Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius

Hyde, Geoffrey J.; Davies, Danielle; Perasso, L.; Cole, Louise; Ashford, A. E.

doi:10.1002/(sici)1097-0169(1999)42:2<114::aid-cm3>3.3.co;2-e

Cited by 18 publications

(33 citation statements)

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“…Morton et al (24) reported that latB affects the kinetics of actin polymerization by specifically binding to actin monomers and shifting the equilibrium to the disassembled state. In the filamentous fungus Pisolithus tinctorius, a perturbation of microfilament organization by latrunculin was shown (15), and in yeast and mammalian cells, receptor-mediated endocytosis was inhibited (3,19). We found that PAF transport into hyphal cells decreased with increasing concentrations of latB, and 50 g of latB/ml abolished internalization (Fig.…”

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confidence: 57%

Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli

Oberparleiter¹,

Kaiserer²,

Haas³

et al. 2003

Antimicrob Agents Chemother

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The Penicillium chrysogenum antifungal protein PAF inhibits the growth of various filamentous fungi. In this study, PAF was found to localize to the cytoplasm of sensitive aspergilli by indirect immunofluorescence staining. The internalization process required active metabolism and ATP and was prevented by latrunculin B, suggesting an endocytotic mechanism.Antimicrobial proteins are produced by many organisms, including humans, amphibians, arthropods, plants, and fungi (6,11,13,32,37). The Penicillium antifungal protein PAF is abundantly secreted into the supernatant of the ␤-lactam-producing mold Penicillium chrysogenum (22). This small, basic, and cysteine-rich protein specifically inhibits the growth of numerous filamentous fungi (16). Although its primary amino acid structure resembles that of antifungal proteins isolated from other molds, e.g., Aspergillus giganteus (AFP), A. niger (ANAFP), and P. nalgiovense (NAF), significant differences exist in their species specificity (12,20,38). Knowledge of the bases of this selectivity is essential for determining strategies by which to overcome the resistance of important pathogens and to design new antifungal agents. Most data on the possible mechanism of action derive from studies on plant antifungal proteins and AFP which support the hypothesis of an interaction with the plasma membrane that would result in its permeabilization (18,34,35). So far, experiments have addressed such an interaction only indirectly. Therefore, we were interested in elucidating the site of action of the antifungal protein PAF from P. chrysogenum in order to gain better insight into putative target structures that might play a role in the activity of the protein. Here we report the localization of PAF in the sensitive molds A. nidulans, A. fumigatus, and A. niger and give evidence for an active transport of the antifungal protein into affected hyphae.Purification of PAF and generation of polyclonal antiserum. PAF was purified as described previously (16), and polyclonal antibodies against PAF were raised in rabbits as reported elsewhere (26). To determine antibody specificity and the optimal antibody concentration, 25 to 200 ng of PAF was immobilized on Biotrace-NT 0.45-m nitrocellulose membrane strips (PALL Corp., Ann Arbor, Mich.) and detected with various dilutions of rabbit anti-PAF serum and alkaline phosphataseconjugated goat anti-rabbit immunoglobulin G (IgG; 1:10,000; Sigma, Vienna, Austria) in accordance with the protocol previously described (21). The polyclonal antiserum reacted specifically with PAF, and no signals were obtained with the control serum that was collected before the first injection (data not shown).Localization of PAF in aspergilli. Fungi were grown overnight on glass coverslips at 30°C in complete medium CM (16) inoculated with 10 6 conidia/ml. The samples were stained in accordance with the protocol of Fischer et al. (9). In brief, prior to fixation, hyphae were treated with 10 to 50 g of PAF/ml in CM for 90 min at room temperature. The samples were incubat...

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confidence: 57%

Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli

Oberparleiter¹,

Kaiserer²,

Haas³

et al. 2003

Antimicrob Agents Chemother

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“…Nickel uptake is thoroughly documented in other fungal cells (Mohan et al, 1984;Joho et al, 1995;Nishimura et al, 1998;Dönmez & Aksu, 2001) and it has been localized within Hebeloma crustuliniforme ectomycorrhizas (Brunner & Frey, 2000). Vacuole vesiculation observed during Ni 2+ exposure also occurs by exposure to acids, cytoskeletal drugs, fixatives and fluorescent probes (Wilson et al, 1990;Cole et al, 1997Cole et al, , 2000Bachewich & Heath, 1999;Hyde et al, 1999), suggesting that this is a general response to toxins.…”

Section: +mentioning

confidence: 99%

“…These results suggest that Ni 2+ effects on MTs are related to the disruption of organelle tubularity and motility in P. involutus. Microtubule disruption causes vacuole vesiculation and mitochondrial fragmentation in other fungal cells (Steinberg et al, 1998;Bachewich & Heath, 1999;Hyde et al, 1999;McDaniel & Roberson, 2000;Khalaj et al, 2001;Fuchs et al, 2002). Interestingly MT disruption also triggers apoptosis through the mitochondrial pathway (Chen et al, 2002;Giacca, 2005. Instead of MTs, various fungal cells rely on the actin cytoskeleton for organelle motility and morphology (Morris & Hollenbeck, 1995; Simon et al, 1995;Gupta & Heath, 1997;Bachewich & Heath, 1998Suelmann & Fischer, 2000).…”

Section: +mentioning

confidence: 99%

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Tuszyńska

2006

New Phytol

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Summary• Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni 2+ ) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil.• Vacuoles, mitochondria and microtubules were labelled with Oregon Green ® 488 carboxylic acid diacetate, 3,3 ′ -dihexyloxacarbocyanine iodide (DiOC 6 (3)) and anti-α -tubulin antibodies, respectively; hyphae were treated with NiSO 4 in the range of 0-1 mmol l − 1 and examined microscopically.• Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni 2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO 4 removal and tubular mitochondria in the presence of NiSO 4 suggesting cellular detoxification.• These results demonstrate that Ni 2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni 2+ exposure suggests cellular detoxification of the metal ion.

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“…MT-dependent processes that, if disrupted, are likely to affect the multicomponent tip growth process include the transportation and organization of vesicles Lehmler et al, 1997;Seiler et al, 1997;Steinberg & Schliwa, 1993Steinberg et al, 1998;Wedlich-Soldner et al, 2000;Wu et al, 1998), nuclei Herr & Heath, 1982;Kaminskyj et al, 1989;McKerracher & Heath, 1986;Meyer et al, 1988;Oakley & Morris, 1980;Oakley & Rinehart, 1985;Steinberg & Schliwa, 1993;Wedlich-Soldner et al, 2000;Wu et al, 1998), vacuoles (Allaway et al, 1997;Herr & Heath, 1982;Hyde et al, 1999;Shepherd et al, 1993;Steinberg et al, 1998), mitochondria Herr & Heath, 1982;Steinberg & Schliwa, 1993;Wu et al, 1998) and the endoplasmic reticulum (Wedlich-Soldner et al, 2002). However, direct structural evidence of MT and organelle interactions is rare (Heath, 1994).…”

Section: Organelle Positioningmentioning

confidence: 99%

The dynamic behaviour of microtubules and their contributions to hyphal tip growth in Aspergillus nidulans

Sampson

Heath

2005

Microbiology

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Creating and maintaining cell polarity are complex processes that are not fully understood. Fungal hyphal tip growth is a highly polarized and dynamic process involving both F-actin and microtubules (MTs), but the behaviour and roles of the latter are unclear. To address this issue, MT dynamics and subunit distribution were analysed in a strain of Aspergillus nidulans expressing GFP-a-tubulin. Apical MTs are the most dynamic, the bulk of which move tipwards from multiple subapical spindle pole bodies, the only clear region of microtubule nucleation detected. MTs populate the apex predominantly by elongation at rates about three times faster than tip extension. This polymerization was facilitated by the tipward migration of MT subunits, which generated a tip-high gradient. Subapical regions of apical cells showed variable tubulin subunit distributions, without tipward flow, while subapical cells showed even tubulin subunit distribution and low MT dynamics. Short MTs, of a similar size to those reported in axons, also occasionally slid into the apex. During mitosis in apical cells, MT populations at the tip varied. Cells with less distance between the tip and the first nucleus were more likely to loose normal MT populations and dynamics. Reduced MTs in the tip, during mitosis or after exposure to the MT inhibitor carbendazim (MBC), generally correlated with reduced, but continuing growth and near-normal tip morphology. In contrast, the actin-disrupting agent latrunculin B reduced growth rates much more severely and dramatically distorted tip morphology. These results suggest substantial independence between MTs and hyphal tip growth and a more essential role for F-actin. Among MT-dependent processes possibly contributing to tip growth is the transportation of vesicles. However, preliminary ultrastructural data indicated a lack of direct MT-organelle interactions. It is suggested that the population of dynamic apical MTs enhance migration of the 'cytomatrix', thus ensuring that organelles and proteins maintain proximity to the constantly elongating tip.

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Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius

Cited by 18 publications

References 0 publications

Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli

Active Internalization of the Penicillium chrysogenum Antifungal Protein PAF in Sensitive Aspergilli

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

The dynamic behaviour of microtubules and their contributions to hyphal tip growth in Aspergillus nidulans

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