THE ISOLATION OF A MAJOR STRUCTURAL ELEMENT OF THE SEA URCHIN FERTILIZATION MEMBRANE

Procedures for isolating the contents of the cortical granules from the ova of the sea urchin, Strongylocentrotus purpuratus, are reported. Dithiothreitol is used to remove the vitelline coat; the "demembranated" eggs are then subsequently activated with butyric acid. By means of these procedures, the hyaline protein and crystalline or paracrystalline material have been isolated from the cortical granules. The crystalline material consists of sheets of cylinders or tubules 150–200 A in diameter. This material is believed to be a major structural element of the fertilization membrane which, in the absence of the vitelline coat, does not form.     

AN UNUSUAL CONFIGURATION OF THE GOLGI COMPLEX IN PIGMENT-PRODUCING "TEST" CELLS OF THE OVARY OF THE TUNICATE,STYELA

The test cell in the ovary of the tunicate Styela contains a large and robust Golgi complex which demonstrates a regional structural differentiation. In one of the regions, branching of the lamellae occurs resulting in a honeycomb or lattice-type arrangement. Small, dense granules or homogeneous material of moderate density may be present within certain of the Golgi cisternae. The close association, or continuity in some cases, between elements of the Golgi complex and immature forms of pigment suggests that the Golgi complex in these cells is involved in pigment formation. These relationships are shown and discussed in terms of possible functional significance.     

THE ULTRASTRUCTURE OF THE VITELLINE BODY IN THE OOCYTE OF THE SPIDER TEGENARIA PARIETINA

The vitelline body in the mature oocyte of the spider Tegenaria parietina is composed of 4 different zones. 1. The central zone contains granular areas, vesicles, and a few lamellae. 2. The lamellar zone consists of numerous concentric lamellae. These sheets, 45 A in thickness, are stacked in groups. The fine structure and the regular arrangement recall those of myelin sheets, retinal rods, and chloroplasts. Between the stacks of lamellae, finely granular masses and various vesicles are to be found. 3. The "zone of transition" consists of a finely granular substance accumulated in abundant masses. This substance is composed of very closely packed granules about 50 to 60 A in diameter. Very often, near the lamellae, the granules show alignment giving a gradual transition from grains to lamellae. 4. The vesicular zone contains ergastoplasm, dense particles, mitochondria, and Golgi material. It is suggested that the peculiar ultrastructure of these cytoplasmic components may be related to an intense metabolic activity.     

COMPARATIVE CYTOPHYSIOLOGY OF STRIATED MUSCLE WITH SPECIAL REFERENCE TO THE ROLE OF THE ENDOPLASMIC RETICULUM

1. The structure and distribution of the components of striated muscle cells vary with the species and with the specialization of muscle fiber function. 2. There appear to be two, easily distinguishable, general categories of striated muscle structure. A. High frequency muscle (represented by flight muscle of higher insects and hummingbird, and cicada tympanal muscle) is characterized by widely spaced, non-branching fibrils of large diameter and short period, little endoplasmic reticulum, and large quantities of large mitochondria (low fibril-sarcoplasm ratio). This structure is correlated with heavy tracheolization or vascularization, high oxidative activity, and dark color as compared with other muscles of the same species. B. Low frequency muscle is characterized, in general, by high fibril-sarcoplasm ratio, relatively long period, few mitochondria increasing with activity and decreasing with absolute power of the fiber. Oxidative capacity and color are proportional to the quantity of mitochondria. These fibers are further differentiated into (a) fibrillar arrangement of contractile material which permits a regular pattern of interfibrillar and segmental reticulum, and (b) afibrillar arrangement of contractile material leading to an unsystematic distribution of reticulum. 3. The endoplasmic reticulum appears as a complex coordination system in the muscle fiber. Peripherally, it links the Z and M lines of the fibrils to the sarcolemma and between the fibrils it links the cross-bands, forming the Grundmembran of earlier authors. By longitudinal linkage, it connects with the sarcolemma at the muscle extremity to form a digital arrangement into which the tendon fibrils are spliced. The extent of its development and its position have a definite relationship to the degree and site of fiber shortening. At present the reticulum is the only structure that one can consider to be an internal conducting system. It may distribute the excitation transversely from fibril to fibril, and lengthwise saltatorially to the symmetry centers of the sarcomeres. 4. The nucleus is the mediating element between the cytoplasmic phases within and without the tubular system of the endoplasmic reticulum. A possible mechanism which correlates nucleus, adenylic acid system, ion exchange, and reticulum with the initiation of contraction is postulated.     

THE FORMATION OF BASAL BODIES (CENTRIOLES) IN THE RHESUS MONKEY OVIDUCT

Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mµ long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.     

THE ULTRASTRUCTURE OF THE KINOCILIUM OF THE SENSORY CELLS IN THE INNER EAR AND LATERAL LINE ORGANS

The bundle of sensory hairs protruding from the top of each receptor cell in the vestibular and lateral line organs in the teleost fish (burbot) Lota vulgaris is composed of a number of stereocilia and one kinocilium located in the periphery of the bundle. The ultrastructure of the kinocilium and its basal body is described. It is found that the kinocilium is morphologically polarized by the asymmetric arrangement of its component fibers and of the basal body by the presence of a basal foot. Peripheral fibers 5 and 6 of the kinocilium and the basal foot of the basal body are oriented away from the stereocilia; that is, in a direction coinciding with the direction of excitatory stimulation. The findings are discussed in terms of directional sensitivity.     

THE DOUBLE REFRACTION OF CHITIN

The double refraction of the chitinous hair of the crayfish is positive with respect to the axis of the hair, and is largely caused by the arrangement of submicroscopic, elongated chitin particles parallel to this axis (form birefringence). Using a series of relatively unreactive liquids and fluid mixtures which permeate the chitin framework, the type of curve relating double refraction and refractive index of the imbibed fluid is found to depend greatly on the chemical nature of the fluid. Either a positive or a negative residual birefringence may be found, depending on the choice of imbibing liquid. Separation of form and crystalline elements in double refraction by means of Ambronn''s imbibition technique is therefore unsafe in a system like chitin, where some type of oriented association of the imbibed molecules with the chitin framework is prevalent.     

Ribosome arrangement during oogenesis of Lacerta sicula Raf

Ribosomal bodies (RB), constituted of crystalline sheets of ribosomes interposed between flat parallel cisternae, are present in the oocyte and follicular cells of Lacerta sicula during the winter rest. During spring, the RB undergo progressive transformation into rough endoplasmic reticulum and free ribosomes, so that these formations are completely lacking during summer. The mechanism underlying the formation of RB and the possible significance that they may have for protein synthesis during oogenesis are discussed.     

THE EFFECT OF TEMPERATURE ON THE MECHANICAL ACTIVITY OF THE GILLS OF THE OYSTER (OSTREA VIRGINICA Gm.)

1. The method is described whereby the rate of flow produced by the gills of the oyster can be measured accurately. 2. The rate of doing work in maintaining a constant current along the glass tube can be expressed by the formula W = 2πlµ S ², where W = ergs/sec., l = length of the tube, µ = viscosity in poises, and S = speed at the axis of the tube. 3. The relationship between the rate of doing work and the temperature cannot be described by the equation of Arrhenius. 4. The optimum temperature for the mechanical activity of the gills lies between 25° and 30°C. Below 5° no current is produced, though the cilia are beating. Ciliary motion stops entirely at the freezing temperature of sea water. 5. The factors responsible for the production of current are discussed. The study of the relations between the variability of the rate of flow and the temperature shows that between 15° and 25°C. the absolute variability remains constant and increases considerably above 25° and below 15°. The rôle of the coordination in the production of current is discussed, and the conclusion is reached that coordination is affected by the changes in temperature.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

FINE STRUCTURE OF THE PINEAL ORGANS OF THE ADULT FROG, RANA PIPIENS

Frontal organs and epiphyses of the pineal system from the adult frog, Rana pipiens, were fixed in s-collidine-buffered osmium tetroxide, embedded in Epon 812, and examined by electron microscopy. Epiphyseal material was also fixed in a variety of ways and subjected to a series of cytochemical tests for light microscopy. An ultrastructure resembling that of lateral eye retina is confirmed in this species. Photoreceptor cells of the epiphysis and frontal organ display many cytological features similar to those of retinal rods and cones in the arrangement of their outer and inner segments and synaptic components. However, in these pineal organs the outer segments are disoriented relative to each other and may display a disarranged internal organization unlike normal retinal photoreceptors. Furthermore, other pineal outer segments often appear degenerate. Since immature stages in the development of new outer segments also appear to be present, adult pineal photoreceptors are probably engaged in a constant renewal of outer segment membranes. The evidence further suggests that macrophages are involved in phagocytosis of degenerated outer segments. Postulated photoreceptor activities and the possibility of secondary pineal functions, such as secretion, are discussed in view of current morphological and cytochemical findings.     

THE CHEMICAL COMPOSITION OF THE CELL WALL OF CHLAMYDOMONAS GYMNOGAMA AND THE CONCEPT OF A PLANT CELL WALL PROTEIN

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.     

Membranous cytochrome c oxidase. A freeze-fracture electron microscopic analysis

Suspensions of membranous cytochrome c oxidase prepared from beef heart mitochondria by Triton extraction were ultra-rapidly cooled (in excess of 10,000 deg.C/s) and analyzed using freeze-fracture and freeze-fracture-etch techniques. The preparations contained non-crystalline and crystalline vesicles as isolated vesicles, vesicles inside other vesicles and stacks of vesicles. In non-crystalline vesicles the particles (about 100 Å diameter) are probably formed by the deviation of hydrophobic fracture planes of the membranes around the large transmembrane enzymes. The intramembrane particles thus formed are compared to particles (about 80 Å diameter) in a vesicle reconstituted from purified enzyme and lipid. Crystalline membranous cytochrome c oxidase vesicles display an unusual fracture pattern in which adjacent crystalline surfaces are separated from each other and from the surrounding ice by fracture steps that are approximately the thickness of a single membrane (100 to 120 Å). In addition adjacent crystalline fracture surfaces have similar low-relief textures, both of which differ significantly from the hydrophobic surfaces normally exposed in membrane fractures. This fracture morphology is interpreted in terms of fractures along hydrophilic surfaces of the membranes. Images of etched crystalline vesicles provide support for this interpretation because etching exposes no new surfaces. It is concluded that the crystalline lattices are derived from the portions of enzymes that protrude from the membrane bilayers and that the interdigitation of the enzymes on the inside surfaces of the vesicles or between vesicles determines the appearance of the crystalline surfaces. The arrangement of the tails of the y-shaped molecules on the cytoplasmic sides of the crystalline membranes can be visualized in micrographs directly and in reconstructions of filtered images. The more complex pattern of arms protruding on the matrix side is obscured by the unidirectional shadowing. Fragmentation of the crystalline membranes during fracturing is indicated by particles sometimes present at the edges of fractured membranes and by deep, irregular pits observed in crystalline surfaces. Particles resting on some crystalline surfaces may be fragments of crystalline membranes removed during fracturing. In other crystalline membranes non-protein is removed during fracturing, leaving globular particles embedded in the lattice, which measure about 118 Å diameter. Comparing these particles to the 3-dimensional arrangement of protein described in the accompanying paper (Frey et al., 1982) suggests that such particles are composed of 2 dimers paired along the a-axis. Intramembrane and fragmentation particles of similar size may also have this protein composition.     

DEVELOPMENT OF EYE COLORS IN DROSOPHILA: SOME PROPERTIES OF THE HORMONES CONCERNED

The substance inducing the production of pigment in the eyes of vermilion brown mutants of Drosophila melanogaster has been shown to be a relatively stable chemical entity possessing true hormone-like activity. A simple method for obtaining hormone solutions has been developed involving extraction of dried wild type Drosophila pupae with ethyl alcohol and water. A logarithmic proportionality has been found to exist between the amount of hormone and the induced eye color. This relationship provides a simple method for the quantitative determination of hormone concentration in given extracts. Larvae and pupae of D. melanogaster contain an intracellular enzyme which inactivates the hormone in the presence of molecular oxygen. The hormone is not oxidized under ordinary conditions with either molecular oxygen or hydrogen peroxide. The hormone has been found to be an amphoteric compound with both acidic and basic groups and with a molecular weight between 400 and 600. The solubility and precipitation reactions of the hormone suggest its amino acid-like nature. However, the instability to heat, acid, and alkali, and its rather restricted occurrence indicate a rather complex specific structure.     

THE FINE STRUCTURE AND DEVELOPMENT OF THE COMPANION CELL OF THE PHLOEM OF ACER PSEUDOPLATANUS

At maturity the companion cell of the phloem of the sycamore Acer pseudoplatanus has a large nucleus, simple plastids closely sheathed with rough endoplasmic reticulum, and numerous mitochondria. The cytoplasm contains numerous ribosomes, resulting in a very electron-opaque cytoplasm after permanganate fixation. Bodies similar to the spherosomes of Frey-Wyssling et al. (4) are collected in clusters and these also contain bodies of an unidentified nature similar to those found by Buttrose (1) in the aleurone cells of the wheat grain. The pores through the wall between the companion cell and sieve tube are complex and develop from a single plasmodesma. Eight to fifteen plasmodesmata on the companion cell side communicate individually with a cavity in the centre of the wall which is linked to the sieve tube by a single pore about twice the diameter of an individual plasmodesma. This pore is lined with material of an electron opacity equivalent to that of material bounding the sieve plate pores. The development of the cell organelles, the possible role played in the phloem tissue by the companion cell, and the function of the complex pores contained in its wall are discussed.     

THE STRUCTURE AND FORMATION OF CILIA AND FILAMENTS IN RUMEN PROTOZOA

The large oligotrich rumen protozoa Diplodinium ecaudatum and Ophryoscolex caudatus have been studied by electron microscopy during interphase and division. The structure of mature cilia is contrasted with that seen during their formation particularly in a tuft where development lags and is arrested. Here the shaft is only a few micra long and is composed of filaments that have circular cross-sections not in the typical circular arrangement. In their diameter and appearance the filaments are similar to filaments associated with the nuclei during division. The macronucleus has within it randomly directed filaments, while the micronucleus contains well aligned filaments and other arrangements typical of an intranuclear mitotic process. An extranuclear filament system is also present and is elaborated during division. The infraciliary filament system is particularly elaborate in these organisms. Filaments ranging from 14 to 22 mµ have been observed with some tendency for a bimodal distribution in diameters of 15 and 21 mµ. Formation of such filaments has been observed and consists of an initial orientation of very fine elements followed by filament formation. The observations are discussed in relation to filament involvements in cell movements. The concepts are discussed that filaments are metastable structures and that the transitions from one state to another are functionally significant.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

PHYSICAL AND ULTRASTRUCTURAL BASIS OF BLUE LEAF IRIDESCENCE IN TWO NEOTROPICAL FERNS

Iridescent blue leaf coloration in two neotropical ferns, Danaea nodosa (L.) Sm. (Marattiaceae) and Trichomanes elegans L. C. Rich. (Hymenophyllaceae), is caused by thin film constructive interference. The ultrastructural basis for the film in D. nodosa is multiple layers of cellulose microfibrils in the adaxial cell walls of the adaxial epidermis. The apparent helicoidal arrangement of the fibrils is analogous to similar color production in arthropods. In T. elegans the blue-green coloration is caused by the remarkably uniform thickness and arrangement of grana in specialized chloroplasts adjacent to the adaxial wall of the adaxial epidermis. The selective advantage of this color production, if any, is unknown but apparently different from that previously studied in Selaginella.     

THE DEGENERATION AND REAPPEARANCE OF MITOCHONDRIA IN THE EGG CELLS OF A PLANT

Details are given of the elimination of mitochondria which occurs during the first 1 to 2 hours of the life of the egg of the fern Pteridium aquilinum (L.) Kuhn. During this phase the only mitochondria present are swollen and appear degenerate, but subsequently the cytoplasm of the egg becomes filled with large mitochondria containing numerous villi. Accompanying the appearance of these mitochondria, many, if not all of which have a peculiar umbo-like form, is the production by the nucleus of conspicuous and complex evaginations. The umbo-mitochondria are believed to be new, and a mechanism is suggested by which they may be generated from the complex evaginations of the nucleus.     

A POSSIBLE MECHANISM FOR THE MORPHOGENESIS OF LAMELLAR SYSTEMS IN PLANT CELLS

A mechanism for the formation of lamellar systems in the plant cell has been proposed as a result of electron microscope observations of young and mature cells of Nitella cristata and the plastids of Zea mays in normal plants, developing plants, and certain mutant types. The results are compatible with the concept that lamellar structures arise by the fusion or coalescence of small vesicular elements, giving rise initially to closed double membrane Structures (cisternae). In the chloroplasts of Zea, the cisternae subsequently undergo structural transformations to give rise to a compound layer structure already described for the individual chloroplast lamellae. During normal development, the minute vesicles in the young chloroplast are aggregated into one or more dense granular bodies (prolamellar bodies) which often appear crystalline. Lamellae grow out from these bodies. In fully etiolated leaves lamellae are absent and the prolamellar bodies become quite large, presumably because of inhibition of the fusion step which appears to require chlorophyll. Lamellae develop rapidly on exposure of the plant to light, and subsequent development closely parallels that seen under normal conditions. The plastids of white and very pale green mutants of Zea similarly lack lamellae and contain only vesicular elements. A specialized peripheral zone immediately below the double limiting membrane in Zea chloroplasts appears to be responsible for the production of vesicles. These may be immediately converted to lamellae under normal conditions, but accumulate to form a prolamellar body if lamellar formation is prevented, as in the case of etiolation and chlorophyll-deficient mutation, or when the rate of lamellar formation is slower than that of the production of precursor material (as appears to be the case in the early stages of normal development).