THE RELATIONSHIP BETWEEN THE FINE STRUCTURE AND DIRECTION OF BEAT IN GILL CILIA OF A LAMELLIBRANCH MOLLUSC

This paper describes the fine structure and its relationship to the direction of beat in four types of cilia on the gill of the fresh-water mussel Anodonta cataracta. The cilia contain nine outer, nine secondary, and two central fibers, such as have been described previously in other material. Each outer fiber is a doublet with one subfiber bearing arms. One particular pair of outer fibers (numbers 5 and 6) are joined together by a bridge. The two central fibers are enclosed by a central sheath; also present in this region is a single, small mid-fiber. The different groups of fibers are connected together by radial links that extend from the outer to the secondary fibers, and from the secondary fibers to the central sheath. The basal body consists of a cylinder of nine triplet fibers. Projecting from it on one side is a dense conical structure called the basal foot. The cylinder of outer fibers continues from the basal body into the cilium, passing through a complex transitional region in which five distinct changes of structure occur at different levels. There are two sets of fibers associated with the basal bodies: a pair of striated rootlets that extends from each basal body down into the cell, and a system of fine tubular fibers that runs parallel to the cell surface. The relationship between fine structure and direction of beat is the same in all four types of cilia examined. The plane of beat is perpendicular to the plane of the central fibers, with the effective stroke toward the bridge between outer fibers 5 and 6, and toward the foot on the basal body.     

Digestive System of Trichodorus porosus

The onchiostyle of Trichodorus porosus has an anterior outer portion, a fine inner spear and a posterior onchiostyle extension. The extension has a ventral lumen and is fused to the pharynx wall. The inner spear enters the dorsal wall of the outer onchiostyle posterior to the guide ring and extends anteriorly inside the anterior portion of the onchiostyle. Muscle cells are absent in the basal position of the esophagus. The glandular portion of the basal part of the esophagus consists mainly of endoplasmic reticulum lined with ribosomes. A sinus empties into the lumen through the dorsal esophageal gland orifice. The configuration of the intesinal lumen is highly variable. The rectum is attached to the dorsal and ventral walls of the body cavity by striated rectal muscle cells.     

Centrin scaffold in Chlamydomonas reinhardtii revealed by immunoelectron microscopy

In the flagellate green alga Chlamydomonas reinhardtii the Ca(2+)-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.     

CHANGES IN FINE STRUCTURE DURING SILK PROTEIN PRODUCTION IN THE AMPULLATE GLAND OF THE SPIDER ARANEUS SERICATUS

The ampullate silk gland of the spider, Araneus sericatus, produces the silk fiber for the scaffolding of the web. The fine structure of the various parts of the gland is described. The distal portion of the duct consist of a tube of epithelial cells which appear to secrete a substance which forms the tunica intima of the duct wall. At the proximal end of the duct there is a region of secretory cells. The epithelium of the sac portion contains five morphologically distinct types of granules. The bulk of the synthesis of silk occurs in the tail of the gland, and in this region only a single type of secretory droplet is seen in the epithelium. Protein synthesis can be stimulated by the injection of 1 mg/kg acetylcholine into the body fluids. 10 min after injection, much of the protein stored in the cytoplasm of the epithelial cells has been secreted into the lumen. 20 min after stimulation, the ergastoplasmic sacs form large whorls in the cytoplasm. Protein, similar in electron-opacity to protein found in the lumen, begins to form in that portion of the cytoplasm which is enclosed by the whorls. The limiting membrane of these droplets is formed by ergastoplasmic membranes which lose their ribosomes. No Golgi material has been found in these cells. Protein appears to be manufactured in the cytoplasm of the tail cells in a form which is ready for secretion.     

The Isolation of Amoeba RNA of Low Protein Content in High Yields

Comparison of six different RNA isolation procedures from amoebae showed that RNA recovery was increased by approximately 65% if pronase treatment was carried out prior to phenol extraction and was decreased by about 55%. and 17%. respectively if chloroform-isoamyl alcohol extraction or dialysis were carried out following phenol extraction. Dialysis was found to decrease contamination from protein or protein degradation products by 6–12 fold. Although pronase digestion appeared to increase protein contamination of the RNA preparation, the fact that chloroform-isoamyl alcohol extraction did not reduce it indicates that the contaminants were not protein. It is concluded that the most efficient scheme for isolating and purifying RNA from the free-living amoebae utilizes pronase digestion prior to phenol extraction and dialysis prior to RNA precipitation.     

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin- section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.     

Ribosomal RNA on the surfaces of nonleukemic mouse ascites tumor cells

RNAs on the cell surfaces of two nonleukemic and two leukemic strains of mouse ascites tumor cells were studied by fractionating the RNAs released from the cell surface by gentle pronase treatment after sucrose density gradient centrifugation, by indirect membrane immunofluorescence that used anti-RNA antibody and by cell electrophoresis. RNA was extracted from the cell supernatants of Ehrlich ascites tumor and sarcoma 180 cells (nonleukemic) that had been treated or not treated with pronase (1 microgram/ml, 37 degrees C, 20 min) followed by sucrose density gradient centrifugation. It was clearly demonstrated that the amounts of ribosomal RNA (18S and 28S) released after pronase treatment were approximately 80% greater than that of nonpronase-treated cells. Ehrlich ascites tumor cells that had been treated with actinomycin D (100 micrograms/kg body weight of mouse, 16 h) in vivo released an amount of ribosomal RNA after pronase treatment only 20% greater than the value for untreated cells. Actinomycin D treatment greatly reduced both the cell surface negative charge and the cell surface immunofluorescence when rabbit anti-RNA antibody was used. Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs. In contrast, L1210 and C1498 cells (leukemic) showed no change in the amounts of ribosomal RNA released after pronase treatment. L1210 cells also showed no change in the surface negative charge after being treated with actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural changes of dinoflagellate chromosomes by pronase and ribonuclease

When cells of the dinoflagellates Prorocentrum micans and Gyrodinium cohnii are exposed to the proteolytic enzyme pronase or alternatively to ribonuclease, the structure of chromosomes is markedly altered. These changes have been observed electron microscopically in thin sections and spreads. Treatment of cells with pronase removed the bulk of nonfibrillar chromosome material completely unmasking fine chromosomal DNA fibres. Pronase had similar effect also on the dense material which is in contact with chromosomes; fibrillar loops protruding from chromosomes were exposed. However, pronase had no effect on the structural integrity of chromosomes. On the contrary, treatment of cells with ribonuclease loosened the package of chromosomal fibres. Thin sections showed that the tight package of longitudinal periodic structures seen in untreated chromosome was relaxed; chromosome extended longitudinally and formed a linear array of balls. When ribonuclease-treated chromosomes were spread, they were substantially more stretched than untreated chromosomes because of uncoiling of two oppositehanded spiral chromatid bundles. The effect of ribonuclease treatment suggests that unknown RNA species have an important role in the maintenance of permanent condensation of dinoflagellate chromosomes. On the other hand, proteins removable by pronase are also present. Most probably they are not linked to the chromosome structure but represent the matrix of nuclear activity.     

Structure and Protein Composition of a Basal‐Body Scaffold (“Cage”) in the Hypotrich Ciliate Euplotes

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal‐body “cage”. Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45‐kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45‐kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double‐layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or “basal” cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or “medial” cage layer. The isolated cage thus resembles a miniature test‐tube rack, able to accommodate varying arrangements of basal‐body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal‐body cages in Euplotes, we have termed this novel 45‐kDa protein “cagein”.     

Cilia on bovine mammary epithelium: ultrastructural observations

Ultrastructural examination of bovine mammary tissues revealed the presence of 9+0 or primary cilia protruding from surfaces of alveolar epithelial and myoepithelial cells. Cilia of epithelial cells protruded approximately 1200 nm into lumina of alveoli and arose from a basal body centriole, the associated centriole of the diplosome, and an accessory rootlet system. Cilia on epithelial cells were more frequently observed than cilia on myoepithelial cells. Occasional cilia made contact with macrophages in the alveolar lumen. The structures were more commonly found in tissues from nonlactating cows, and most were observed in the ventral portion of the mammary gland.     

Basal-body-associated macromolecules: a continuing debate

Controversy over the possibility that centrioles/basal bodies contain nucleic acids has overshadowed results demonstrating other macromolecules in the lumen of these organelles. Glycogen particles, which are known to be present within the lumen of the centriole/basal body of sperm cells, have now been found in basal bodies of protists belonging to three different groups. Here, we extend the debate on a role for RNA in basal body/centriole function and speculate on the origin and the function of centriolar glycogen.     

Les Tentacules d'Ephelota; Etude au Microscope Electronique.

SUMMARY. The central canal of the suctorial tentacle of Ephelota is limited by a fine pellicle composed of numerous longitudinal fibrils and bearing 16–18 membrano-fibrillar ridges arranged radially in the lumen of the canal. This structure resembles that of the myonemes in the heterotrichous ciliate Stentor.
The prehensile tentacle of Ephelota contains 4–6 axial protein fibers each consisting of a lamello-fibrillar bundle and isolated from one another by thin intracytoplasmic membranes.
In both types of tentacle the cytoplasmic portion is immediately limited by a very thin pellicle which is continuous with the "epiplasmic membrane" and covered by the alveolar cuticle which envelops the entire body of the ciliate.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE FLAGELLAR APPARATUS OF BRYOPSIS MAXIMA (CHLOROPHYCEAE)1

The flagellar apparatus in male gametes of the siphonaceous green alga, Bryopsis maxima Okamura, was studied and compared with that of other green biflagellate cells. The proximal portions of two basal bodies are connected by a single striated proximal band, unique among the biflagellate reproductive cells of green algae studied. Anterior to the flagellar bases is a pair of distal bands different from the single structure in other biflagellate cells. These bands which arise from the distal portion of each basal body, extend upward in the papilla and curve down toward the lower edges of the basal bodies. They seem to have no direct association with each other. Two pairs of distinct flagellar roots, one consisting of 3–5 microtubules and the other of a partially striated fiber of undetermined numbers of microtubules, diverge from the basal body region and extend towards the cell posterior. Their component microtubules are disorganized into single or smaller groups midway over the cell length. The uniqueness of the flagellar apparatus is briefly discussed.     

Fine structure of adult Litomosoides carinii (Nematoda: Filarioidea)

The fine structure of the body wall and the intestine of male and female Litomosoides carinii was studied in sections through the middle and posterior regions of the worms. In the sublateral cells of the female hypodermis the organelles are arranged in regular layers. The zone with the basal labyrinth is the most prominent layer. The intestinal epithelium of the female worm varies considerably in thickness. The cytoplasm of these cells contains many large droplets. The lateral hypodermal chords of the male worm are very narrow bands. The muscle cells of the male worm are of the circomyarian type, without an afibrillar portion. The intestinal lumen of the male worm is very narrow, and the epithelial cells contain very large droplets. In both sexes the hypodermis contains bacteria. Larger glycogen deposits were absent in the hypodermal chords and in the muscle cells of the midbody regions.     

Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli.

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of divalent cation such that pronase digestion of the reassociated material gave protein Bp.     

ULTRASTRUCTURE OF THE FLAGELLAR APPARATUS OF PYROBOTRYS (CHLOROPHYCEAE)1

The flagellar apparatus of Pyrobotrys has a number of features that are typical of the Chlorophyceae, but others that are unusual for this class. The two flagella are inserted at the apex, but they extend to the side of the cell toward the outside of the colony, here designated as the ventral side. Four basal bodies are present, two of which extend into flagella. Four microtubular rootlets alternate between the functional and accessory basal bodies. In each cell, the two ventral rootlets are nearly parallel, but the dorsal rootlets are more widely divergent. The rootlets alternate between two and four microtubules each. A striated distal fiber connects the two functional basal bodies in the plane of the flagella. Two additional, apparently nonstriated, fibers connect the basal bodies proximal to the distal fiber. Another striated fiber is associated with each four-membered rootlet near its insertion into the flagellar apparatus. A fine periodic component is associated with each two-membered rootlet. A rhizoplast-like structure extends into the cell from each of the functional basal bodies. The arrangement of these components does not reflect the 180° rotational symmetry that is usually present in the Chlorophyceae, but appears to be derived from a more symmetrical ancestor. It is suggested that the form of the flagellar apparatus is associated with the unusual colony structure of Pyrobotrys.     

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD alpha-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD alpha-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this alpha-zein reveals a putative mutation alanine-->valine (Ala-->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 alpha-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 alpha-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 alpha-zein and the fl2 alpha-zein (Val-->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 alpha-zein (Val-->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 alpha-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.     

DNA dependent RNA polymerase from Ehrlich ascites tumor cells. V. Characterization of a factor repressing RNA polymerase II as a ribonucleoprotein.

Previously we reported the isolation of a factor, named the R-protein, which strongly repressed RNA polymerase II [EC 2.7.7.6] of Ehrlich ascites tumor cells. In the present work this factor was found to contain much RNA (ratio of RNA to protein, 2.3 to 1). The RNA was G:C rich, with a very high content of guanylic acid (about 38%). On equilibrium density gradient centrifugation in Cs2SO4 solution, the RNA became distributed above free RNA, but after digestion of the R-protein with pronase the RNA cosedimented with free RNA. Thus the R-protein is a complex of RNA and protein.     

The pineal-paraphyseal complex of sea turtles. I. Light microscopic description

The pineal-paraphyseal complex of sea turtles is an impressively large structure which projects dorsally and anteriorly above the prosencephalon. The complex was examined by light microscopy in several age classes of green sea turtles (Chelonia mydas) and from juvenile loggerhead turtles (Caretta caretta). The paraphysis is extensively fused to the distal portion of the pineal body, suggesting an interrelated function for these two tissues. No duct or canal was observed connecting the pineal lumen to the third ventricle. Two pineal cell types are described which appear to correspond to the neuroglial supportive cells and the secretory rudimentary photoreceptor cells of other amniotic vertebrates. A possible luminal secretion in the form of apical protrusions is produced by the latter cell type. No typical photoreceptive outer segments were observed.