Involvement of the Golgi Apparatus in the Synthesis and Secretion of Hydroxyproline-rich Cell Wall Glycoproteins

Pulse labeling of carrot root phloem parenchyma (Daucus carota L. cv. Nantes) tissue with ¹⁴C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identity of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron-microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as ¹⁴C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major site of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell-wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma membrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants.     

SILICEOUS SCALE PRODUCTION IN CHRYSOPHYTE AND SYNUROPHYTE ALGAE. I. EFFECTS OF SILICA-LIMITED GROWTH ON CELL SILICA CONTENT,SCALE MORPHOLOGY,AND THE CONSTRUCTION OF THE SCALE LAYER OF SYNURA PETERSENII1

The cells of synurophyte flagellates (algal class Synurophyceae, formerly included in the Chrysophyceae) are enclosed within a regularly imbricate layer of ornamented siliceous scales. Scale morphology is of critical taxonomic importance within this group of algae, and the scales are valuable indicator microfossils in paleolimnological studies. The data presented here demonstrate that scale morphology and the integrity of the scale layer can exhibit extreme variability in culture as a function of the cellular quota of silica under silica-limited growth. Silica-limited, steady-state populations of the colonial flagellate Synura petersenii Korsh. were maintained over a range of specific growth rates (μ= 0.11–0.69 days^?1) and silica cell quotas (Q_si= 0.13–2.40 pmoles Si · cell¹). Scale morphology and the organization of the scale layer became increasingly aberrant as silica stress increased. Under severe stress, scale deposition was completely suppressed so that cells appeared scale-free. This depression of scale deposition was reversible; populations of silica-starved, scale-free cells rapidly regenerated new scale layers when placed in batch culture and spiked with dissolved silica. During recovery from silica stress, cell division was repressed for 24 h while mean cell silica quota increased 25-fold. The first new scales appeared within 2 h after the silica addition, and development of the new scale layer proceeded in an approximately synchronous manner, residting in normal scale layers on virtually all cells after 48 h of recovery in Sirich medium. Silica content of silica-replete Synura cells is comparable to freshwater diatoms of siynilar size, but Synura has much greater potential quota variability than diatoms and no apparent threshold silica requirement. Silica-limited growth kinetics and competition between diatoms and Synura for silica are discussed. The results suggest that morphological variability of siliceous scales in natural populations of synurophyte flagellates may result from silica stress and that the experimental approach developed here has great potential value as a means for circumscribing ecotypic variation in scale morphology. Results also demonstrate that scale production can be uncoupled from cell division, suggesting that cell cycle regulation of silica biomineralization in the Synurophyceae may be fundamentally different from that of diatoms (algal class Bacillariophyceae). This experimental system has application in the future study of the intracellular membrane systems and the regulatory processes involved in silica biomineralization.     

Schistosoma mansoni: cytochemistry and morphology of the gastrodermal Golgi Apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.     

STUDIES IN SOME FOLIOSE RED ALGAE OF THE PACIFIC COAST. I. CRYPTONEMIACEXE1

Pacific coast species of the red algae Halymenia and Cryptonemia are described Of the 8 previously reported species of Halymenia 3 (H. abyssicola Dawson, H. megaspore Dawson, H refugiensis Dawson) hive been placed in synonymy with other species. Halymenia hollenbergii from southern California is described as new; H cocinea is transferred from Schizymenia (and includes Aeodes gardneri Kylin) and H. templetonii is a transfer from Weeksia. Five species of Cryptonemia are described; one of them, C. taylorii from the Revillagigedo Archipelago, as new. Wider distribution ranges are given for the remaining species, C. ovalifolia, C. obovata, C. bore alis and C. angustata. Keys to genera of the west coast Cryptonemiaceae and to the species of Halymenia and Crypronemia are provided.     

THE BENTHIC ALGAE OF SOUTHERN BAFFIN ISLAND. I. EPIPELIC COMMUNITIES IN RIVERS1

The epipelic algae found in 9 rivers of southern Baffin Island were investigated during the 1972 growing season. The overall assemblage consisted of 240 taxa, of which 200 belonged to the Bacillariophyta and, only 17 to the Chlorophyta. Members of the Bacillariophyta accounted for S7–100% by numbers and 44–100% by volume of the algae at most localities. The dominant taxa were Achnanthes kriegeri Krasske, A. marginulata Grun., and Tabellaria flocculosa (Roth.) Kütz. The Chlorophyta comprised. 0–7% by numbers and 0–30% by volume of the algae, with Cosmarium tinctum Ralfs, Cylindrocystis spp., and Mougeotia sp. being most common. The standing crop in the different rivers commonly exceeded 8 × 10⁶ cells/cm² (8 × 10⁹μ³/cm²), and a maximum growth rate of 3.2 × 10⁵ cells/cm²/day (3.2 × 10⁸μ/cm²/day) was observed. Temperature and light are considered important, factors in the regulation of algal numbers, while nutrient supply in the overlying water, grazing by herbivores, wave action, and flooding appeared to have little effect.     

STUDIES ON CARTILAGE : III. The Occurrence of Collagen within Vacuoles of the Golgi Apparatus

Electron microscopic observations on chondrocytes from rabbit ear cartilage 72 hours after papain has been given intravenously show that many vacuoles in the Golgi apparatus contain a material with a speckled appearance. Some Golgi vacuoles also contain a material with a banded pattern which can be identified as a collagen. The significance of this observation is discussed.     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS : I. The Structure of the Cell Wall of Cylindrotheca fusiformis Reimann and Lewin

An electron microscope study on the cell wall of the diatom Cylindrotheca fusiformis was carried out using stereoscopic and sectioning techniques. Material prepared by an enzyme treatment or by a mechanical method showed that the wall consists of two major components: a silica shell and organic material. Vapor of hydrofluoric acid was employed to remove the silica and thereby reveal the arrangement of the organic material. An attempt was made to increase the contrast of the organic component by "staining." Uranylacetate not only increased the electron opacity of the organic material but also apparently decreased the electron opacity of the silica shell. In ultrathin sections of complete cells, the structure as revealed by stereoscopy could be confirmed and extended. Every part of the silica shell is tightly enclosed by organic material. In the valve region the silica enclosed in this way is located between other layers of organic material. The whole cell wall is surrounded by a mucilaginous substance which stains with ruthenium red.     

Golgi Apparatus Mediated Polysaccharide Secretion by Outer Root Cap Cells of Zea mays. III. Control by Exogenous Sugars

Sugars supplied to germinating seedlings of maize (Zea mays L.) regulate the secretion of polysaccharides by the outer cells of the root cap. The polysaccharide secreted by these cells adheres to the root tip as a droplet and the size of the droplet was used to quantitate polysaccharide secretion. The polysaccharide contains glucose, galacrose, and galacturonic acid residues with smaller quantities of mannose, arabinose, xylose, fucose and rhamnose. These sugars supplied to maize seedlings had marked effects on the rate of polysaccharide secretion by root tips. The effects on secretion were independent of the growth rates of the roots. Glucose, fucose and xylose increased droplet size 1.5–2 fold (as did sucrose, maltose, lacrose, fructose and ribose) whereas galactose, arabinose and galacturonic acid were inhibitory. Mannose increased dropler size 5–7 fold. The marked effect of mannose on polysaccharide secretion was due to an increased rate of secretion combined with a longer phase of extrusion of polysaccharide into the forming droplet. The effect of mannose was partially reversed by inorganic phosphate and other sugars (except for fucose which had no effect or promoted secretion in the presence of mannose). In contrast to sucrose, mannose stimulated secretion in a maize variety having a high sugar endosperm (high endogenous sugar). The results suggest that regulation of secretion by mannose is due to an alteration of normal sugar metabolism; whereas stimulation of secretion by sucrose and other sugars may be due to an increased availability of sugars for metabolism.     

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

The various splice variants of the three SERCA- and the two SPCA-pump genes in higher vertebrates encode P-type ATPases of the P_2A group found respectively in the membranes of the endoplasmic reticulum and the secretory pathway. Of these, SERCA2b and SPCA1a represent the housekeeping isoforms. The SERCA2b form is characterized by a luminal carboxy terminus imposing a higher affinity for cytosolic Ca²⁺ compared to the other SERCAs. This is mediated by intramembrane and luminal interactions of this extension with the pump. Other known affinity modulators like phospholamban and sarcolipin decrease the affinity for Ca²⁺. The number of proteins reported to interact with SERCA is rapidly growing. Here, we limit the discussion to those for which the interaction site with the ATPase is specified: HAX-1, calumenin, histidine-rich Ca²⁺-binding protein, and indirectly calreticulin, calnexin, and ERp57. The role of the phylogenetically older and structurally simpler SPCAs as transporters of Ca²⁺, but also of Mn²⁺, is also addressed.All cells invest a considerable part of their total energy budget in active transport to keep up transmembrane (TM) ion gradients (Rolfe and Brown 1997). Prokaryotes already evolved P-type ion-transport ATPases/ion pumps to that aim (Axelsen and Palmgren 1998). The name P-type refers to the transient transfer of the γ-phosphate group of ATP to a highly conserved aspartate group in the enzyme forming a phospho-intermediate. This autophosphorylation is an important step in the pump’s catalytic cycle (Kuhlbrandt 2004). Based on amino-acid sequence comparisons and on the exon/intron layout of the corresponding genes, three types of P-type Ca²⁺ pumps can be discerned in Eumetazoa: the SERCA-, the SPCA-, and the PMCA-type. Whereas ancestral representatives of each type are recognized in some Eubacteria and Archaea, it is also remarkable that some Eukaryotes have apparently lost either SERCA or SPCA pumps. Yeast for instance lacks SERCA pumps whereas plants thrive well without SPCAs (Mills et al. 2008). The SERCA pumps, which are found in the endoplasmic reticulum (ER) or in the sarcoplasmic reticulum (SR) of eukaryotic cells and the evolutionary older secretory pathway ATPases (SPCA) found in the Golgi apparatus, are closely related to each other and together belong to the P_2A subfamily. They form the topic of this review. The plasma-membrane Ca²⁺-pumps (PMCA), on the other hand, appear to be phylogenetically the oldest of the three and form the P_2B-subfamily branch. PMCAs are addressed in an article by Brini and Carafoli (2009). Some further information on the evolution of the three types of ATPases was recently reviewed by Palmgren and Axelsen (1998) and Vangheluwe et al. (2009). Of the three families, only SERCA pumps translocate two Ca²⁺ ions and hydrolyze one ATP for each catalytic turnover. They possess two Ca²⁺-transport sites: site I and site II; the numbers specify the sequence of filling of the respective sites. The single Ca²⁺-binding site of the SPCA and PMCA pumps structurally corresponds to site II of SERCA (Toyoshima 2009).     

The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus

Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.     

WOUND HEALING AND COLLAGEN FORMATION : I. Sequential Changes in Components of Guinea Pig Skin Wounds Observed in the Electron Microscope

The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed.     

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION : I. During Mouse Fetal Liver Development

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.     

MICROTUBULES IN THE FORMATION AND DEVELOPMENT OF THE PRIMARY MESENCHYME IN ARBACIA PUNCTULATA : I. The Distribution of Microtubules

Prior to gastrulation, the microtubules in the presumptive primary mesenchyme cells appear to diverge from points (satellites) in close association with the basal body of the cilium; from here most of the microtubules extend basally down the lateral margins of the cell. As these cells begin their migration into the blastocoel, they lose their cilia and adopt a spherical form. At the center of these newly formed mesenchyme cells is a centriole on which the microtubules directly converge and from which they radiate in all directions. Later these same cells develop slender pseudopodia containing large numbers of microtubules; the pseudopodia come into contact and fuse to form a "cable" of cytoplasm. Microtubules are now distributed parallel to the long axis of the cable and parallel to the stalks which connect the cell bodies of the mesenchyme cells to the cable. Microtubules are no longer connected to the centrioles in the cell bodies. On the basis of these observations we suggest that microtubules are a morphological expression of a framework which opeartes to shape cells. Since at each stage in the developmental sequence microtubules appear to originate (or insert) on different sites in the cytoplasm, the possibility is discussed that these sites may ultimately control the distribution of the microtubules and thus the developmental sequence of form changes.     

INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL : I. Role of the Peripheral Elements of the Golgi Complex

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-¹⁴C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.     

Microbiological Aspects of Ethylene Oxide Sterilization: I. Experimental Apparatus and Methods

A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.