Uptake and compartmentalisation of fluorescent probes byPisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Cole, Louise; Hyde, Geoffrey J.; Ashford, A. E.

doi:10.1007/bf02539802

Cited by 56 publications

(46 citation statements)

References 48 publications

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“…Improved staining, but restricted to thin rhizomorphs, was obtained for lucifer yellow carbohydrazide (LYCH ; 20 µg ml −" diH # O). As previously recorded (Cole, Hyde & Ashford 1997), this dye stained fungal cell walls and dolipore septa (Fig. 1).…”

Section: Test Of Stainssupporting

confidence: 83%

Visualisation of ectomycorrhizal rhizomorph structure using laser scanning confocal microscopy

Schweiger

Rouhier

Söderström

2002

Mycological Research

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A method for the observation of the three-dimensional structure of intact ectomycorrhizal rhizomorphs is described. The method is based on a combination of clearing the material with KOH followed by staining with congo red and subsequent imaging under a laser scanning confocal microscope (LSCM). The images obtained are of a much higher three-dimensional resolution than those obtained previously by use of conventional light microscopical techniques. The structure of highly differentiated and undifferentiated rhizomorphs is described. Applications of the method are briefly discussed.

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Section: Test Of Stainssupporting

confidence: 83%

Visualisation of ectomycorrhizal rhizomorph structure using laser scanning confocal microscopy

Schweiger

Rouhier

Söderström

2002

Mycological Research

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“…In this report, we established three strains expressing the fusion protein of EGFP with AoVam3p, a putative vacuolar t-SNARE in Aspergillus oryzae. In contrast to conventional methods, in which the vacuolar lumen is stained with either CFDA derivatives (2,6,7,34,42) or CPY-EGFP (23,24), EGFP-AoVam3p visualizes vacuolar membranes and therefore allowed us to observe vacuolar membrane dynamics at high resolution easily and reproducibly. Furthermore, several aspects of fungal vacuoles that had not been paid much attention previously, such as vacuoles in aerial and glass surface hyphae, as well as the internalization of EGFP fluorescence in the vacuolar lumen in old hyphae, were demonstrated for the first time using our observation system.…”

Section: Discussionmentioning

confidence: 99%

“…First, vacuoles in filamentous fungi display approximately predictable pleomorphism in the different regions of the mycelia; vacuoles are typically rare or absent in the apical region, whereas ovoidspherical vacuoles are present in the subapical region, tubular vacuoles in the more distal region, and large spherical vacuoles in the basal zone (14). Second is the presence of highly motile tubular vacuoles that can be visualized by vacuolar fluorescent probes such as 6-carboxyfluorescein diacetate (CFDA) derivatives (2,6,7,34,42) or N-(3-triethylammoniumpropyl)-4-(pdiethylaminophenyl-hexatrienyl)pyridinium dibromide (FM4-64) (10) and by the fusion protein of carboxypeptidase Y (CPY) with enhanced GFP (EGFP) (23,24). These characteristic vacuolar morphologies indicate that the vacuoles may have particular roles in filamentous fungi.…”

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confidence: 99%

Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae

2006

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Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra-and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both ⌬vam3 and ⌬pep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.Vacuoles are acidic compartments that are important for metabolite storage and cytosolic ion and pH homeostasis (for a review, see reference 18). In the unicellular yeast Saccharomyces cerevisiae, roles of and transport pathways to vacuoles have been extensively studied. Especially, many studies have been focused on the syntaxin family t-SNAREs (target organelle soluble N-ethyl-maleimide-sensitive factor [NSF] attachment protein receptors), which are central molecules of intracellular vesicular transport. t-SNAREs that reside on the target organelle membrane mediate the fusion of transport vesicles with the organelles via the specific interaction with vesicle SNAREs on the transport vesicles (29). S. cerevisiae Pep12p is the syntaxin family t-SNARE on the late endosome/ prevacuolar compartment and receives Golgi-derived vesicles (3). Vam3p is the vacuolar membrane syntaxin that regulates the vesicular traffic to and the homotypic fusion of vacuoles (44). Since S. cerevisiae Vam3p localizes exclusively on the vacuolar membrane, Vam3p is frequently used for visualization of vacuoles (45, 46) and as a marker of vacuoles in subcellular fractionation experiments. AtVam3p is a Vam3p-homologue protein in Arabidopsis thaliana (30), and its fusion protein with...

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“…Also, no biochemical assay for any endocytotic fusion or transport event has been performed with yeast membranes in vitro (Vida and Emr, 1995). A similar situation exists with fungal hyphae, where membrane-impermeant probes either did not penetrate fungal walls or were not internalized by fluid-phase endocytosis (Cole et al, 1997).…”

Section: Figs 8 Andmentioning

confidence: 99%

Endocytosis and Membrane Turnover in the Germ Tube ofUromyces fabae

Hoffmann

Mendgen

1998

Fungal Genetics and Biology

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Hoffmann, J. and Mendgen, K. 1998. Endocytosis and membrane turnover in the germ tube of Uromyces fabae. Fungal Genetics and Biology 24, 77-85. We have used the fluorescent dye FM4-64 as a tracer to demonstrate bulk membrane internalization (endocytosis) and redistribution of the dye within the cytoplasm of the germ tube of the rust fungus Uromyces fabae. Staining of the hyphal membrane was detected 4 s after application of FM4-64 and reached a maximum after 1 min. The highest fluorescence intensity occurred in the apex. Subsequently, staining of the plasma membrane decreased and a subapical region of the fungal protoplast (5-20 m from the tip) displayed increasing fluorescence with a maximum after 5 min. Fluorescence in the subapical region was redistributed to an area in the hyphal tip, which corresponds to the accumulation of apical vesicles, after 10-15 min and subsequently to a cytoplasmic region in front of the two nuclei (35-45 m from the tip). We conclude from our measurements of membrane fluorescence that the turnover time from endocytosis to secretion of the dye amounts to 15 min. The uptake of the dye into the cytoplasm, but not membrane loading, could be inhibited completely with 5 mM NaN 3 or by a temperature shift to 4°C. This is the first evidence for endocytosis in a fungal germ tube. Academic PressIndex Descriptors: tip growth; hyphal apex; Spitzenkö rper; apical vesicle cluster.

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Uptake and compartmentalisation of fluorescent probes byPisolithus tinctorius hyphae: evidence for an anion transport mechanism at the tonoplast but not for fluid-phase endocytosis

Cited by 56 publications

References 48 publications

Visualisation of ectomycorrhizal rhizomorph structure using laser scanning confocal microscopy

Visualisation of ectomycorrhizal rhizomorph structure using laser scanning confocal microscopy

Vacuolar Membrane Dynamics in the Filamentous Fungus Aspergillus oryzae

Endocytosis and Membrane Turnover in the Germ Tube ofUromyces fabae

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