The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) I. An ultrastructural analysis

Summary The endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), is complex. It consists of a proliferating ER network, a perinuclear complex of 14–18 dictyosomes and 8–12 vacuoles and an anterior contractile vacuole complex. The ER network extends from the nuclear envelope outwards, ensheafhs a dictyosome, extends out through a lobe of the chloroplast and terminates in the thin zone of peripheral cytoplasm between the chloroplast and plasmamembrane. The individual dictyosome is polar with distinct cis- and trans-faces. The cis-face is closely associated with transition vesicles emerging from the adjacent ER. Large vesicles emerge from peripheral swellings of terminal cisternae. The dictyosome-associated ER is connected to the peripheral vacuolar system. During cell division and cytokinesis, changes in the endomembrane system occur. Dictyosomes divide and quickly separate to form perinuclear complexes around the daughter nuclei. Each dictyosome undergoes morphological changes during this wall precursor-producing stage. ER lines the furrow zone and is closely associated with phycoplast microtubules. A discussion of the endomembrane system in membrane flow mechanics is provided.Abbreviations ER endoplasmic reticulum - OsFeCN Osmium ferricyanide     

Ultrastructure of the early stages of carposporophyte development in the red algaChondria tenuissima (Rhodomelaceae,Ceramiales)

The ultrastructure of the early stages of carposporophyte development in the marine red algaChondria tenuissima has been studied. The diploid carposporophyte grows on the gametophyte. Apical gonimoblast cells develop into diploid carpospores. The basal gonimoblast cells cease to divide and undergo considerable cytoplasmic changes before they become incorporated into the expanding fusion cell. Nucleus and plastids degenerate gradually, while mitochondria remain intact. The smooth endoplasmic reticulum becomes prominent, it seems to produce small vesicles with electron dense contents. Simultaneously, numerous mucilage sacs are formed, presumably from dilating ER cisternae. The contents of the mucilage sacs are secreted by exocytosis. The pit connections between gonimoblast cells flare out. They remain as isolated bodies without connection to a wall after fusion. Secondary pit connections occur between vegetative gametophyte cells and sterile carposporophyte cells. There are three different morphological types of pit connections.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) II. A cytochemical analysis

Summary Cytochemical analysis of the endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), reveals distinct compartmentalization. Phosphatase localization shows that: IDPase is located throughout all cisternae of the dictyosome and vesicles associated with the contractile vacuole. Other alkaline phosphatases like TPPase, ATPase and ITPase were localized within the trans-face cisternae and vesicles of the contractile vacuole. IMPase was localized at the plasmamembrane and not within the endomembrane system. Acid phosphatases, incl. CMPase, NADPase and -glycerophosphatase, were localized in vesicles emerging from the central terminus of the trans-face of the dictyosome and in the peripheral vacuolar network. Silver proteinate labeling was noted in the dictyosome, contractile vacuole and on the anterior plasmamembrane. A summary of endomembrane compartmentalization and a putative interpretation of membrane flow and economy are presented.Abbreviations ER endoplasmic reticulum - IDPase inosine 5-diphosphatase - ITPase inosine 5-triphosphatase - ATPase adenosine 5-triphosphatase - TPPase thiamine pyrophosphatase - CMPase cytidine 5-monophosphatase - NADPase -nicotinamide adenine diphosphatase - AcPase acid phosphatase     

The structure,composition and distribution of proteinaceous crystalloids in vegetative cells of the red algaWrangelia plumosa

Summary High concentrations of proteinaceous crystalloids accumulate in vegetative cells of the red algaWrangelia plumosa Harvey but disappear prior to sporogenesis. The distinctly structured crystalloids lack a bounding membrane and appear to autopolymerize within the cytoplasm. Chemical analysis of isolated crystalloids showed the presence of all amino acids except cysteine and cysteic acid. Carbohydrate accounted for 7.5% of the preparation. The crystalloids appear to have a storage function during growth and development.     

Rice protein-body formation: all types are initiated by dilation of the endoplasmic reticulum

The ultrastructure of protein deposition in the starchy endosperm of developing rice (Oryza sativa L.) grains was examined in conventionally fixed (glutaraldehyde and osmium tetroxide) tissues and also in thick sections (0.3 m) of zinc iodide-osmium tetroxide post-fixed tissue. Three types of previously characterised protein body were observed and it was shown that each type was initiated by dilations of the endoplasmic reticulum. Crystalline type protein bodies were initiated by a ribosome-free dilation from rough cisternal endoplasmic reticulum and developed by inclusion of protein from dictyosome-derived vesicles. The large spherical and small spherical protein bodies developed within the cisternae of the rough endoplasmic reticulum.Abbreviations Cr crystalline protein body - DAF days after fertilization - ER endoplasmic reticulum - Ls large spherical protein body - Ss small spherical protein body - ZIO zinc iodide-osmium tetroxide     

The nuclear reticulum in placental cells ofLilium regale is a part of the endomembrane system

Summary Placental cells line the ovarian transmitting tract inLilium regale and produce exudates for secretion. Sections through the highly lobed nuclei of these cells reveal the presence of membrane profiles which form vesicles with varying dimensions in cross section. Computer reconstruction of the nucleus reveals that the vesicle profiles form a complex reticulum of tubular cisternae, which spans the whole nucleus, enclosing a maze of continuous lumen space. Connections between the vesicles and the inner nuclear envelope are visible at various points along the nuclear envelope. This complex network of tubules which constitutes the reticulum arises from the inner nuclear membrane. The nuclear reticulum dramatically increases the inner-envelope surface area, comprising 82% of the total membrane perimeter of inner nuclear envelope and nuclear reticulum. The inner nuclear envelope invaginates into the nucleus forming the nuclear reticulum and the outer nuclear envelope evaginates into the endoplasmic reticulum (ER), indicating that there is a continuity between the lumens of the nuclear reticulum and the ER. The nuclear reticulum is labelled with zinc iodide-osmium tetroxide, a staining pattern identical to that seen in the ER. Positive reaction to the zinc iodide-osmium tetroxide indicates that the nuclear reticulum is a site for Ca²⁺ deposition. The nuclear reticulum forms an extension of the endomembrane system which reaches deep into the nucleoplasm. The lumenal continuity of this system means that there is a channel for communication from the cytoplasm into the nucleoplasm, and that this channel sequesters calcium.Abbreviations ER endoplasmic reticulum - TEM transmission electron microscope - ZIO zinc iodide-osmium tetroxide     

Mutant invertase proteins accumulate in the yeast endoplasmic reticulum

Summary Intercompartmental transport of secreted proteins in yeast was analysed using invertase mutants. Deletions and insertions at the BamHI (position +787) or the Asp718 (position +1159) sites of the SUC2 gene led to mutant proteins with different behaviour regarding secretion, localization and enzyme activity. The deletion mutants showed accumulation of core glycosylated material in the endoplasmic reticulum (ER) a decrease of secreted protein by 5%–30% and loss of enzyme activity. The secreted material was localized in the culture medium and not — as is normal for invertase-in the cell wall. No delay in transport from the Golgi to the cell surface was observed, indicating that the rate-limiting step for secretion is at the ER-Golgi stage. Two insertion mutants, pIPA and pIPB, retained enzyme activity. Mutant pIPB showed 10% secretion, while 60%–70% secretion was observed for pIPA. While the non-secreted material accumulated in the ER, the secreted material was present in the cell wall. The results suggest that the presence of structures incompatible with secretion leads to ER accumulation of mutated invertase.     

Structure,differentiation, and multiplication of Golgi apparatus in fungal hyphae

Summary Golgi apparatus in subapical regions of hyphae consist of paranuclear dictyosomes with 4–5 cisternae each. Transverse and tangential sections provide ultrastructural evidence for a three-dimensional architectural model of the Golgi apparatus and a stepwise mechanism for dictyosome multiplication. The dictyosomes are polarized, with progressive morphological and developmental differentiation of cisternae from the cis to the trans pole. Small membrane blebs and transition vesicles provide developmental continuity between the nuclear envelope and the adjacent dictyosome cisterna at the cis face. Cisternae are formed as fenestrated plates with extended tubular peripheries. The morphology of each cisterna depends on its position in the stack, consistent with a developmental gradient of progressive maturation and turnover of cisternae. Mature cisternae at the trans face are dissociated to produce spheroid and tubular vesicles. Evidence in support of a schematic sequence for increasing the numbers of dictyosomes comes from images of distinctive and unusual forms of Golgi apparatus in hyphal regions where nuclei and dictyosomes multiply, as follows: (a) The area of the nuclear envelope exhibiting forming-face activity next to a dictyosome expands, which in turn increases the size of cisternae subsequently assembled at the cis face of the dictyosome. (b) As subsequent large cisternae are formed and mature as they pass through the dictyosome, an entire dictyosome about twice normal size is built up. The number of cisternae per stack remains the same because of continuing turnover and loss of cisternae at the trans face, (c) This enlarged dictyosome becomes separated into two by a small region of the nuclear envelope next to the cis face that acquires polyribosomes and no longer generates transition vesicles, (d) As a consequence, assembly of new dictyosomes is physically separated into two adjacent regions, (e) As.the enlarged cisternae are lost to vesiculation at the trans pole, they are replaced by two separate stacks of cisternae with typical normal diameters, (f) The net result is two adjacent dictyosomes where one existed previously. Dictyosome multiplication is thus accomplished as part of the normal developmental turnover of cisternae, without interrupting the functioning of the Golgi apparatus as it continues to produce new secretory vesicles from mature cisternae at the trans face. Coordination of Golgi apparatus multiplication with nuclear division ensures that each daughter nucleus receives a complement of paranuclear dictyosomes.     

The fine structure and cytology of the association between the parasitic red algaHolmsella australis and its red algal hostGracilaria furcellata

Summary Holmsella australis Noble andKraft ms. is a colourless red algal parasite, forming whitish pustules on its photosynthetic red algal host,Gracilaria furcellata Harvey. In the infected region, host cortical tissue continues to grow and enclose the expanding pustule. Filaments of both host and parasite grow apically, the cells being connected by primary pit connections (PCs). Secondary PCs form between cells of the same species, and in addition,H. australis initiates the formation of secondary PCs with cells ofG. furcellata. All three types of secondary PC are morphologically distinct. In hostparasite PCs the surface adjoining the host cell is similar in structure to a host-host PC, while that adjoining the parasite cell has the structure of a parasite-parasite PC. The plasma membrane is continuous between the cells of the unrelated host and parasite. In addition, a cap membrane is typically produced only on the host surface, though occasionally the parasite side is enclosed by a cap membrane as well. Cap membranes are absent from parasite-parasite PCs (making them intracellular), while host-host PCs are typically extracellular, both cells producing cap membranes. The presence or absence of a cap membrane in certain positions appears to vary, and suggests that cells may be able to regulate its presence. Since transport of nutrients would be expected to occur from host to parasite cells, and between parasite cells, the morphological evidence presented here suggests the PCs may be the pathway.     

Giardia lamblia: identification and characterization of Rab and GDI proteins in a genome survey of the ER to Golgi endomembrane system

To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell.     

The structure and formation of host-parasite pit connections between the red algal alloparasiteHarveyella mirabilis and its red algal hostOdonthalia floccosa

Summary Harveyella mirabilis is a colourless red algal alloparasite which grows on and within its photosynthetic hostOdonthalia floccosa. Cells ofHarveyella establish secondary pit connections (PCs) with other parasite cells and with cells of the host. Small, uninucleate conjunctor cells are produced by parasite cells and remain connected to them by PCs. Conjunctor cells may fuse with either an adjacent host or parasite cell, with the parasite-conjunctor cell PC becoming either a host-parasite or parasite-parasite secondary PC. Occasionally the conjunctor cell does not fuse with an adjacent cell (either host or parasite) and degenerates. The secondary pit plug which forms between a parasite cell and its conjunctor cell always develops with two structurally distinct surfaces characteristic of a host-parasite pit plug. Only if the conjunctor cell fuses with another parasite cell will the structure of the pit plug be altered to that of a parasite-parasite pit plug. Fungal hyphae also invade the region of infection, andHarveyella cells respond by producing nonfunctional conjunctor cells that grow towards adjacent hyphae. Evidence suggests that secondary PCs may be induced to form mechanically, by the physical presence of another cell, rather than in direct response to a message received from an adjacent cell. The mechanism of secondary PC formation described here is similar to that reported for the closely related alloparasiteHolmsella and may be common to a number of red algal parasitic associations. Helen Margaret Quirk, B. Sc. (Hons), M. Sc. (1953–1982), student, research assistant and friend, died after a long illness on October 24, 1982.     

Glutamine synthetase activity,ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.Abbreviations NR Nitrate reductase - GS Glutamine synthetase - GOGAT Glutamate syntase - MSX l-methionine-dl-sulphoximine     

Unusual cell structures in tumor-like formations of Gracilaria (Rhodophyta)

This paper deals with electron microscopic observations on cultivated plants of the marine red alga Gracilaria verrucosa which developed simple galls; also sea collected material, without galls, had been studied. The galls showed unusual but characteristic cell structures, caterpillar-like bodies, containing rows of fusiform bodies. These were found mostly in the cytoplasm near the plastids, in one case connected with the endoplasmic reticulum, occasionally even inside the nucleus, and are described here, as far as we know, for the first time. It does not seem probable that the caterpillar-like bodies represent mitochondria or bacteria, but the hypothesis that fusiform bodies are related to virus-like structures is discussed. The normal tissues as well as the gall tissue of the laboratory plants contained, besides plastids typical for the red algae, another type of plastids characterized by tubular thylakoids.This work was supported by grants from Consiglio Nazionale delle Ricerche (Rome) and Ministero dell'Agricoltura e delle Foreste of Italy.     

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

Summary Covisualizations with wide-field computational opticalsectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is antigenicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-₁ integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of ₁ integrin and an antibody against the matrix region of ₁ integrin. The latter antibody also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - GF Gaussian filtering - ML maximum likelihood (algorithm or method) - PBS phosphate-buffered saline     

Specialized calciferous cells in the marine algaRhodogorgon carriebowensis and their implications for models of red algal calcification

Summary Calcification inRhodogorgon carriebowensis J. Norris et Bucher was associated with a particular cell type in the cortex. Calciferous cells were 4–6 times the length of cortical assimilatory cells. The distal two-thirds of the calcifying cell was invested with a thick wall that stained with periodic acid Schiff. Thick fibrils formed a reticulum and surrounded grains of calcium carbonate that ranged in shape from rhombohedral to subspherical and were up to 200 nm in greatest dimension. The proximal third of the cell was a tapering uncalcified stalk. The narrow base of the cell was attached to the subtending cell of the fascicle by a normal septum with a pit plug. The cell within the calcified wall matrix was usually flattened and had a very small volume. Cellular contents were dense; even when organelles could be discerned, they could not be identified. X-ray microanalysis revealed that other elements commonly found mixed with calcium carbonate are virtually absent from mineral deposits inR. carriebowensis, but electron diffraction study showed d-spacings that varied from those of pure calcite. Current models of red algal calcification are discussed in light of the findings on this alga.Abbreviations CaCO³ calcium carbonate - DIG differential interference contrast - PAS periodic acid Schiff - SEM scanning electron microscopy - TEM transmission electron microscopy     

Ultrastructure of cell division in the marine red algaLomentaria baileyana

Summary Mitosis in the marine red algaLomentaria baileyana (Rhodymeniales, Rhodophyta) was studied with the electron microscope. Nucleus associated organelles known as polar rings (PRs) migrate to establish the division poles at prophase. At prometaphase, shallow invaginations in the nuclear envelope (NE) form on two sides of each PR and soon rupture. The gaps that are consequently formed contain several small fragments of NE. A larger region of NE remains intact between the two gaps. By metaphase several cisternae of perinuclear endoplasmic reticulum (PER) have enclosed most of the nucleus but remain absent from the polar regions. The nucleolus disperses partially and a typical metaphase plate of chromosomes is formed. Each PR has disjoined into separate proximal and distal portions. MTs converge widely on all regions of the polar area, but do not extend into the cytoplasm. Some MTs end near or at the chromosomes while others extend slightly farther past the chromosomes or diagonally to the NE. As chromosomes move to opposite poles at anaphase, they are accompanied by nucleolar material. An interzonal midpiece (IZM) is created as the pole to pole distance increases and the NE remains intact except for the polar gaps. Following detachment from the IZM, the daughter nuclei are separated by a large central vacuole as a cleavage furrow develops and eventually constricts to form two cells following pit connection formation. It is suggested that mitosis inLomentaria represents an evolutionary intermediate between that seen in the higher and lower groups of red algae. This conclusion is in agreement with conventional morphological and light microscopic criteria used to placeLomentaria in theRhodymeniales, which is considered to be the next to most advanced order in theRhodophyta.     

Immunological detection and localization of a calsequestrin-like protein in red beet and cucumber cells

Summary Calsequestrin is a calcium binding protein present in the sarcoplasmic reticulum (SR) of animal muscle cells and is thought to be essential for the rapid uptake and release of Ca²⁺, and thus for the regulation of Ca²⁺-dependent cellular functions. Higher plant cells of red beet (Beta vulgaris L.) and cucumber (Cucumis sativus L.) contain a polypeptide of about M_r 55000 that cross-reacts with a monoclonal antibody raised against calsequestrin from rabbit skeletal muscle SR. In beet this protein changes its apparent molecular weight with pH as indicated in Western immunoblotting. Although this protein bound calcium it was not the dominant calcium-binding protein in red beet. Washing of beet root tissue leads to a slight increase of this polypeptide in microsomal fractions as indicated by immunoblotting. After immunoblotting to partially purified cell membrane fractions this polypeptide appeared to be predominantly associated with endoplasmic reticulum-enriched fractions. Immunogold labelling of ultrathin sections of cucumber hypocotyl using the anti-calsequestrin antibody showed that gold particles were very largely confined to the cytosol and often in close proximity to the ER. Clusters of up to nine gold particles were observed, often over small vesicular areas, as observed in some animal tissues. These results indicate that red beet and cucumber cells contain a protein which may be related to animal calsequestrin. It appears to be associated with the ER and could be involved in cellular calcium regulation.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

Calcium oxalate crystals in the red algaAntithamnion kylinii (Ceramiales): cytoplasmic and limited to indeterminate axes

Summary The red alga,Antithamnion kylinii Gardner, was found to have needle-shaped inclusions about 10 m long and less than 0.4 m thick. They ranged in abundance from one or a few in young cells to hundreds in fully enlarged cells. Under polarized light, the inclusions were birefringent, indicating crystallinity. Solubility tests suggested that the inclusions were composed of calcium oxalate: they dissolved in 1 N hydrochloric acid and in a saturated solution of aqueous cupric acetate, but they were not soluble in 10 N acetic acid or 5.25% sodium hypochlorite. X-ray microanalysis confirmed the presence of calcium. Calcium oxalate crystals were present in cells of indeterminate axes, but cells of determinate lateral filaments lacked them. Light and electron microscopic study demonstrated that the crystals were associated with the parietal cytoplasm. Calcium oxalate crystals were also present inA. defectum Kylin, but they were not found in ten more distantly related taxa.