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Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

The genus Aspergillus represents a diverse group of fungi that are among the most abundant fungi in the world. Germination of a spore can lead to a vegetative mycelium that colonizes a substrate. The hyphae within the mycelium are highly heterogeneous with respect to gene expression, growth, and secretion. Aspergilli can reproduce both asexually and sexually. To this end, conidiophores and ascocarps are produced that form conidia and ascospores, respectively. This review describes the molecular mechanisms underlying growth and development of Aspergillus. © CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.

To adapt to changes in the environment, cells have to dynamically alter their phenotype in response to, for instance, temperature and oxygen availability. Interestingly, mitochondrial function in Saccharomyces cerevisiae is inherently temperature sensitive; above 37 °C, yeast cells cannot grow on respiratory carbon sources. To investigate this phenomenon, we studied the effect of cultivation temperature on the efficiency (production of ATP per atom of oxygen consumed, or P/O) of the yeast respiratory chain in glucose-limited chemostats. We determined that even though the specific oxygen consumption rate did not change with temperature, oxygen consumption no longer contributed to mitochondrial ATP generation at temperatures higher than 37 °C. Remarkably, between 30 and 37 °C, we observed a linear increase in respiratory efficiency with growth temperature, up to a P/O of 1.4, close to the theoretical maximum that can be reached in vivo. The temperature-dependent increase in efficiency required the presence of the mitochondrial glycerol-3-phosphate dehydrogenase GUT2. Respiratory chain efficiency was also altered in response to changes in oxygen availibility. Our data show that, even in the absence of alternative oxidases or uncoupling proteins, yeast has retained the ability to dynamically regulate the efficiency of coupling of oxygen consumption to proton translocation in the respiratory chain in response to changes in the environment.

Summary Mushroom-forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno-localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales. It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction.

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.

Bacteriophage p2 belongs to the most prevalent lactococcal phage group (936) responsible for considerable losses in industrial production of cheese. Immunization of a llama with bacteriophage p2 led to higher titers of neutralizing heavy-chain antibodies (i.e., devoid of light chains) than of the classical type of immunoglobulins. A panel of p2-specific single-domain antibody fragments was obtained using phage display technology, from which a group of potent neutralizing antibodies were identified. The antigen bound by these antibodies was identified as a protein with a molecular mass of 30 kDa, homologous to open reading frame 18 (ORF18) of phage sk1, another 936-like phage for which the complete genomic sequence is available. By the use of immunoelectron microscopy, the protein is located at the tip of the tail of the phage particle. The addition of purified ORF18 protein to a bacterial culture suppressed phage infection. This result and the inhibition of cell lysis by anti-ORF18 protein antibodies support the conclusion that the ORF18 protein plays a crucial role in the interaction of bacteriophage p2 with the surface receptors of Lactococcus lactis .

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

The ultrastructure of the septal pore cap (SPC) of Asterodon, Asterostroma and Coltricia were examined to establish the taxonomic position of these genera. Asterostroma has dolipores with perforate SPCs and is classified in the Lachnocladiaceae. In contrast, Asterodon and Coltricia have dolipores with imperforate SPCs and belong to the Hymenochaetaceae. Other selected species of genera belonging to the Hymenochaetaceae like Hydnochaete, Coltriciella, Inonotus, Onnia, and Cyclomyces also contained imperforate SPCs. Coltriciella, Inonotus and Cyclomyces moreover presented a lamella of endoplasmic reticulum above the imperforate SPC after chemical fixation. Such a lamella could rarely be observed in Coltricia only after high-pressure freezing and freeze substitution. Cryofixed fungal cells of Cyclomyces and Coltricia showed differences in the architecture of the matrix of the SPC. Coltricia showed a more layered matrix structure than the SPC of Cyclomyces. In addition, transmission- and scanning electron microscopy revealed an indent in the centre of the imperforate SPC of Cyclomyces, indicating a reduced thickness, and resulting into a tented profile in cross-sections.

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hypal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani. (C) 2000 Academic Press.

In the past decades, high-throughput technologies were developed with the aim to unravel the secret of life. In the genomics project many genomes were sequenced, including the human genome. We ended up with a lot of data but no real clue if and at what point of time a certain illness will manifest, finally not the gene but the presence or absence of the responsible protein will be decisive. Therefore, proteomics came into reach and focus of research. Again we ended up with a wealth of data but still the final answer cannot be found. Finally, not only the presence of the protein is important but also its modification and, even more importantly, its location, whether it is ‘just’ stored in a cell or whether it is active at the place the where action is needed. The future will therefore be structomics.

For scarce antigens or antigens which are embedded in a dense macromolecular structure, on-section labeling, the first method of choice, is not always successful. Often, the antigen can be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method. Most of these methods lead to loss of ultrastructural details and, hence, labeling at electron microscope resolution does not add essential information. The scope of this paper is to compare five permeabilization methods for pre-embedding labelling for electron microscopy. We aim for a method that is easy to use and suitable for routine investigations. For our ongoing work, special attention is given to labeling of the cell nucleus. Accessibility of cytoplasmic and nuclear antigens is monitored with a set of different marker antibodies. From this investigation, we suggest that prefixation with formaldehyde/glutaraldehyde is necessary to stabilize the ultrastructure before using a detergent (Triton X-100 or Brij 58) to permeabilize or remove the membranes. The experimental conditions for labeling should be checked first with fluorescence or fluorescence-gold markers by fluorescence microscopy. Then either ultrasmall gold particles (with or without fluorochrome) with silver enhancement or, if the ultrasmall gold particles are obstructed, peroxidase markers are advised. The most promising technique to localize scarce antigens with good contrast is the combination of a pre-embedding peroxidase/tyramide-FITC or -biotin labeling followed by an on-section colloidal gold detection.

Black pigmented conidia of Aspergillus niger give rise to micro-colonies when incubated in liquid shaken medium. These micro-colonies are heterogeneous with respect to gene expression and size. We here studied the biophysical properties of the conidia of a control strain and of strains in which the fwnA, olvA or brnA gene is inactivated. These strains form fawn-, olive-, and brown-coloured conidia, respectively. The δolvA strain produced larger conidia (3.8 μm) when compared to the other strains (3.2-3.3 μm). Moreover, the conidia of the δolvA strain were highly hydrophilic, whereas those of the other strains were hydrophobic. The zeta potential of the δolvA conidia in medium was also more negative when compared to the control strain. This was accompanied by the near absence of a rodlet layer of hydrophobins. Using the Complex Object Parametric Analyzer and Sorter it was shown that the ratio of individual hyphae and micro-colonies in liquid shaken cultures of the deletion strains was lower when compared to the control strain. The average size of the micro-colonies of the control strain was also smaller (628 μm) than that of the deletion strains (790-858 μm). The size distribution of the micro-colonies of the δfwnA strain was normally distributed, while that of the other strains could be explained by assuming a population of small and a population of large micro-colonies. In the last set of experiments it was shown that relative expression levels of gpdA, and AmyR and XlnR regulated genes correlate in individual hyphae at the periphery of micro-colonies. This indicates the existence of transcriptionally and translationally highly active and lowly active hyphae as was previously shown in macro-colonies. However, the existence of distinct populations of hyphae with high and low transcriptional and translational activity seems to be less robust when compared to macro-colonies grown on solid medium. © CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands.

A fungal culture (FCAS11) was isolated from coastal sediments of the Arabian Sea during the anoxic season. Multigene phylogenetic analyses confidentially place the organism as a novel species within the recently defined class Tritirachiomycetes, subphylum Pucciniomycotina, phylum Basidiomycota. We named the new species Tritirachium candoliense and provide the first description of a member of this class from a marine environment. DNA sequences and morphological characters distinguish T. candoliense from previously described Tritirachium species. Its growth characteristics, morphology, and ultrastructural features showed that under anoxic conditions the species grows slowly and produces mainly hyphae with only few blastoconidia. Electron microscopy revealed differences when the culture was exposed to anoxic stress. Notable ultrastructural changes occur for example in mitochondrial cristae, irregularly shaped fat globules and the presence of intracellular membrane invaginations. We assume that the growth characteristics and substrate utilization patterns are an adaptation to its source location, the seasonally anoxic environment of the Arabian Sea.

A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.

As part of a comparative study of the structure and function of pore structures in heterobasidiomycetous yeasts, dikaryotic hyphae of Schizophyllum commune were subjected to chemical fixation, freeze fracturing, maceration, and freeze substitution, and were subsequently prepared for scanning electron microscopy. The interior of the hyphal cell was visualized and revealed the perforated septal pore cap or parenthesome, mitochondria, vacuoles, and tubular endoplasmic reticulum. The septal pore cap showed connections with tubular endoplasmic reticulum. This tubular endoplasmic reticulum covered the dolipore septal surface. The results presented here complement and extend the ultrastructural image of the septal pore cap obtained from transmission electron micrographs.Key words: septal pore cap, Schizophyllum commune, freeze fracture, maceration, scanning electron microscopy.

Field campaigns in Antarctica, Greenland and the Italian glaciers aiming to explore the biodiversity of these disappearing environments identified several undescribed yeast strains unable to grow at temperature above 20 degrees C and belonging to unknown species. Fourteen of these strains were selected and grouped based on their morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal RNA demonstrated that the strains belong to unknown species related to Leucosporidium antarcticum. The new genus Glaciozyma is proposed and two new species are described, namely Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp. nov. Additionally, re-classification of Leucosporidium antarcticum as Glaciozyma antarctica is proposed. Strains of Glaciozyma form a monophyletic clade and a well separated lineage within class Microbotryomycetes (Pucciniomycotina, Basidiomycota). The description of Glaciozyma genus and the re-classification of L. antarcticum reduce the polyphyletic nature of the genus Leucosporidium.

This chapter aims to present an overview of yeast cell biology, biochemical structure and composition of cell walls in various yeast species, septal pore ultrastructure, and other subcellular characteristics, and a phylogenetic framework to these observations. Yeast cells have ultrastructural features typical of other eukaryotic cells, which include the presence of membrane-bound organelles. Through the use of microscopic techniques, yeast cells have been the topic of detailed structural research for many decades. Because of their relatively small size, electron microscopy is an essential tool for the study of the cellular ultrastructure of the cell. Yeast cells contain all subcellular structures typical of eukaryotes. These structures include the cytoskeleton, a nucleus and various subcellular organelles such as the endoplasmic reticulum (ER), the Golgi apparatus, mitochondria, and microbodies. Like plants, yeast cells contain a vacuole, and the plasma membrane is surrounded by a rigid cell wall. However, chloroplasts are absent in all fungi, which include the yeasts. Fluorescence microscopy methods have become an attractive tool for studying living yeast cells due to the availability of novel fluorescent dyes and the introduction of genes that encode fluorescent proteins. Upon introduction of hybrid genes encoding a fluorescent protein fused to a yeast protein, any subcellular structure can be specifically marked and its kinetics analyzed in real time in living yeast cells.

Mushroom-forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno-localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.

Repellents of the maize pathogen Ustilago maydis are involved in formation of hydrophobic aerial hyphae and in cellular attachment. These peptides, called Rep1-1 to Rep1-11, are encoded by the rep1 gene and result from cleavage of the precursor protein Rep1 during passage of the secretion pathway. Using green fluorescent protein as a reporter, we here show that rep1 is expressed in filaments and not in the yeast form of U. maydis. In situ hybridization localized rep1 mRNA in the apex of the filament, which correlates with the expected site of secretion of the repellents into the cell wall. We also produced a synthetic peptide, Rep1-1. This peptide reduced the water surface tension to as low as 36 mJ m-2. In addition, it formed amyloid-like fibrils as was shown by negative staining, by thioflavin T fluorescence, and by x-ray diffraction. These fibrils were not soluble in SDS but could be dissociated with trifluoroacetic acid. The repellents in the hyphal cell wall had a similar solubility and also stained with thioflavin T, strongly indicating that they are present as amyloid fibrils. However, such fibrils could not be observed at the hyphal surface. This can be explained by the fact that the Rep1-1 filaments decrease in length at increasing concentrations. Taken together, we have identified the second class of fungal proteins that form functional amyloid-like filaments at the hyphal surface.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.

The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina ) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani . Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.

Septal pore caps occur in many filamentous basidiomycetes located at both sides of the dolipore septum and are at their base connected to the endoplasmic reticulum. The septal pore cap ultrastructure has been described extensively by the use of electron microscopy, but its composition and function are not yet known. To enable biochemical and functional analyses in the future, we here describe an enrichment method for perforate septal pore caps from Rhizoctonia solani. Our method is based on the combined use of French press and isopycnic centrifugation, using a discontinuous sucrose gradient followed by a treatment with Triton X-100. Enrichment was monitored by the use of scanning electron microscopy and transmission electron microscopy. Using the same isolation method, smaller septal pore caps were isolated from two other basidiomycetes as well. Furthermore, we showed pore-occluding material co-purified with the septal pore caps. This observation supports the hypothesis that septal pore caps play a key role in the plugging process of the septal pores in filamentous basidiomycetes.

Laser microdissection has been proven a successful technique to isolate single cells or groups of cells from animal and plant tissue. Here, we demonstrate that laser microdissection is suitable to isolate subcellular parts of fungal hyphae. Dolipore septa of Rhizoctonia solani containing septal pore caps were cut by laser microdissection from sections of mycelium and collected by laser pressure catapulting. Subsequently, microdissected septa were visualised using a wheat germ agglutinin labelling of cell walls, septa and septal pore caps and scanning electron microscopy. The use of laser microdissection on fungal cells opens new ways to study subcellular fungal structures and the biochemical composition of hyphal cells.

Colonization of apples by ballistoconidium-forming fungi causes a new disorder, here named 'white haze'. White haze may occur in mild form in the field, but only becomes problematic after Ultra-Low Oxygen storage, and, therefore, may be considered as a postharvest disorder. All isolates, obtained using the spore-fall method, were morphologically identified as anamorphs of smut fungi belonging to the genus Tilletiopsis. Sequence analysis of the D1/D2 and the ITS domains of the rDNA revealed nine novel taxa scattered among the Exobasidiomycetidae (Ustilaginomycetes). Field experiments confirmed the erratic incidence of white haze over the years, and the development of the disorder seems to be enhanced at lower temperatures and a high relative humidity. Several scab fungicide treatments showed diminishing effects on the incidence of white haze.

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) in Saccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama VHH antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion. Eukaryotic cells, such as yeasts, have several quality control systems to ensure that only correctly folded proteins are trans- ported along their secretory pathway (9, 13, 20). These quality control systems are necessary because protein folding in vivo is much more complex than protein folding in vitro. The nascent polypeptide chain emerges from the ribosome and is translo- cated across the endoplasmic reticulum (ER) membrane, thereby being gradually exposed to the lumen of the ER. Since

β1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, β1,3-glucan, and chitin. It has been postulated that the synthesis of β1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of β1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular β1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of β1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of β1,6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.

The interactions between cells of lower fungi may occur through plasmodesmata-like structures, which have been visualized electron microscopically in fungal septa. Developing zygospores of the zygomycetous speciesRhizopus sexualis andGilbertella persicaria contain transcellular strands in septa which separate young gametangia from the suspensors. These simple, unbranched strands occur at intervals in the gametangial wall, and resemble plasmodesmata (Hawker et al.1966). The structures were found to be connected with the endoplasmic reticulum. Plasmodesmata-like structures have also been demonstrated in septa of hyphae of the ascomycetous yeastsGeotrichum candidum (Kirk and Sinclair1966) and inEndomycopsis fibuliger (Takada et al.1965). Septa ofGalactomyces geotrichum exhibit unbranched plasmodesmata-like structures, extending through the cell wall. The width of these channels is40-60 nm, with a desmotubule of10 nm diameter (Marchant1976) which connects endoplasmic reticulum on either side of the septum. Plasmodesmata-like structures occur in few fungi, while in the majority of fungi with septate hyphae the protoplasm of neighbouring cells is connected through the septal pore channel.

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae , a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.

The septal pore cap (SPC) of Trichosporon sporotrichoides CBS 8245 is vesicular-tubular, connected with flat-tubular endoplasmic reticulum (ER), and stains densely with zinc/iodine/osmium tetroxide, as does the ER. The SPC of Schizophyllum commune CBS 340.81 is more complex, about 600 nm in diameter, with perforations of 80--120 nm diameter, and stains less densely with zinc/iodine/osmium tetroxide than the ER. In high-pressure frozen and freeze-substituted hyphae of T. sporotrichoides the ER is present parallel to the dolipore septa, and electron-dense material occurs opposite the septal pore channel; the SPC rarely showed smooth vesicular-tubular membranes, suggesting that this is an ephemeral function of the SPC. The SPC of S. commune has a smooth outer and inner membrane, which enclose a matrix with a palisade-like substructure. A thin layer of electron-dense material covers the inner surface of the SPC of S. commune, from which beaded filamentous structures connect the SPC and the pore-occluding material. These filamentous structures may maintain the intracellular position of the SPC and possibly play a role in plugging the septal pore channel. The septal pore swellings of T. sporotrichoides contain more 1,6--glucan than the septum, and intracellular glucans are also present near the septal pore channel. This cytosolic 1,6--glucan in T. sporotrichoides may serve as a matrix to keep the tubular membranous structures of the SPC together. In contrast, 1,6--glucan is not observed in the SPC and in the pore-occluding material of S. commune, and hyphal septa of this species show less labelling of 1,6--glucan than the septal swelling. The evolutionary transition from simple to more complex types of SPCs may have resulted in a requirement for different components to maintain the morphological integrity and cell biological function.

The yeast Saccharomyces cerevisiae has a number of properties which makes it an important organism for the production of heterologous proteins for research, medical and industrial use. Yeast is particularly amenable to genetic manipulation and a number of auxotrophic markers and strong (inducible) promoters are known. It can secrete proteins into the culture medium and, in contrast to prokaryotes, it can also glycosylate proteins, which is of particular importance for the production of heterologous eukaryotic proteins, which are glycosylated by their naturel hosts. Yeast can grow rapidly on simple, inexpensive media to high cell densities, and knowledge on large scale fermentation and downstream processing is extensive. Furthermore, as it has been used in the brewing and baking industries for a long time, it has become a GRAS (Generally Regarded As Safe) organism. So far, it has been successfully used for the production of a number of single-chain heterologous proteins (Romanos et al.,1992).

Pseudomonas glumae PG1 is able to secrete lipase into the extracellular medium. The lipase is produced as a precursor protein, with an N-terminal signal sequence. A second open reading frame (ORF) was found immediately downstream of the lipase structural gene, lipA, a situation found for the lipases of some other Pseudomonas species. Inactivation of this ORF resulted in a lipase-negative phenotype, indicating its importance in the production of active extracellular lipase. The ORF, lipB, potentially encodes a protein of 353-amino-acid residues, having a hydrophobic N-terminal (amino acids 1 to 90) and a hydrophilic C-terminal part. As a first step in determining the role of LipB, its subcellular location was determined. The protein was found to fractionate with the inner membranes. The expression of fusions of lipB fragments with phoA revealed an N(in)-C(out) topology for the LipB protein, which was confirmed by protease accessibility studies on EDTA-permeabilized cells and on inverted inner membrane vesicles. These and other results indicate that most of the LipB polypeptide is located in the periplasm and anchored to the inner membrane by an N-terminal transmembrane helix, located between amino acids 19 and 40.

Penicillium chrysogenum strains were constructed which express a mutant acyltransferase lacking the putative targeting signal for microbody proteins. The mutated enzyme was located in vacuoles and in neighbouring cytoplasm. Although acyltransferase was expressed in vivo and was active in vitro, the mutants did not produce penicillin. The results demonstrate the involvement of microbodies in penicillin production.

A combination of cryofixation without pre-treatment, freeze-substitution and low-temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P.chrysogenum colonies were grown between dissected-dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze-substitution media, but it was reduced after embedding in Epon 812.

Portland Press, 1998. £39.50/$65.50 (326 pages)ISBN 1 85578 060 7Glauert and Lewis take the reader from fixation to embedding of biological specimens in ten chapters. In the first three chapters, the reader is introduced to the criteria of ultrastructural preservation, artifacts and hazards. The popular chemical fixatives are described in detail, together with how they interact with biological components. The user will find practical tips about fixation methods, such as immersion, injection, perfusion and surface application, improvement of fixing a biological sample, fixation of cells pellets, and fixatives used in cytochemical and immunological techniques.Chapter 4 describes organic solvents required for dehydrating fixed specimens. Besides the removal of water and lipids, swelling and shrinkage occur. Swelling of the specimen is partially prevented by adding Ca2+, subjecting the specimen to 70% ethanol or by performing freeze-substitution. Chapters 5 and 6 elaborate the advantages and disadvantages of epoxy and acrylic resins. Many details are given concerning embedding in different epoxy resins, and about monomers, hardeners, accelerators, flexibilizers, plasticizers and their hazards. In addition, there are also water-miscible epoxy resins that do not require dehydration. The next section introduces the acrylic resins and proceeds to give their advantages and disadvantages. It is important to know that LR White has a shelf life of 12 months, and the reader is advised to purchase uncatalysed LR White. Chapter 8 describes extensively the Lowicryl resins K4M, K11M, HM20 and HM23. This is followed by an introduction to the polyester resins, water-miscible melamine resins and reversible waxy media. Chapter 10 summarizes the preparation methods and shows processing schedules for ultrastructural study, general cytochemistry, enzyme cytochemistry and immunocytochemistry. The book ends with an appendix of suppliers, their addresses and a subject index.The chapters are clearly written, well illustrated with many molecular structures, electron micrographs, drawings, photos of the essential equipment, tables of hazards and instructions for handling hazardous chemicals (e.g. milk powder is a handy absorbent for mopping up fixative spillages). Handy tips are given in the tables showing how to prepare fixatives, buffers and embedding media as well as the schedules of fixation, dehydration, infiltration and embedding. In addition, the reader is referred to other original publications and additional books for further reading at the end of each chapter.The fledgling electron microscopist may well be dazzled by Glauert and Lewis and might find it easier to read a more practical electron microscopy book (e.g. Ref. 1xPractical Electron Microscopy: A Beginner’s Illustrated Guide. Hunter, E.E. CrossrefSee all ReferencesRef. 1). The reader should also refer to books about microorganisms (e.g. Ref. 2xElectron Microscopy in Microbiology. Hoppert, M. and Holzenburg, A. See all ReferencesRef. 2); articles about pre-embedding immunolabelling (e.g. Ref. 3xHumbel, B.M. et al. Microscopy Research and Technique. 1998; 42: 43–58Crossref | Scopus (45)See all ReferencesRef. 3); and more electron microscopy methods (e.g. Ref. 4xElectron Microscopy Methods and Protocols. Hajibagheri, N. CrossrefSee all ReferencesRef. 4). However, almost all you could wish to know about fixation, dehydration, and embedding is in this book, making it a useful asset.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis. © 2011 Elsevier Ltd. All rights reserved.