciae adherentes supplant Z lines where myofibrils insert into the transverse borders (intercalated discs) of the cells. The concentration of these myocardial components at the level of the Z lines suggests that a particular specialization of structural and physiological activities exists in the Z-level regions of the myoplasm. In particular, it appears that the combination of intermediate filaments, T tubules, and Z-level SR elements forms a series of parallel planar bodies that extend across each myocardial cell to impart transverse rigidity. The movement and compartmentation of calcium ion (Ca²⁺) would seem especially active near the Z lines of the myofibrils, in view of the preferential location there of Ca²⁺-sequestering myocardial structures such as T tubules, junctional SR, extended junctional SR and Z tubules.     

The membrane systems of the cardiac muscle cell of Tmetonyx cicada O. Fabricius (Crustacea,Amphipoda)

Summary The membrane systems of the cardiac muscle cell of the amphipod Tmetonyx cicada (O. Fabricius) are described. The sarcolemma invaginates and forms a transverse network of tubules at the level of the Z band. Narrow longitudinal tubules branch from the network and connect to another transverse network of tubules at the H band level, where dyadic and triadic junctions are formed with the sarcoplasmic reticulum. Adjacent myofibrils are normally separated by a well developed double layer of the sarcoplasmic reticulum. In areas where the myofibrils closely approach the outer sarcolemma, peripheral couplings have been found at the level of the H band.     

Gap junction distribution in the Drosophila wing disc mutants vg,l(2)gd,l(3)c43hs1, and l(2)gl4

The density of gap junctions in four Drosophila melanogaster mutants with abnormal wing disc development has been determined using quantitative electron microscopy and compared with the gap junction density in wild-type wing discs. No appreciable differences relative to wild-type controls were found in the cell death mutant vestigial or in the mildly hyperplastic mutant lethal giant disc which could not be accounted for in terms of altered lateral plasma membrane surface density or as an extension of the gap junction growth which normally occurs during the third larval stage of development in wild-type wing discs. However, both the severely hyperplastic mutant l₍₃₎c43^hs1 and the neoplastic mutant lethal giant larva have significant reductions in the gap junction surface density, the number of gap junctions, and the gap junction areal fraction of the lateral plasma membrane compared with wild-type controls. These differences cannot be attributed to altered lateral plasma membrane surface densities which are not significantly different from wild-type control wing discs. The reduced gap junction density in severely hyperplastic and neoplastic wing discs suggests that alterations in the number or distribution of gap junctions may be as disruptive to normal growth and development as their complete absence.     

Ultrastructure of Trypanosoma conorhini in the Crithidial Phase*

SYNOPSIS. The ultrastructure of the crithidial phase of T. conorhini culture has been studied. The structure of the plasma membrane is not easy to make out; only at a few points in highly magnified picit is seen as a double osmiophilic membrane with an intermedilayer of low density. Sub-pellicular tubules are present also around the flagellar pocket. The nucleus usually has a single large nucleolus, but sometimes this may be double. The flagellum has the sual 2 central and 9 double peripheral fibers, the latter having lateral arms. The structure of the kinetoplast is similar to that of other rypanosomatids but the division of the organelle seems to be more complex than has been described: the central, DNA-bearing lamellae duplicate, forming a double transverse band; 1 of these bands probably migrates toward the side before invagination of the membrane completes the division. Mitochondria are long tubular structures with few cristae, disposed chiefly along the periphery of the cell. A communication between the kinetoplast and tubular mitochondria is very frequent. The endoplasmic reticulum is poorly developed, represented chiefly by smooth membranes surrounding vesicles, rough-surfaced membranes being scanty in most cells. Several inclusions may be found, some probably lipids, others being of unknown nature.     

A Reanalysis of the Two-Component Phloem Loading System in Beta vulgaris

Kinetic analysis of [¹⁴C]sucrose loading into sugar beet leaf discs revealed the presence of two transport components. At low exogenous sucrose concentrations, a saturable component, which exhibited Michaelis-Menten characteristics, was the main mode of transport. At concentrations greater than 50 millimolar, phloem loading was dominated by a linear component which appeared to operate as a first order kinetic transport process. Over the exogenous sucrose concentrations employed, influx could be described by the equation v = V_maxS/(S + K_m) + kS. Influx via both processes was strongly pH-dependent. Evidence is presented that the linear component was not explicable in terms of simple diffusion, or exchange diffusion, into either mesophyll or minor vein phloem tissue. Extensive metabolic conversion of sucrose was not a factor contributing to influx at high external sucrose concentrations. At present, it is believed that both components operate in parallel at the membrane bounding the sieve element-companion cell complex. The saturable component is identified with sucrose-H⁺ cotransport. While the significance of the linear component has been established, its nature remains to be elucidated.     

ULTRASTRUCTURAL CHANGES IN THE MITOTIC APPARATUS AT THE METAPHASE-TO-ANAPHASE TRANSITION

As the metaphase HeLa cell approaches anaphase, pericentriolar spindle tubules fragment and become encapsulated by a unit membrane. By early anaphase, the encapsulated forms appear to have expanded, giving rise to polar spherical aggregates. Some of these elements show ribosomes on their bounding membrane, and some of them localize on the condensed chromatin during reformation of the nuclear membrane. It thus is suggested that these elements are newly derived cisternae of the endoplasmic reticulum (ER). Similar transformations are seen in later anaphase in the interzonal region, and it may be that the ER serves as a storage depot for some fraction of depolymerized microtubules. The time and location of the pericentriolar transitions are consistent with their being intimately involved in the mechanics of chromosome separation.     

THE OCCURRENCE OF A SUBUNIT PATTERN IN THE UNIT MEMBRANES OF CLUB ENDINGS IN MAUTHNER CELL SYNAPSES IN GOLDFISH BRAINS

Observations additional to those previously reported (34) on boutons terminaux and club endings on Mauthner cell lateral dendrites, primarily as seen in sections of permanganate-fixed material, are described. Certain new findings on OSO₄-fixed endings are also included. The boutons terminaux are closely packed in the synaptic bed with ~ 100 to 150 A gaps between their contiguous unit membranes and a few interspersed glial extensions. Their synaptic membrane complexes (SMC) appear as pairs of unit membranes separated by ~ 100 to 150A clefts. They contain many vesicles and unoriented mitochondria, but no neurofilaments. The club endings after KMnO₄ fixation are, as after OSO₄ fixation (34), again seen surrounded by a layer of extracellular matrix material. These endings contain relatively few synaptic vesicles, a few unit membrane limited tubules ~ 300 A in diameter, and mitochondria oriented perpendicular to the SMC. Neurotubules and neurofilaments are not clearly seen. These components are also virtually absent in the Mauthner cytoplasm. No ribosomes are seen in the KMnO₄-fixed material. The unit membranes of the SMC of club endings show up clearly in essentially the same junctional relations described after formalin-OSO₄ fixation (34). In addition, the synaptic discs in transverse section show a central beading repeating at a period of ~ A associated with scalloping of the cytoplasmic surfaces. In oblique views, dense lines are seen repeating at a period of ~ 90 A. In frontal views a hexagonal array of close-packed polygonal facets is seen. These repeat at a period of ~ 95 A. Each has a central dense spot <25 A in diameter. Similar subunits are seen in the unit membranes of synaptic vesicles.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

Associations between microbodies and a system of cytoplasmic tubules in oil-body cells of Marchantia

Summary Preliminary observations on differentiating oil-body cells of Marchantia sp. revealed that the cytoplasm possesses conventional cytoplasmic microtubules as well as a greater number of other tubules, which average 35 nm in diameter and appear in cytoplasmic regions rich in endoplasmic reticulum. These tubules, at a stage of active synthesis of oil, increase in number, may form well-organized bundles traversing the cytoplasm close to the vacuole, and show a preferential spatial relationship to elongated microbodies. One layer of densely arrayed cytoplasmic tubules surrounds the microbodies partially or totally. Fine links bridge the tubules to one another or some of them to the microbody bounding membrane.     

Sodium pump molecular activity and membrane lipid composition in two disparate ectotherms,and comparison with endotherms

Previous research has shown that the lower sodium pump molecular activity observed in tissues of ectotherms compared to endotherms, is largely related to the lower levels of polyunsaturates and higher levels of monounsaturates found in the cell membranes of ectotherms. Marine-based ectotherms, however, have very polyunsaturated membranes, and in the current study, we measured molecular activity and membrane lipid composition in tissues of two disparate ectothermic species, the octopus (Octopus vulgaris) and the bearded dragon lizard (Pogona vitticeps), to determine whether the high level of membrane polyunsaturation generally observed in marine-based ectotherms is associated with an increased sodium pump molecular activity relative to other ectotherms. Phospholipids from all tissues of the octopus were highly polyunsaturated and contained high concentrations of the omega-3 polyunsaturate, docosahexaenoic acid (22:6 (n–3)). In contrast, phospholipids from bearded dragon tissues contained higher proportions of monounsaturates and lower proportions of polyunsaturates. Sodium pump molecular activity was only moderately elevated in tissues of the octopus compared to the bearded dragon, despite the much greater level of polyunsaturation in octopus membranes. When the current data were combined with data for the ectothermic cane toad, a significant (P=0.003) correlation was observed between sodium pump molecular activity and the content of 22:6 (n–3) in the surrounding membrane. These results are discussed in relation to recent work which shows a similar relationship in endotherms.     

Endoplasmic reticulum derived decorated tubules in the sieve elements ofNymphaea

Summary Bundles of decorated tubules found in the sieve elements ofNymphaea have been studied with the transmission electron microscope. Comparatively straight tubules (100 nm in diameter) arise from the endoplasmic reticulum during early stages of sieveelement development and subsequently associate into bundles of up to 100 tubules that parallel the longitudinal cell axis. From the start of their formation the tubules are structurally distinct from other ER profiles due to their dense decoration with particles. High magnifications reveal an orderly array of the particles (about 24 surround a 100 nm tubule) and suggest a modification of their membrane so that it is no longer dissolvable into a regular three-layered structure. Later during sieve-element ontogeny the decorated tubules get invaginated by smooth ER membranes, thereby squeezing out the intratubular (extracytoplasmic) space. As a result a double mantle is formed that surrounds a plasmatic cylinder. Decorated 100 nm tubules with inner membranes are present in enucleate mature sieve elements ofNymphaea alba andN. tuberosa. Considerably larger tubules (about 200 nm in diameter) were found inN. Candida andN. tetragona and occasionally also inNuphar and Barclaya, two other genera from the same family. The decoration of the tubules and their subsequent invagination by smooth membranes are discussed with respect to the controlled autolysis of sieve elements.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Disc formation in cholesterol-free liposomes during phase transition

Cryogenic transmission electron microscopy (cryo-TEM) images of lysolipid-containing thermosensitive liposomes (LTSL) revealed that open liposomes and bilayer discs appeared when liposomes were cycled through the gel (L_β′) to liquid-crystalline (L_α) phase transition. The amount of bilayer discs generated was dependent on the combined presence of PEG-lipid and lysolipid in the membrane. We hypothesize that micelle-forming membrane components stabilize the rim of bilayer openings and membrane discs that form when liposomes are cycled through T_C.