COMPOSITION AND SYNTHESIS OF THE PECTIN AND PROTEIN COMPONENTS OF THE CELL WALL OF CLOSTERIUM ACEROSUM (CHLOROPHYTA)

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na₂CO₃-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na₂CO₃ extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans- Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

Synthesis and Transport of Hydroxyproline-rich Components in Suspension Cultures of Sycamore-Maple Cells

Plant cell walls contain a glycoprotein rich in hydroxyproline. To determine how Acer pseudoplatanus L. cells transport this glycoprotein to the wall, the pulse-chase technique was used to follow changes in specific radio-activity of hydroxyproline and proline in isolated, mitochondrial, Golgi, microsomal, soluble protein, and wall fractions. The turnover rates or changes in specific radioactivity of cytoplasmic hydroxyproline in these cell fractions indicated that the bulk of this hydroxyproline was transferred not by the Golgi apparatus but by a smooth membranous component.     

Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy.

The membrane compartments responsible for Golgi functions in wild-type Saccharomyces cerevisiae were identified and characterized by immunoelectron microscopy. Using improved fixation methods, Golgi compartments were identified by labeling with antibodies specific for alpha 1-6 mannose linkages, the Sec7 protein, or the Ypt1 protein. The compartments labeled by each of these antibodies appear as disk-like structures that are apparently surrounded by small vesicles. Yeast Golgi typically are seen as single, isolated cisternae, generally not arranged into parallel stacks. The location of the Golgi structures was monitored by immunoelectron microscopy through the yeast cell cycle. Several Golgi compartments, apparently randomly distributed, were always observed in mother cells. During the initiation of new daughter cells, additional Golgi structures cluster just below the site of bud emergence. These Golgi enter daughter cells at an early stage, raising the possibility that much of the bud's growth might be due to secretory vesicles formed as well as consumed entirely within the daughter. During cytokinesis, the Golgi compartments are concentrated near the site of cell wall synthesis. Clustering of Golgi both at the site of bud formation and at the cell septum suggests that these organelles might be directed toward sites of rapid cell surface growth.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Antibodies to hepatic endosomes. Identification of two endosome antigens

Endosome fractions were prepared from rat liver homogenates, and antibodies were raised in rabbits against the integral membrane proteins. Immunofluorescent studies showed that these antibodies identified primarily intracellular structures in liver sections, isolated hepatocytes and HepG-2 cells. Immunoelectron microscopy using protein A-gold confirmed that endocytic multivesicular structures, especially those located at the biliary pole of the hepatocyte, were labeled. Biochemical analysis showed that approximately 12 endosome antigens were present. A major 43 kDa glycosylated antigen corresponded to the asialoglycoprotein receptor subunit. A further antigen identified in endosomes was a 115 kDa polypeptide pI 4.3 previously identified as a major calmodulin-binding protein. The antigens identified in rat liver endosomes were different to those previously shown by other studies to be present in the Golgi apparatus and lysosomes.     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

A high molecular weight polypeptide cross-reacting with the antibodies to the dynein heavy chain localizes to the subset of Golgi complex in higher plant cells

Antibodies were produced against fragments of the microtubule-binding domain and the motor domain of the dynein heavy chain from Dictyostelium discoideum to probe whole cell extracts of root meristem cells of wheat Triticum aestivum. In plant extracts, these antibodies cross-reacted with a polypeptide of high molecular weight (>500 kDa). The antibodies bound to protein A-Sepharose precipitated high molecular weight polypeptide from cell extracts. Immunofluorescence showed that the antibodies identified various aggregates inside cells, localized at the perinuclear area during interphase to early prophase, at the spindle periphery and polar area during mitosis, and in the interzonal region during phragmoplast development. Some aggregates were also co-labeled by markers for the Golgi apparatus. Thus, we found in higher plant cells a high molecular weight antigen cross-reacting with the antibodies to motor and microtubule-binding domains of dynein heavy chains. This antigen is associated with aggregates distributed in the cytoplasm in cell cycle-dependent manner. A subset of these aggregates belongs to the Golgi complex.     

High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

Antiviral activity of Lactobacillus brevis towards herpes simplex virus type 2: role of cell wall associated components

The aim of this research was to study the mechanisms of Lactobacillus brevis antiviral activity towards HSV-2 and to identify the bacterial components responsible for the inhibiting effect. Bacterial extract and cell walls were prepared by lysozyme digestion of L. brevis cells untreated or treated with LiCl to remove S-layer proteins. Bacterial extract and cell wall fragments showed a dose dependent inhibitory effect on HSV-2 multiplication. In order to characterize the inhibitory activity of L. brevis, the bacterial extract was subjected to different physical and chemical treatments. The inhibitory activity was resistant to high temperature and proteases digestion and appeared to be associated with compounds with a molecular weight higher than 10 kDa. DNA, RNA and lipids isolated from bacterial cells were devoid of inhibitory effect. The antiviral activity of both bacterial extract and cell wall fragments obtained from L. brevis cells after the S-layer removal was significantly reduced compared to untreated cells suggesting that the inhibitory activity is likely due to a heat-resistant non-protein cell surface bacterial component.     

VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells.

In simple epithelial cells, apical and basolateral proteins and lipids in transit to the cell surface are sorted in the trans-Golgi network. We have recently isolated detergent-insoluble complexes from Madin-Darby canine kidney cells that are enriched in glycosphingolipids, apical cargo and a subset of the proteins of the exocytic carrier vesicles. The vesicular proteins are thought to be involved in protein sorting and include VIP21-caveolin. The vesicular protein VIP36 (36 kDa vesicular integral membrane protein) has been purified from a CHAPS-insoluble residue and a cDNA encoding VIP36 has been isolated. The N-terminal 31 kDa luminal/exoplasmic domain of the encoded protein shows homology to leguminous plant lectins. The transiently expressed protein is localized to the Golgi apparatus, endosomal and vesicular structures and the plasma membrane, as predicted for a protein involved in transport between the Golgi and the cell surface. It is diffusely localized on the plasma membrane but can be redistributed by antibody modulation into caveolae and clathrin-coated pits. We speculate that VIP36 binds to sugar residues of glycosphingolipids and/or glycosylphosphatidyl-inositol anchors and might provide a link between the extracellular/luminal face of glycolipid rafts and the cytoplasmic protein segregation machinery.     

The t complex polypeptide 1 (TCP-1) is associated with the cytoplasmic aspect of Golgi membranes

The t complex polypeptide 1 (TCP-1) is a protein of unknown function expressed in large amounts during spermatogenesis. Rat monoclonal antibodies recognizing TCP-1 have been prepared and used to immunoprecipitate and Western blot a 57 kd protein from germ cell and tissue culture cell extracts. In tissue culture cells, indirect immunofluorescent localization of antigen indicated a perinuclear distribution similar to that of the Golgi apparatus. Analysis of the TCP-1 distribution in tissue culture cells showed that the polypeptide was associated with the cytoplasmic aspect of membranes of the trans-Golgi network (TGN). The distribution in spermatids suggested that TCP-1 was localized to structures often associated with the developing acrosome. The TCP-1 antigenic epitopes are highly conserved, allowing the protein to be identified in cells across a wide variety of vertebrate species and tissues. These experiments suggest that TCP-1 may be essential for transport of proteins through the exocytic pathway in all cells and required in large amounts for acrosome formation in developing spermatids.     

Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa.

The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.     

Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.     

Immunocytochemical localization of polygalacturonase during tracheary element differentiation in<Emphasis Type="Italic"> Zinnia elegans</Emphasis>

Polygalacturonase (PG) is a cell wall-associated protein that degrades pectin. A ZePG1 cDNA encoding a putative PG was isolated from Zinnia elegens L. and a rabbit antibody specific to the ZePG1 protein was generated. The level of the ZePG1 protein was up-regulated when tracheary element differentiation was initiated. Using gold-labeled secondary antibodies for light and electron microscopy, ZePG1 protein was localized in cultured Zinnia cells. This protein was preferentially distributed on tracheary elements (TEs). At the subcellular level, the protein was localized on secondary wall thickenings, primary walls, Golgi bodies and vesicles. Thus, the putative role of the ZePG1 protein might be the degradation of pectic substances before lignification. Some non-TE cells also accumulated ZePG1 protein on primary walls, Golgi bodies and vesicles. The accumulation of ZePG1 protein on primary walls seems to be at the elongating tips of non-TE cells. In plants, ZePG1 protein was localized on the secondary wall thickenings of differentiating TEs and phloem regions. These results suggest that the expression of the ZePG1 protein is highly regulated both spatially and temporally during in vitro and in situ TE differentiation.Abbreviations GST Glutathione-S-transferase - PATAg Periodic acid–thiocarbohydrazide–silver proteinate - PG Polygalacturonase - TE Tracheary element     

A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.     

Isolation and partial characterization of uronic acid-containing glycoproteins from Mucor rouxii

Five different fractions containing uronic acids associated with protein were isolated from the cytoplasm of the filamentous form of Mucor rouxii. A signle fraction was isolated from the cell wall by hot sodium dodecyl sulfate followed by ion exchange column chromatography. Two cytoplasmic entities (peaks I and II) were not adsorbed to DEAE Bio-Gel A. The molecular mass of peaks I to V ranged from 16.5 to 210 kDa. The protein-uronic acid ratios were different for each fraction. The cell wall fraction showed a molecular mass of 16.5 kDa, similar to that of peak II but with differences in chromatographic behavior and protein-uronic acid ratio. The possible role of these molecules as acceptors of sugar residues during polyuronide chain growth is discussed.