Copper accumulation in the cell-wall-deficient slime variant of Neurospora crassa. Comparison with a wild-type strain.

The copper-uptake process in the cell-wall-deficient slime variant of the fungus Neurospora crassa was compared with that in a wild-type strain. In both organisms investigated most of the copper is taken up from the culture medium during the exponential growth period. The wild-type strain, however, accumulates much more copper than does the slime variant. The influence of the copper concentration in the culture medium on the amounts of copper accumulated intracellularly suggests separate ways of copper import used by the two morphologically different N. crassa strains. Copper analyses of three different cytosolic fractions as a function of growth time or exogenous copper concentration indicate both strains to share a very similar copper metabolism. All the data presented are consistent with a detoxification function of the low-Mr copper-binding fraction of N. crassa. Both copper-metallothionein and oxidized glutathione (GSSG) are co-eluted with this fraction. The possible involvement of glutathione in metallothionein biosynthesis is discussed.     

Chitin synthase activity from the slime variant of Neurospora crassa.

Chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity from the wall-less variant of Neurospora crassa (slime) was partially characterized. The slime enzyme activity was found to be similar to that reported for slime-like and wild-type chitin synthase activities with respect to the following: specific activity, particulate cell-fraction localization, activation by N-acetylglucosamine, apparent Km with respect to substrate, pH optimum and ion requirement. It appears that the phenotype of slime cannot be solely accounted for by the absence of chitin synthase enzyme activity.     

Method for identification of intracellular free flavin species in the photosensitive fungus Neurospora crassa

Establishing the relative intracellular proportions of flavins in Neurospora crassa (and in other organisms) in vivo may be hampered by degradation of flavins after homogenization of the cells. The system described here allows separation and identification of intracellular free and bound flavins under conditions restrictive for the FAD-degrading enzyme(s). A "protective buffer" containing 0.1 M citrate adjusted to pH 4.0 with K2HPO4, 5 mM ATP, and 0.5 mM EDTA prevents FAD from rapid enzymatic cleavage in crude cell lysates of the Neurospora crassa mutant "slime."     

Bioelectrorheological model of the cell. 3. Viscoelastic shear deformation of the membrane.

An analytical electromechanical model of a spherical cell exposed to an alternating electric field was used to calculate shear stress generated in the cellular membrane. Shape deformation of Neurospora crassa (slime) spheroplasts was measured. Statistical analysis permitted empirical evaluation of creep of the cellular membrane within the range of infinitesimal stress. Final results were discussed in terms of various rheological models.     

A specific insulin receptor and tyrosine kinase activity in the membranes of Neurospora crassa

Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.     

Identification of the First Fungal Annexin: Analysis of Annexin Gene Duplications and Implications for Eukaryotic Evolution

Annexin homologues have been found in animals, plants, and distinct protist lineages. We report the identification of the first fungal annexin, encoded by the anx14 gene of the filamentous ascomycete Neurospora crassa. Annexins have a complex evolutionary history and exhibit a large number of gene duplications and gene losses in various taxa, including the complete loss of annexin sequences from another ascomycete, the budding yeast Saccharomyces cerevisiae. Surprisingly, the N. crassa annexin homologue is most closely related to the annexin homologue of the slime mold Dictyostelium discoideum, suggesting a phylogenetic link between cellular slime molds and true fungi. Both of these annexin homologues are closely related to the family of annexin homologues present in animals, an observation consistent with the existence of the animal–fungal clade. These data further suggest that the gene duplications that generated the family of annexin sequences present in animals, fungi, and slime molds began prior to the divergence of these taxa. Received: 10 December 1997 / Accepted: 17 April 1998     

The Neurospora crassa DCC-1 protein, a putative histidine kinase, is required for normal sexual and asexual development and carotenogenesis

Two-component signaling pathways based on phosphoryl group transfer between histidine kinase and response regulator proteins regulate environmental responses in bacteria, archaea, plants, slime molds, and fungi. Here we characterize a mutant form of DCC-1, a putative histidine kinase encoded by the NCU00939 gene of the filamentous fungus Neurospora crassa. We show that this protein participates in the regulation of processes such as conidiation, perithecial development, and, to a certain degree, carotenogenesis. Furthermore, DCC-1 is suggested to exert its effect by promoting cyclic AMP production, thereby placing this protein within the context of a signaling pathway.     

(1,3)-β-D-Glucan Synthase Activity in Mycelial and Cell Wall-less Phenotypes of the fz,sg, os-1 ("Slime") Mutant Strain of Neurospora crassa

Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa. Experimental Mycology 19, 35-47. The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-βD-glucan synthase activity. fz, sg, os-1 segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, K_m app, V_max, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.     

Isolation and characterization of a DNA-uptake-stimulating protein from the culture medium of Neurospora crassa slime strain

A protein fraction was purified to homogeneity from the culture medium of the wall-less (slime) strain of Neurospora crassa (FGSC 1118), which proved to be identical with DNA-uptake-stimulating factor (designated DUSF), which has been described earlier [Schablik, M. and Szabó, G. (1981) FEMS Microbiol. Lett. 10, 395-397]. The quantity of DUSF is measured by the amount of [3H]DNA uptake by Neurospora cells at standard conditions. Its relative molecular mass was 230,000. It has an isoelectric point of pH 5.5. This protein consists of two identical subunits, relative molecular mass 110,000.     

Blue light modulation of ion transport in the slime mutant of Neurospora crassa

Blue light is the primary entrainment signal for a number of developmental and morphological processes in the lower eucaryote Neurospora crassa. Blue light regulates photoactivation of carotenoid synthesis, conidiation, phototropism of perithecia and circadian rhythms. Changes in the electrical properties of the plasma membrane are one of the fastest responses to blue light irradiation. To enable patch-clamp studies on light-induced ion channel activity, the wall-less slime mutant was used. Patch-clamp experiments were complemented by non-invasive ion-selective measurements of light-induced ion fluxes of slime cells using the vibrating probe technique. Blue light usually caused a decrease in conductance within 2-5 minutes at both negative and positive voltages, and a negative shift in the reversal potential in whole-cell patch-clamp measurements. Both K+ and Cl- channels contribute to the inward and outward currents, based on the effects of TEA (10 mM) and DIDS (500 microM). However, the negative shift in the reversal potential indicates that under blue light the Cl- conductance becomes dominant in the electrical properties of the slime cells due to a decrease of K+ conductance. The ion-selective probe revealed that blue light induced the following changes in the net ion fluxes within 5 minutes: 1) decrease in H+ influx; 2) increase in K+ efflux; and 3) increase in Cl- influx. Ca2+ flux was unchanged. Therefore, blue light regulates an ensemble of transport processes: H+, Cl-, and K+ transport.     

Purification and characterization of flavokinase from Neurospora crassa

The ATP-dependent phosphorylation of riboflavin to FMN by flavokinase is the key step in flavin biosynthesis. Flavokinase has been purified from a fungal source for the first time. The enzyme purified from a cell wall lacking mutant of Neurospora crassa, slime, is a monomer of M(r) 35.5 kDa with maximal activity at alkaline pH and high temperature (55 degrees C). The K(m) for both substrates is the lowest reported for flavokinase from any source so far (120 nM for riboflavin and 210 nM for MgATP2-). The enzyme exhibits preference for Mg2+ over Zn2+ as the essential activator and is also significantly activated by several cations. Activation by orthophosphate may be physiologically relevant for the intracellular regulation of flavokinase.     

Reorganization of plasma membrane lipid domains during conidial germination

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted.Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30 °C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.     

Isolation and biochemical properties of two types of microbody from Neurospora crassa cells.

Cells of the Neurospora crassa slime mutant grown in sucrose medium exhibited low activities of glyoxysomal marker enzymes isocitrate lyase (ICL), malate synthetase (MS), and malate dehydrogenase. Transfer of the cells to a medium containing acetate as sole carbon source ("acetate medium") induced a strong increase in the activities of these enzymes in both the soluble and the crude particulate cell fraction. Soluble isocitrate lyase activity increased rapidly after a lag phase of about 45 minutes. Addition of 0.1 mM cycloheximide to the acetate medium 3 hours after transfer of the cells halted the rise of isocitrate lyase activity in either cell fraction, but the inhibition of the incorporation of ICL activity into the particulate cell fraction was delayed by 1 hour. Addition of 20 g/l glucose resulted in the immediate decrease of both soluble and particulate ICL activities. Transfer to acetate medium induced no change in the activities of other microbody marker enzymes such as catalase, uricase or D-amino acid oxidase. Resolution of crude homogenates of "slime" cells by sucrose density gradient centrifugation yielded two major protein bands: A mitochondrial band at a density of 1.180 kg/l showing maximum activites of fumarase, isocitrate dehydrogenase and cytochrome c oxidase, and a microbody-rich band which obviously consisted of two types of organelles with different biochemical properties. Maximum activities of ICL and MS sedimented at a density of 1.21 kg/l while the peaks of particulate uricase and catalase activities were recovered at 1.24 kg/l.     

Bioelectrorheological model of the cell. 2. Analysis of creep and its experimental verification

The electrorheological model of the cell proposed in Part 1 of this work was used to analyze changes in time of the shape of a cell acted on by a constant-amplitude external alternating electric field, with lossiness of the media taken into account. Shear stress in the cell membrane was determined. This model was then subjected to preliminary experimental verification using Neurospora crassa (slime) spheroplasts subjected to an external alternating electric field of constant frequency (3 MHz) and varying magnitude for different periods of time. Reversible viscoelastic cell deformation and fatigue (stiffening) of the material were observed. A satisfactory fit of the experimental data to Burgers' rheological model was found, and the values of the elastic moduli E1 = 9 X 10(2) N/m2, E2 = 2 X 10(2) N/m2 and viscosities eta 1 = 8 X 10(4) Ns/m2; eta 2 = 7 X 10(3) Ns/m2 were evaluated.     

Determination of electric parameters of cell membranes by a dielectrophoresis method.

Marszalek, P., J. J. Zielinsky, and M. Fikus (1989. Bioelectrochem. Bioenerg. 22:289-298) have described a novel design for measuring the complete dielectrophoretic spectrum of a single cell. From the analysis of the dielectrophoretic spectrum, the membrane conductivity, sigma membr, and the membrane dielectric permittivity, epsilon membr, of the cell may be determined according to the theory of dielectrophoresis described by Sauer, F. A. (1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). At Fo, the net force experienced by a single shell sphere in a nonuniform periodic field is zero, and the sphere ceases to move in the field. In other words, at Fo, the effective polarizability, chi eff, of the sphere (the polarizability of sphere minus the polarizability of the medium) is equal to zero. For biological cells in high conductivity medium, e.g., the isotonic saline, sigma membr falls below 2 x 10(-6) S m-1, where Fo becomes insensitive to sigma membr, and the method becomes impractical. In a low conductivity medium, 0.3 M sucrose, sigma membr of cells is generally higher and the method may be applied. Assuming a membrane thickness of 9 nm, epsilon membr of Neurospora crassa slime cells was determined to be in the range of 8.3-9.4 epsilon o, and of myeloma Tib9 to be 9.4 epsilon o, epsilon o being the dielectric permittivity of the free space. The values for the slime cells were compared with values obtained by the dielectric spectroscopy method which measures average values for cells in suspension.     

Ribosomal RNA genes of Neurospora crassa: multiple copies and specificities

Ribosomal RNA genes were isolated from the germinated conidial and mycelial cells of N. crassa by repeated cycles of 3H-DNA:rRNA reactions followed by hydroxyapatite chromatography. Specificity of multiple copies of those rDNAs with respect to N. crassa cell types was studied. The fraction of N. crassa germinated conidial in vitro labelled 3H-DNA recovered in the presence of rRNA isolated from the same cell type was about 2.2%, when compared with approximately 1.2% rDNAs obtained in mycelial cells. These isolated rDNAs reacted specifically to 26S and 17S rRNAs of eukaryotic (N. crassa) organisms and did not react with 4S tRNAs. rRNA:rDNA reassociation kinetics studies indicate that 90% of the rRNA genes were homogeneous and not identical with the other 10% rRNA genes isolated from N. crassa mycelia. These studies suggest that the possible heterogeneity of rDNA sequences of N. crassa cannot be attributed to inclusion of any tDNA sequences as has been shown in the heterogeneity of rDNA sequences of the bacterium Escherichia coli. The heterogeneity of multiple copies of N. crassa rDNAs could be due to differences in internal or external spacer regions of N. crassa rRNA genes.     

Characterization of a cobalt-resistant mutant of Neurospora crassa with transport block

A cobalt-resistant strain of Neurospora crassa (cor) was obtained by repeated subculturing of the wild type on cobalt-containing agar medium. N. crassa cor is twentyfold more resistant to cobalt ions compared with the wild type. Resistance was stable on repeated subculturing of cor on cobalt-free media. N. crassa cor is also cross-resistant to nickel (fourfold), but not to zinc or copper. Higher concentrations of iron and magnesium ions are required to reverse growth inhibition due to cobalt toxicity in N. crassa cor, compared with the wild type. Germinating conidia and mycelia of the cor strain accumulated lower levels of cobalt ions compared with the parent N. crassa. The partial transport block for cobalt uptake is shown to be primarily due to decreased surface binding of cobalt to mycelia and cell walls. Efflux of mycelial cobalt was also observed in wild type and cobalt-resistant N. crassa. The characteristics of cor in comparison with wild type N. crassa are discussed in relation to the mechanisms of cobalt resistance.