Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus.

Do fathead minnows, Pimephales promelas Rafinesque, alter their club cell investment in responses to variable risk of infection from Saprolegnia?

Fish in the Superorder Ostariophysi possess large epidermal club cells that release chemical cues warning nearby conspecifics of danger. Despite the long-held assumption that such club cells evolved under the selective force of predation, recent studies demonstrated that predation has no effect on club cell investment. Rather, club cells have an immune function and cell production may be stimulated by skin-penetrating pathogens and parasites. The current work investigates whether fathead minnows, Pimephales promelas, alter their club cell characteristics based on variation in infection risk. In a 2 × 3 design, we exposed minnows to infective cysts of two oomycete species (Saprolegnia ferax and S. parasitica) at three different concentrations (2, 20 or 200 cysts L(-1)). Club cell characteristics (number and size) were quantified 12 days after exposure. Saprolegnia parasitica is thought to be more pathogenic than S. ferax, hence we predicted greater club cell investment and a larger turnover rate of cells by minnows exposed to S. parasitica than S. ferax. We also predicted that minnows exposed to higher numbers of cysts should invest more in club cells and have a higher turnover rate of cells. We found no difference in club cell density or size between fish exposed to the two Saprolegnia species; however, fish exposed to high concentrations of pathogens had smaller club cells than those exposed to low concentrations, indicating a higher rate of turnover of cells in the epidermis.

Synthesis and antifungal properties of compounds which target the alpha-aminoadipate pathway.

Fungi synthesize lysine via the alpha-aminoadipate pathway, which is not found in plants or animals. This pathway has been proposed as a target for antifungal agents, but until now no reports have appeared to test this proposal. Hampering studies on the susceptibility of filamentous fungi such as those of the clinically important genus Aspergillus is the fact that growth quantitation is notoriously difficult. We have used the recently-reported XTT-based method of biomass quantitation to measure the susceptibility of Aspergillus nidulans strain A28 to growth suppression by novel compounds designed to target early steps in the alpha-aminoadipate lysine biosynthesis pathway, specifically those steps involving (R)-homocitrate and (2R,3S)-homoisocitrate. Three compounds show moderate inhibition of fungal growth, which can be partially restored by the presence of lysine in the growth medium.

Septum position is marked at the tip of Aspergillus nidulans hyphae.

Aspergillus nidulans hyphae have long tip cells that are separated from short basal cells by septa. Basal cells average 40 microm long with three or four nuclei. Septation follows parasynchronous mitoses in the tip cell and seems to occur at premarked sites, but how these sites are established is unclear. A. nidulans strains with the hypA1 mutation are wildtype at 28 degrees C but if shifted to 42 degrees C, their tip cells insert septa with a wildtype spacing, apparently triggered by an aberrant mitosis. Tip cell septa are trilamellar, like wildtype, but lack a central pore. Like wildtype, tip cell septation requires a minimum cell size and is inhibited by actin and microtubule poisons. In a hypA1 background, tip cell septation is blocked by nim (never in mitosis) mutants, but not by bim (blocked in mitosis) mutants. Future septation sites appear to be established during tip growth, before their activation in basal regions.

hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.

Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth.

Saprolegnia ferax contains an integrin homologue, identified by crossreactivity with antiserum to the consensus sequence of human/chick/Xenopus cytoplasmic domain beta 1-integrin, which is highly conserved. In non-reduced samples, this integrin was larger than the reported size range for beta 1-integrins, at 178 kDa. In reduced samples, there was a reducing agent-concentration-dependent conversion from 178 kDa to 120 kDa, well within the reported size range for beta 1-integrins in other organisms. The integrin antiserum stained plasma membrane-associated patches, which had a shallow tip-high gradient. This population was reduced and its distribution perturbed in hyphae whose growth rate was reduced by half with tetrapentyl ammonium chloride. The expected integrin function in cytoplasm-cell wall attachment was shown by differential resistance to plasmolysis-induced separation, which positively correlated with integrin abundance. However, when there was separation, remnants of cytoplasm stayed attached to the wall. These were enriched in actin and integrin. Saprolegnia also has a spectrin homologue identified by crossreactivity with an erythrocyte spectin antibody, which has a size (246 kDa) similar to other organisms. This spectrin had a superficially similar distribution to that of integrin, but it did not participate in cytoplasm-wall anchoring. These data suggest that Saprolegnia hyphae have a plasma membrane which is strengthened by spectrin, and cytoplasm which is attached to the cell wall by integrin.

A comparison of techniques for localizing actin and tubulin in hyphae of Saprolegnia ferax.

We have evaluated protocols for immunofluorescence (IF) staining of the potentially interacting actin filaments (F-actin) and microtubules in hyphae of Saprolegnia ferax, using rhodamine-phalloidin (RP) and freeze-substitution electron microscopy (FSEM), respectively, as standards for their distribution. Saprolegnia has four distinguishable cortical F-actin populations with characteristic organizations and RP- and actin-IF-staining affinities, all of which could be labeled with both probes after some protocols. Other protocols stained only some of the populations. Cortical F-actin was always more reproducibly and sharply stained with RP than IF, indicating that the former is the probe of choice for F-actin in these cells. Although no single IF protocol revealed all of the F-actin and microtubule populations, showing the potential need to optimize protocols for specific antibodies, simultaneous localization was readily achieved by dual labeling with RP and tubulin IF. Tubulin IF patterns differed from FSEM: mitotic spindles were revealed but not the more abundant prophase microtubule arrays, and the cytoplasmic microtubules were subapically displaced and bundled into long cables. These cables, which apparently linked nuclei, indicate a previously undetected involvement in nuclear spacing. The tubulin antibody successfully used for IF failed to recognize any proteins in immunoblots, indicating that immunoblots may not always be a useful indicator of success with IF.

Nuclear migration in a nud mutant of Aspergillus nidulans is inhibited in the presence of a quantitatively normal population of cytoplasmic microtubules.

Nuclear migration was studied in germinating conidia of a temperature-sensitive mutant of the fungus Aspergillus nidulans. At the restrictive temperature motility was demonstrably impaired because significantly fewer nuclei migrated into the germ tube relative to a population of similarly sized germlings grown at the permissive temperature. Further comparison of these populations showed that the mutant was leaky in that an increasing number of nuclei migrated as the total nuclear content increased in each germling. The restrictive temperature also induced elevated mitotic asynchrony and increased numbers of nuclei per germling. Serial section-based reconstruction of the microtubules in a freeze-substituted germling showed that they were not attached to the nucleus-associated organelles, were approximately parallel to the long axis of the germ tube, and seemed to be randomly distributed between the central and peripheral cytoplasm. Five germlings from each temperature were selected for quantitative analysis of cytoplasmic microtubules. All 10 germlings had typical nuclear migration phenotypes. No significant temperature-related difference in microtubule density was found. We conclude that inhibition of nuclear migration in the mutant is the effect of some defect other than the failure of cytoplasmic microtubules to assemble to their normal population density. We also suggest that nuclear motility is not dependent on mitosis-related microtubules.

Basal body loss during fungal zoospore encystment: evidence against centriole autonomy.

The controversial question of the possible autonomy of centrioles, as shown by the persistence of all or part of them in the generative cell line throughout the life cycle of organisms, remains unresolved. All previous reports on shedding or withdrawal of cilia and flagella showed that their basal bodies (= centrioles) were retained in the cells where they may, or may not, subsequently disassemble. We show that in the fungus Neocallimastix sp. the basal bodies are discarded with the flagella when zoospores encyst. This shedding of basal bodies argues against centriolar persistence in any form and thus against their autonomy and endosymbiotic origin.