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Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.

Lipoplexes and polyplexes, that is, assemblies of cationic lipids and polymers with nucleic acids, respectively, are popular nanocarriers for delivery of genes or siRNA into cells for therapeutic or cell biological purposes. Although endocytosis represents a major mechanism for their cellular entry, very little is known about parameters that govern early events in the initial interaction of such delivery devices with the cell surface. Here, we demonstrate that prior to entry, poly- and lipoplexes are captured by thin, actin-rich filopodial extensions, protruding from the cell surface. Subsequent additional recruitment and local clustering of filopodia-localized syndecans, presumably driven by multivalent interactions with the polycationic nanocarriers, appear instrumental in their processing to the cell body. Detailed microscopic analyses reveal that the latter relies on either directional surfing along or retraction of the filopodia. By interfering with actin polymerization or inhibiting the motor protein myosin II, localized at the base of filopodia, our data reveal that the binding of the nanocarriers to and subsequent clustering of syndecans initiates actin retrograde flow, which moves the syndecan-bound nanocarriers to the cell body. At the present experimental conditions, inhibition of this process inhibits nanocarrier-mediated transfection by 50–90%. The present findings add novel insight to our understanding of the mechanism of nanocarrier-cell surface interaction, which may be instrumental in further improving delivery efficiency. In addition, the current experimental approach may also be of relevance to improving our understanding of cellular infection by viruses and pathogenic bacteria, given a striking parallel in filopodia-mediated processing of these infectious particles and nanocarriers.

The hydrophobin SC4 was isolated from the medium of a dikaryon from Schizophyllum commune with disrupted SC3 genes. Although not glycosylated, its biophysical properties were similar to those of SC3. As the hydrophobins SC3 from S. commune and ABH1 and ABH3 from Agaricus bisporus, SC4 self-assembled at hydrophilic-hydrophobic interfaces into an SDS insoluble amphipathic film with a typical rodlet structure at its hydrophobic face, and also proved to be a powerful surfactant. Similar rodlet structures were observed in the fruiting body plectenchyma. By immunodetection SC4 could be localized lining air channels within this tissue. A similar localization was found for the ABH1 hydrophobin in fruiting bodies of A. bisporus. Probably, these hydrophobin coatings prevent collapse of air channels allowing efficient gas exchange even under wet conditions.

The presence of hydrogenosomes in phylogenetically distinct anaerobic eukaryotes implies that they have been acquired independently, and previously reported differences in ultrastructure among taxa have suggested that some hydrogenosomes have different origins. Of particular interest are reports that Neocallimastix frontalishydrogenosomes resemble microbodies in possessing a single membrane, in contrast to those in ciliates and trichomonads which have two and thus resemble mitochondria. In this investigation we have clearly demonstrated that N. frontalishydrogenosomes possess two, rather than one, closely apposed membranes and in some preparations cristae‐like structures were observed. These observations have led us to reject the microbody hypothesis and provide some indirect support for a possible mitochondrion origin as proposed for other hydrogenosomes. N. frontalishydrogenosomes were shown to lack an associated genome as previously demonstrated for trichomonad hydrogenosomes. This might be explained by assuming that a mitochondrial genome encoding proteins for aerobic function is no longer necessary for either organelle.

Growth of Penicillium simplicissimumon anisyl alcohol, veratryl alcohol or 4‐(methoxymethyl)phenol, is associated with the synthesis of relatively large amounts of the hydrogen peroxide producing flavoprotein vanillyl‐alcohol oxidase (VAO). Immunocytochemistry revealed that the enzyme has a dual location namely in peroxisomes and in the cytosol. The C‐terminus of the primary structure of VAO displays a WKL‐COOH sequence which might function as a peroxisomal targeting signal type 1 (PTS1). As VAO activity results in production of hydrogen peroxide also the subcellular location of a recently characterized co‐inducible catalase‐peroxidase was studied. As VAO, this hydroperoxidase is also distributed throughout the cytosol and peroxisomes.

The protein import capacity of peroxisomes in methylotrophic yeasts was studied using Pichia pastoriscontaining one or two extra copies of the gene encoding the peroxisomal protein alcohol oxidase. The alcohol oxidase overproduced in this strain was only partially imported and assembled into the active, octameric form of the protein. The excess remained in the cytosol as protein aggregates composed of monomers. These results are discussed in view of the possible application of peroxisomes as storage compartments for heterologous proteins.

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.

Growth of Penicillium simplicissimumon anisyl alcohol, veratryl alcohol or 4-(methoxymethyl)phenol, is associated with the synthesis of relatively large amounts of the hydrogen peroxide producing flavoprotein vanillyl-alcohol oxidase (VAO). Immunocytochemistry revealed that the enzyme has a dual location namely in peroxisomes and in the cytosol. The C-terminus of the primary structure of VAO displays a WKL-COOH sequence which might function as a peroxisomal targeting signal type 1 (PTS1). As VAO activity results in production of hydrogen peroxide also the subcellular location of a recently characterized co-inducible catalase-peroxidase was studied. As VAO, this hydroperoxidase is also distributed throughout the cytosol and peroxisomes.

The protein import capacity of peroxisomes in methylotrophic yeasts was studied using Pichia pastoriscontaining one or two extra copies of the gene encoding the peroxisomal protein alcohol oxidase. The alcohol oxidase overproduced in this strain was only partially imported and assembled into the active, octameric form of the protein. The excess remained in the cytosol as protein aggregates composed of monomers. These results are discussed in view of the possible application of peroxisomes as storage compartments for heterologous proteins.

Abstract: Hydrogenosomal proteins always contain an amino-terminal extension which is believed to be a hydrogenosomal targeting signal. In the anaerobic fungus Neocallimastix frontalis these putative targeting signals are 27 amino acids long, are enriched in Ala, Leu, Ser and Arg, and have an Arg at position –2 relative to amino-acid 1 of the mature protein. These features are typically observed in mitochondrial targeting signals. Here we show that the 27 amino-acid leader sequence of the hydrogenosomal malic enzyme of N. frontalis was capable of targeting the enzyme to mitochondria of the methylotrophic ascomycete yeast Hansenula polymorpha. The same protein without this leader sequence remained cytosolic. These data suggest a close relationship between the protein import machineries of mitochondria and hydrogenosomes in fungi and provide further support for the notion that these two organelles share a common evolutionary origin.

Understanding etiology of diabetes and finding alternatives for insulin therapy in Type I diabetic patients demands a full understanding of the Islets of Langerhans. Electron microscopic (EM) imaging of islets is a powerful method to analyze islet composition, cellular interactions, hormone content and physiological condition in a single analysis. However, single high-resolution images of complete islets can not be made. Our aim is to create high-resolution EM-maps of cross-sections of islets and share this data in a Google-earth like approach. This will allow examination of important tissue, cellular and (macro)molecular characteristics of Islets between man and animal models in health and disease.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in teh cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped cyrstal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultra-structure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a= 7.6 nm, b= 4 nm, c= 4.2 nm, γ= 75°. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.