Klaas Sjollema

Immunolabeling artifacts and the need for live-cell imaging

Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.

Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance in Saccharomyces cerevisiae

Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of peroxisome numbers. This was not observed in glucose-grown dnm1 cells, but was evident in cells grown in the presence of oleate. Similar observations were made in cells lacking Fis1p, a protein involved in Dnm1p function. Fluorescence microscopy of cells producing Dnm1-GFP or GFP-Fis1p demonstrated that both proteins had a dual localization on mitochondria and peroxisomes. Quantitative analysis revealed a greater reduction in peroxisome number in oleate-induced vps1 cells relative to dnm1 or fis1 cells. A significant fraction of oleate-induced vps1 cells still contained two or more peroxisomes. Conversely, almost all cells of a dnm1 vps1 double-deletion strain contained only one, enlarged peroxisome. This suggests that deletion of DNM1 reinforces the vps1 peroxisome phenotype. Time-lapse imaging indicated that during budding of dnm1 vps1 cells, the single peroxisome present in the mother cell formed long protrusions into the developing bud. This organelle divided at a very late stage of the budding process, possibly during cytokinesis.

Salt shock-inducible Photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP+ reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain

Relative to ferredoxin:NADP(+) reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700(+), suggest lack of function of the truncated FNR in the plastoquinone-cytochrome b(6)f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated DeltapetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275-4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b(6)f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.

Hydrophobins line air channels in fruiting bodies of Schizophyllum commune and Agaricus bisporus

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.

Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome

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 frontalis hydrogenosomes 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. frontalis hydrogenosomes 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. frontalis hydrogenosomes 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.

Thioalkalispira microaerophila gen. nov., sp nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake

An anaerobic enrichment medium (pH 10) with thiosulfate as electron donor and nitrate as electron acceptor was inoculated with sediment from soda lake Fazda (Wadi Natrun, Egypt); a novel strain, ALEN 1(T), was isolated from the subsequent enrichment culture. Cells of strain ALEN 1(T) had a spiral morphology (0.3-0.45 x 1-4 microm), were motile and had a single polar flagellum. Sphaeroplasts were formed by the cells and were rapidly lysed during prolonged aerobic incubation of cultures. Cells of strain ALEN 1(T) contained a membrane-associated yellow pigment. The metabolism of this novel organism was obligately chemolithoautotrophic, and thiosulfate or sulfide were utilized as electron donors. Washed cells of strain ALEN 1(T) oxidized thiosulfate, sulfide, polysulfide and elemental sulfur to sulfate. Best growth was observed when the strain was grown under micro-oxic conditions (1-2% O2 in gas phase), whereas growth was inhibited under fully oxic conditions. Nitrate was reduced to nitrite without growth of the novel organism, but other nitrogen oxides were not utilized as electron acceptors. Strain ALEN 1(T) was alkaliphilic and moderately halophilic. It grew between pH 8 and 10.4 (optimum around pH 10) with a salt concentration of between 0.3 and 1.5 M Na+ (optimum 0-5 M). The maximum growth rate (0.08 h(-1)) of the organism was achieved in a thiosulfate-limited micro-oxic continuous culture (pH 10). Phylogenetic analyses of the 16S rDNA sequences of strain ALEN 1(T) and its closest relatives demonstrated that this strain formed a deep branch within the gamma-Proteobacteria, with no obvious association to any described cluster of species/genera. On the basis of its unique physiological properties and distinct phylogenetic position, it is proposed that strain ALEN 1(T) (= DSM 14786(T) = UNICEM 212(T)) represents a novel genus within the gamma-Proteobacteria, for which the name Thioalkalispira is proposed. It is also proposed that the type species of this novel genus be named Thioalkalispira microaerophila.

The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa

Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.

Nonviral Gene Delivery Vectors Use Syndecan-Dependent Transport Mechanisms in Filopodia To Reach the Cell Surface

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.