Nair, Rakesh, Raina, Sheetal, Keshavarz, Tajalli and Kerrigan, Mark J.P. (2011) Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi. Fungal Biology, 115 (4-5). pp. 326-334. ISSN 1878-6146 (online)

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Abstract

A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca2+]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 Ca2+-sensitive dye to quantify and image [Ca2+]i in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca2+ 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 [Ca2+]i levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150 mg L−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.

Item Type:	Article
Uncontrolled Keywords:	calcium, confocal, elicitor, filament tracer, FLUOstar optima, morphology, penicillium chrysogenum
Subjects:	Q Science > Q Science (General) Q Science > QR Microbiology
Pre-2014 Departments:	School of Education
Related URLs:	Publisher Organisation
Last Modified:	14 Oct 2016 09:19
URI:	http://gala.gre.ac.uk/id/eprint/7537

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