Growth rate of Aspergillus nidulans hyphae is independent of a prominent array of microtubules

Abstract

Roles for the microtubule (MT) cytoskeleton in fungal growth include mitosis and nuclear migration but otherwise are less clearly understood. Confocal microscopy was used to quantify MT abundance and growth rate in hyphae of a haploid Aspergillus nidulans strain containing green fluorescent protein (GFP)-α-tubulin. There was no correlation between growth rate and MT abundance for 112 growing hyphae in an untreated population. However, 109 nongrowing hyphae from the same group had lower average MT abundance. Results for untreated cells were compared with cells treated for 30-120 min with the MT drugs benomyl and taxol, the actin drug latrunculin B, and with solvents used for the drug treatments. Compared with their respective controls, MT abundance was significantly increased by dimethyl sulfoxide (DMSO), significantly reduced by benomyl, and moderately increased by latrunculin, but was unaffected by ethanol. In the same cells, growth rates were significantly increased by ethanol and taxol, significantly reduced by latrunculin, and unaffected by DMSO. Average hyphal growth rate in the first 60 min following 1 μg/ml benomyl treatment was statistically similar to untreated cells, despite the absence of visible MTs after 2 min of treatment. However, growth rate was significantly reduced by 2.5 μg/ml benomyl over the same time period, implying additional effects at the higher concentration. For individual hyphae in each treatment, growth rates varied over short time periods; treatment with 0.1% ethanol substantially increased this variability. Growth rates of taxol-treated hyphae decreased following fluorescence observation, suggesting a possible application to cancer chemotherapy. Overall, there was no correlation between cytoplasmic MT abundance and A. nidulans growth rate within 2 h of cytoskeletal drug or solvent treatment.Abbreviations DIC differential interference contrast GFP green fluorescent protein MT microtubule TEM transmission electron microscopy

Rapid tip-directed movement of Golgi equivalents in growing Aspergillus nidulans hyphae suggests a mechanism for delivery of growth-related materials

“…These were consistent with Aspergillus GEs imaged with TEM, which have single pleiomorphic cisternae (Beckett et al, 1974;Kaminskyj & Boire, 2004; Kurtz et al, 1994), similar to those of Pisolithus (Xu et al, 2004) and Schizophyllum (Rupeš et al, 1995). Fluorescent structures are luminous, which enhances detection and can increase apparent size (Hubbard & Kaminskyj, 2007). The apparent size of A. nidulans GEs is also consistent with results from plants, which have numerous, motile Golgi bodies (Boevink et al, 1998).…”

“…, which we had shown previously depolymerizes all cytoplasmic MTs within 2 min but does not prevent hyphal tip growth within 1 h (Hubbard & Kaminskyj, 2007), had reduced tip growth rates, but significantly increased anterograde GE movement (Fig. 4b).…”

“…BFA was stored at 220 uC as a 5 mg ml 21 stock in 100 % methanol, and used at 10 mg ml 21 in 0.5 % methanol. The inhibitor concentrations were similar to or lower than those typically used (benomyl, BFA and latrunculin), or were the lowest for which a response was detected (taxol) as described in Hubbard & Kaminskyj (2007).…”

“…GFP expression was induced by growth on complete medium (CM) containing 1 % (v/v) glycerol or 1 % ethanol as the sole carbohydrate source, or on Difco nutrient agar supplemented with 0.5 % threonine. Anhydrous ethanol or solid threonine was added to cool, autoclaved media.For microscopy, freshly harvested spores were inoculated onto sterile dialysis tubing overlying solid medium, and grown overnight at 28 uC (Hubbard & Kaminskyj, 2007). For drug-treated cells, the dialysis tubing and overlying hyphae were mounted in a microscope slide chamber in~100 ml liquid medium that contained solvents and cytoskeleton-selective inhibitors as required, and allowed to recover for 30 min before observation, as described in Hubbard & Kaminskyj (2007).…”

“…BFA was stored at 220 uC as a 5 mg ml 21 stock in 100 % methanol, and used at 10 mg ml 21 in 0.5 % methanol. The inhibitor concentrations were similar to or lower than those typically used (benomyl, BFA and latrunculin), or were the lowest for which a response was detected (taxol) as described in Hubbard & Kaminskyj (2007).Confocal microscopy. A. nidulans hyphae were imaged with a Zeiss META 510 laser scanning confocal microscope with a Plan Apochromat 663, numerical aperture 1.2 multi-immersion objective equipped with phase-contrast optics.…”