title: Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi.
abstract: A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca(2+)](i) and morphology in filamentous fungus is presented. Using a simple protocol, two fluorescent dyes, Fluo-4-AM and Cell trace calcein red-orange-AM were loaded into the filamentous fungus Penicillium chrysogenum. The present study investigates the applicability of using Ca(2+)-sensitive dye to quantify and image [Ca(2+)](i) in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca(2+) signalling in elicited cultures. The dye loading was optimised and investigated at different pH loading conditions. It was observed that the fluorophore was taken up throughout the hyphae, retaining cell membrane integrity and no dye compartmentalisation within organelles was observed. From the fluorescent plate-reader studies a significant rise (p<0.001) in the relative fluorescence levels corresponding to [Ca(2+)](i) levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150mgL(-1)) which was dependent upon extracellular calcium. Concurrently a novel application of dye-loaded hyphae for morphological analysis was also examined using the imaging software Filament Tracer (Bitplane). Essential quantitative mycelial information including the length and diameter of the segments and number of branch points was obtained using this application based on the three-dimensional data.
authors.affiliation: Applied Biotechnology Research Group, Department of Molecular and Applied Biosciences, School of Life Sciences, University of Westminster, London, UK.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Nair
authors.forename: Sheetal
authors.initials: S
authors.lastname: Raina
authors.forename: Tajalli
authors.initials: T
authors.lastname: Keshavarz
authors.forename: Mark J P
authors.initials: MJ
authors.lastname: Kerrigan
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry*; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods*; D025282:Mycelium / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000378:metabolism*; D014966:Xanthenes / Q000737:chemistry*
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014966:Xanthenes; D002118:Calcium; C007740:fluorexon