Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate (P i ) connected by high-energy bonds. PolyP occurs in a wide range of organisms, including prokaryotes and eukaryotes. PolyP has various biological functions; for example, it acts as a P i reservoir, as an alternative source of highenergy bonds, and as a buffer against alkaline conditions and metals (25). Furthermore, polyP also has regulatory functions such as competence for transformation (18), motility (36, 37), gene expression under stressed conditions (24,35,46), and protein degradation in amino acid starvation (28, 29) in prokaryotic organisms. The regulatory functions of polyP in eukaryotes are less clear, but some important facts are known. Recently, involvement of polyP in apoptosis (43) and enhancement of the mitogenic activities of acidic and basic fibroblast growth factors by polyP (45) have been suggested to occur in mammalian cells.The presence of polyP in cells can be visualized by staining with toluidine blue O (TBO) or 4Ј,6-diamidino-2-phenylindole (DAPI). DAPI is usually used for DNA detection, because blue fluorescence is apparent when the stained tissues are viewed under UV light. However, DAPI-polyP fluoresces yellow at high concentrations when viewed under UV (48). These staining methods have often been used for detecting polyPaccumulating bacteria in activated...

Phialocephala fortinii is a dark septate fungal endophyte that colonizes roots of many host species. Its effect on plant growth varies from being pathogenic to beneficial. The basic biology of this species has received little research, and thus the main objectives of this study were to determine cytological features of hyphae, including the nature of the vacuolar system, and whether polyphosphate was present in vacuoles. Both living hyphae and hyphae that had been rapidly frozen and freeze substituted before embedding were studied. A complex system of vacuoles, including a motile tubular vacuolar system, elongated vacuoles, and spherical vacuoles, was demonstrated in living hyphae by the fluorescent probe Oregon Green 488 carboxylic acid diacetate, using laser scanning confocal microscopy. The motile tubular vacuolar system was more prevalent at the hyphal tip than in more distal regions, whereas elongated vacuoles and spherical vacuoles were more abundant distal to the tip. All vacuoles contained polyphosphate as shown by labelling embedded samples with recombinant polyphosphate binding domain of Escherichia coli exopolyphosphatase, containing Xpress tag at the N-terminal end, followed by anti-Xpress antibody and a secondary antibody conjugated either to a fluorescent probe for laser scanning confocal microscopy or colloidal gold for transmission electron microscopy. The polyphosphate was dispersed in vacuoles. This was confirmed by staining embedded samples with 4',6-diamidino-2-phenylindole and viewing with UV light using epifluorescence microscopy. These cytological methods showed that the tubular vacuolar system had lower concentrations of polyphosphate than the spherical vacuoles. Lipid bodies were present around vacuoles.

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Various categories of mycorrhizas are recognized primarily by the structural changes that occur between fungi and roots. In all mycorrhiza categories, cytological modifications of root cells accompany the establishment of the functional symbiosis, and among these are alterations in the organization of the cytoskeleton. Using immuno labelling combined with confocal scanning laser microscopy, this study documents changes in microtubules (MTs) in root cells of ectendomycorrhizas and monotropoid mycorrhizas; in addition, ectomycorrhizas were reinvestigated to determine the effect of fungal colonization on host root cells. In Pinus banksiana L. – Laccaria bicolor (Maire) Orton ectomycorrhizas, MTs were present in epidermal and cortical cells adjacent to the Hartig net. The remaining cortical MTs had a different organization when compared with those of cortical cells of control roots. MTs were present in Hartig net hyphae. In ectendomycorrhizas formed when roots of P. banksiana were colonized by the ascomycete, Wilcoxina mikolae var. mikolae Yang & Korf, MTs were present adjacent to intracellular hyphae and host nuclei, but few cortical MTs were present. MTs were present within Hartig net and intracellular hyphae. In field-collected roots of Monotropa uniflora L., MTs were associated with fungal pegs, intracellular extensions of inner mantle hyphae within epidermal cells. The close association between MTs and fungal pegs may be related to the formation of the highly branched host-derived wall that envelops each fungal peg. The development of exchange interfaces in the three systems studied involve changes in the organization of microtubules.Key words: cytoskeleton, microtubules, Hartig net, mycorrhizas, immunolocalization, confocal microscopy.