Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.     

Injury-induced vesiculation and membrane redistribution in squid giant axon

Injury of isolated squid giant axons in sea water by cutting or stretching initiates the following unreported processes: (i) vesiculation in the subaxolemmal region extending along the axon several mm from the site of injury, followed by (ii) vesicular fusions that result in the formation of large vesicles (20-50 micron diameter), 'axosomes', and finally (iii) axosomal migration to and accumulation at the injury site. Some axosomes emerge from a cut end, attaining sizes up to 250 microns in diameter. Axosomes did not form after axonal injury unless divalent cations (Ca2+ or Mg2+) were present (10mM) in the external solution. The requirement for Ca2+ and the action of other ions are similar to that for cut-end cytoskeletal constriction in transected squid axons (Gallant, P.E. (1988) J. Neurosci. 8, 1479-1484) and for electrical sealing in transected axons of the cockroach (Yawo, H. and Kuno, M. (1985) J. Neurosci. 5, 1626-1632). Axosomes probably consist of membrane from different sources (e.g., axolemma, organelles and Schwann cells); however, localization of axosomal formation to the inner region of the axolemma and the formation dependence on divalent cations suggest principal involvement of cisternae of endoplasmic reticulum. Patch clamp of excised patches from axosomes liberated spontaneously from cut ends of transected axons showed a 12-pS K+ channel and gave indications of other channel types. Injury-induced vesiculation and membrane redistribution seem to be fundamental processes in the short-term (minutes to hours) that precede axonal degeneration or repair and regeneration. Axosomal formation provides a membrane preparation for the study of ion channels and other membrane processes from inaccessible organelles.     

Subaxolemmal cytoskeleton in squid giant axon. I. Biochemical analysis of microtubules, microfilaments, and their associated high-molecular- weight proteins

Using the squid giant axon, we analyzed biochemically the molecular organization of the axonal cytoskeleton underlying the axolemma (subaxolemmal cytoskeleton). The preparation enriched in the subaxolemmal cytoskeleton was obtained by squeezing out the central part of the axoplasm using a roller. The electrophoretic banding pattern of the subaxolemmal cytoskeleton was characterized by large amounts of two high-molecular-weight (HMW) proteins (260 and 255 kD). The alpha, beta-tubulin, actin, and some other proteins were also its major constituents. The 260-kD protein is known to play an important role in maintaining the excitability of the axolemma (Matsumoto, G., M. Ichikawa, A. Tasaki, H. Murofushi, and H. Sakai, 1983, J. Membr. Biol., 77:77-91) and was recently designated "axolinin" (Sakai, H., G. Matsumoto, and H. Murofushi, 1985, Adv. Biophys., 19:43-89). We purified axolinin and the 255-kD protein in their native forms and further characterized their biochemical properties. The purified axolinin was soluble in 0.6 M NaCl solution but insoluble in 0.1 M NaCl solution. It co-sedimented with microtubules but not with actin filaments. In low-angle rotary-shadowing electron microscopy, the axolinin molecule in 0.6 M NaCl solution looked like a straight rod approximately 105 nm in length with a globular head at one end. On the other hand, the purified 255-kD protein was soluble in both 0.1 and 0.6 M NaCl solution and co-sedimented with actin filaments but not with microtubules. The 255-kD protein molecule appeared as a characteristic horseshoe-shaped structure approximately 35 nm in diameter. Furthermore, the 255-kD protein showed no cross-reactivity to the anti-axolinin antibody. Taken together, these characteristics lead us to conclude that the subaxolemmal cytoskeleton in the squid giant axon is highly specialized, and is mainly composed of microtubules and a microtubule-associated HMW protein (axolinin), and actin filaments and an actin filament-associated HMW protein (255-kD protein).     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

The vas deferens of the spider crab,Libinia emarginata

Sperm enter the anterior vas deferens individually in the spider crab male. There they become surrounded by secretion products from the cells of the vas deferens, and are compartmentalized into spermatophores of varying size. The anterior vas deferens can be divided into three regions. The epithelium of the anterior vas deferens varies regionally from low to high columnar. The cytoplasm contains vast arrays of rough endoplasmic reticulum and Golgi complexes but few mitochondria. Intercellular spaces contain septate junctions, gap junctions and vesicles. Once the spermatophores have been formed in the anterior vas deferens, they are moved posteriorly to the middle vas deferens where they are stored and surrounded by seminal fluids. The epithelial cells of the middle vas deferens contain large amounts of rough endoplasmic reticulum and Golgi complexes. Numerous micropinocytotic vesicles appear, forming at the cell surface and within the apical cytoplasm. Their suggested function is the resorption of secretion products of the anterior vas deferens which initiated compartmentalization of the spermatozoa into spermatophores. The posterior vas deferens functions primarily as a storage center for spermatophores until they are released at the time of copulation. Seminal fluid surrounding the spermatophores is produced in this region as well as in the middle vas deferens. The cells of this region contain vast arrays of vesicular rough endoplasmic reticulum and Golgi complexes. The cells are multinucleate. Microtubules are numerous throughout the length of the cells and appear to insert on the plasma membrane.     

Environmental cadmium is not sequestered by metallothionein in rainbow trout

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca²⁺ transport which is mediated by a (Ca²⁺ + Mg²⁺)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-M_r in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-M_r phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca²⁺ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.     

Ultrastructure of the peritrophic membrane-secreting cells in the cardia of the blowfly, Lucilia cuprina

Scanning and transmission electron microscopy are used to reveal the internal anatomy and ultrastructure of the cardia which is the source of the triple layered peritrophic membrane in the blowfly Lucilia cuprina. Within the cardia, rings of secretory cells (formation zones) and non-secretory tissue (valvula cardiaca) interlock to secrete and mould the layers of membrane. Formation zone cells have abundant rough endoplasmic reticulum, Golgi and secretory vesicles. A portion of midgut just posterior to the formation zone is covered by close-packed microvilli connected by septate-like junctions. The cuticle-lined valvula cardiaca is rich in smooth endoplasmic reticulum, glycogen and microtubules. The oesophageal cuticle is unusual in containing tubular structures. The ultrastructural features of the separate components of the cardia are discussed in terms of their secretory and non-secretory roles; modified midgut cells secrete chitin and protein whereas modified foregut tissue (valvula cardiaca) appears to be adapted to provide structural integrity (extensive junctions, microtubules), movement (muscles, possibly microtubules), a store of energy (glycogen deposits) and possibly a lipidic secretion (from smooth endoplasmic reticulum) to lubricate the passage of the membranes.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Ultrastructure of the pineal gland of the tropical bat Rousettus leschenaulti

The ultrastructure of the pineal organ was studied in the tropical megachiropteran Rousettus leschenaulti. The pineal lies deep beneath the hemispheres adjacent to the third ventricle and is traversed by the habenular commissure anteriorly. Its parenchyma consists of a uniform population of light and occasional dark pinealocytes which appear to differ only in the degree of cytoplasmic staining. Pinealocytes are characterized by well developed Golgi bodies associated with numerous small vesicles, many mitochondria and polyribosomes, and frequent subsurface cisternae. Lipid droplets and elements of smooth endoplasmic reticulum are scant. Cisternae of granular endoplasmic reticulum are occasionally dilated. A distinct feature is the abundance of clear vesicles in the pinealocyte pericapillary terminals, which also frequently contain granular vesicles and a very large vacuole. The pineal is further characterized by the presence of a small number of glial cells and myelinated nerve fibers. A broad perivascular space investing numerous capillaries contains glial-cell and pinealocyte processes, collagen fibrils and abundant unmyelinated nerve fibers. Tortuous extensions of the perivascular space enter the pineal parenchyma where they come in close proximity to branched intercellular channels or canaliculi characterized by specialized junctions and microvilli. Differences between the pineal of the non-hibernating megachiropteran Rousettus and that of the hibernating microchiropteran bats, and structural similarities to the pineal of tropical rodents are discussed.     

Oogenesls in the terrestrial hermit crab,coenobita clypeatus (decapoda,anomura): II. Vitellogenesis

Oocytes from the land hermit crab, Coenobita clypeatus, in various stages of vitellogenesis were examined by light and electron microscopy. Early vitellogenic oocytes are characterized by accumulations of discrete vesicles of endoplasmic reticulum in the perinuclear cytoplasm. As oocytes develop, the endoplasmic reticulum becomes abundant, and numerous Golgi complexes are seen. There is a well developed Golgi-endoplasmic reticulum interaction. Within the confines of the reticulum are discrete intracisternal granules, which can be seen coalescing into electron-dense yolk bodies. Lipid accumulation is seen throughout the cytoplasm. Coincident with the burst of intra-oocytic metabolism are oolemma modifications and micropinocytosis, which provide ultrastructural evidence for extra-oocytic yolk production. The mature oocyte contains numerous yolk and lipid vesicles of varying electron density that comprise both intra- and extra-oocytic substrates.     

Effect of gibberellic Acid and actinomycin d on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells

Analysis of structural changes in barley aleurone cells during germination or following incubation of isolated layers in gibberellic acid with or without actinomycin D revealed extensive development of rough endoplasmic reticulum. Following the assembly of stacked rough endoplasmic reticulum, vesiculation occurred mainly in basal regions of the cell, resulting in a polar distribution of rough endoplasmic reticulum vesicles. It is postulated that these vesicles are involved in protein secretion, because smooth vesicles, derived from the rough endoplasmic reticulum, apparently become appressed to the plasma membrane. The increased α-amylase in the ambient medium and in cell homogenates correlated directly with formation and subsequent vesiculation of the rough endoplasmic reticulum. Furthermore, when cells were treated with actinomycin D and gibberellic acid, α-amylase synthesis was inhibited by 45% and secretion by 63%. These cells were characterized cytologically by large areas of disarrayed segments of fragmented rough endoplasmic reticulum, corresponding to a high intracellular level of α-amylase. In addition, small lipid bodies common to the segmented regions of rough endoplasmic reticulum were surrounded by fine fibrous material, short segments of rough endoplasmic reticulum, and free ribosomes, suggesting that actinomycin D had interfered with development and organization of rough endoplasmic reticulum.     

A fine-structural analysis of mouse molar odontoblast maturation.

The first mandibular molars of the Swiss albino mice, 1 through 4 days of age, were fixed in glutaraldehyde or Karnovsky's fixative. The tissues were postfixed in OSO4, dehydrated and embedded in Epon. The prepolarizing, polarizing and secretory odontoblasts were described. The prepolarizing cells, located in the vicinity of the cervical loop, were mesenchymal-like in morphology. The cells of the polarizing stage possessed organelles indicative of protein synthesis. The nucleus was located proximally. Aperiodic fibers were evident in the wide basement membrane. The secretory odontoblasts were long, slender, polarized cells closely adjoining one another. Each odontoblast possessed six morphologically discernible regions: (1) an infranuclear region, limited in size and containing few cellular organelles; (2) a nuclear region, housing the oval nucleus and a few associated lamellae of rough endoplasmic reticulum as well as a limited number of mitochondria; (3) a supranuclear rough endoplasmic reticulum region, possessing an abundance of these organelles as well as some mitochondria and secretory vesicles; (4) a Golgi region, occupying the middle third of the cell, housing the elements of an extensive Golgi apparatus which was surrounded by peripherally located profiles of rough endoplasmic reticulum; additionally, this region contained smooth endoplasmic reticulum, mitochondria, numerous secretory granules and vesicles and occasional intracellular collagen fibers; (5) an apical rough endoplasmic reticulum region, containing a rough endoplasmic reticulum component that was less extensive than its supranuclear counterpart; in addition, this region was the one richest in mitochondria and contained a plethora of secretory vesicles and granules; (6) the odontoblastic process, a region mostly void of organelles, containing various secretory products, some of which appeared to be in the process of being released extracellularly into the surrounding dentin matrix.     

Calreticulin: not just another calcium-binding protein

In this paper we review some of the rapidly expanding information about calreticulin, a Ca²⁺-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca²⁺-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca²⁺ storage protein in the endoplasmic reticulum.Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum     

LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

Cross-linker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method

The elaborate cross-connections among membranous organelles (MO), microtubules (MT), and neurofilaments (NF) were demonstrated in unifixed axons by the quick-freeze, deep-etch, and rotary-shadowing method. They were categorized into three groups: NF-associated cross-linker, MT-associated cross-bridges, and long cross-links in the subaxolemmal space. Other methods were also employed to make sure that the observed cross-connections in the unfixed axons were not a result of artifactual condensation or precipitation of soluble components or salt during deep-etching. Axolemma were permeablized either chemically (0.1% saponin) or physically (gentle homogenization), to allow egress of their soluble components from the axon; or else the axons were washed with distilled water after fixation. After physical rupture of the axolemma or saponin treatment, most of the MO remained intact. MT were stabilized by adding taxol in the incubation medium. Axons prepared by these methods contained many longitudinally oriented NF connected to each other by numerous fine cross-linkers (4-6 nm in diameter, 20-50 nm in length). Two specialized regions were apparent within the axons: one composed of fascicles of MT linked with each other by fine cross-bridges; the other was in the subaxolemmal space and consisted of actinlike filaments and a network of long cross-links (50-150 nm) which connected axolemma and actinlike filaments with NF and MT. F-actin was localized to the subaxolemmal space by the nitrobenzooxadiazol phallacidin method. MO were located mainly in these two specialized regions and were intimately associated with MT via fine short (10-20 nm in length) cross-bridges. Cross-links from NF to MO and MT were also common. All these cross-connections were observed after chemical extraction or physical rupture of the axon; however, these procedures removed granular materials which were attached to the filaments in the fresh unextracted axons. The cross-connections were also found in the axons washed with distilled water after fixation. I conclude that the cross- connections are real structures while the granular material is composed of soluble material, probably protein in nature.     

Ultrastructural distribution of Ca++ within neurons

Summary We used the oxalate-pyroantimonate technique to determine the ultrastructural distribution of Ca⁺⁺ in neurons of the rat sciatic nerve. The content of the precipitate was confirmed by X-ray microanalysis and appropriate controls. In the cell bodies of the dorsal root ganglia, Ca⁺⁺ precipitate was found in the Golgi, mitochondria, multivesicular bodies and large vesicles of the cytoplasm but not in lysosomes, and was prominently absent from regions of rough endoplasmic reticulum and ribosomes. It was seen in the nucleus but not in the nuclear bodies or nucleolus.Within the axon itself, Ca⁺⁺ precipitate was also found sequestered in mitochondria and smooth endoplasmic reticulum. In addition Ca⁺⁺ precipitate found diffusely throughout the axoplasm exhibited a discrete and heterogeneous distribution. In myelinated fibers the amount of precipitate decreased predictably in the axoplasm beneath the Schmidt-Lanterman clefts and in the paranodal regions at the nodes of Ranvier. This correlated with the presence of dense precipitate in the Schmidt-Lanterman clefts them-selves and in the paranodal loops of myelin.Intracytoplasmic ionic Ca⁺⁺ is maintained at 10^–7 M by balanced processes of influx, sequestration and extrusion. The irregular distribution of Ca⁺⁺ precipitate in the axoplasm of myelinated fibers suggests that there may be specific regions of preferential efflux across the axolemma.     

The possible role of fixed membrane surface charges in acetylcholine release at the frog neuromuscular junction

Summary Bass and Moore [Proc. Nat. Acad. Sci. 55:1214 (1966)] proposed that the vesicles containing acetylcholine undergo Brownian motion in the nerve terminals. Acetylcholine is released whenever a vesicle touches the inner face of the axolemma of the nerve terminal. The frequency at which contact is made is limited by an energy barrier that must be overcome before the vesicle can touch the axolemma. The energy barrier has two components. (1) An electrostatic repulsion between positive, fixed charges on the vesicles and a relatively positive potential at the face of the axolemma that is generated by the resting potential. (2) A layer of water molecules held to the vesicle by the surface charge. This model is inconsistent with experimental data. A modification of the model is presented. Both the vesicle and the inner face of the axolemma are assumed to have fixed, negative surface charges that are responsible for the energy barrier. By a series of simplifications, the model leads to two predictions. (1) A plot of the ln (miniature end plate potentials/sec) as a function of the concentration of ions in the axoplasm)^–0.5 should give a straight line. (2) A plot of ln (end plate potential amplitudes) as a function of (extracellular Ca⁺⁺ concentration)^–0.5 should give a straight line. These predictions are shown to agree reasonably well with experimental data.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Development of secretory activity in the seminal vesicle of the male migratory grasshopper,Melanoplus sanguinipes (fabr.) (Orthoptera : Acrididae)

This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing patterns of its secretion during sexual maturation and after allatectomy in Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). In epithelia from seminal vesicles of newly fledged males, the rough endoplasmic reticulum is well developed, and Golgi complexes are elaborate, which indicates the gland is metabolically active. The cells also contain large glycogen deposits and the lumen microvilli are well differentiated. These ultrastructural features are more dominant in 24-hr-old adults where the cytoplasm is clearly differentiated into basal and apical regions. Basally, the cytoplasm is dominated by rough endoplasmic reticulum, large Golgi complexes, glycogen deposits and numerous mitochondria, while the apical cytoplasm is filled with large secretory and/or lysosomal vesicles. Between days 3 and 7, the ultrastructural features change little other than the rough endoplasmic reticulum cisternae, which become vesicular. Analysis by isoelectric focusing shows that the amount of secretory protein increases with age until day 3, at which time the gland contains its full complement of secretion. In seminal vesicles from allatectomized insects, ultrastructural features of cells and isoelectric focusing patterns of the secretion arc identical to those from normal males.