Comparison of membranes imaged with fluorescent probes FM4-64 and DiOC6 to GFP-tagged ER, Golgi and microtubules in Aspergillus nidulans.

Michelle Hubbard and Susan Kaminskyj

Author address: 

Dept. Biology, Univ Saskatchewan, Saskatoon, SK S7N 5E2, Canada

Abstract: 

Fungal tip growth depends on precisely targeted secretion of endomembrane-derived vesicles. The Aspergillus nidulans hypA and hypB morphogenesis loci have roles in ER-Golgi transport via the COPII and COPI pathways, respectively. To facilitate future work in mutant phenotype studies, we imaged A. nidulans endomembranes with the lipophilic dyes FM4-64 and DiOC6 using confocal microscopy. Arrays were compared with GFP-tagged ER, Golgi and microtubule patterns. Effective DiOC6 concentrations were 1000x lower than FM4-64. FM4-64 patterns changed over time, first staining the cell membrane and later being internalized; DiO6 staining of endomembranes was rapid. In growing wildtype cells there was overall agreement between FM4-64 and DiOC6 patterns after 1 h incubation in FM4-64, except that DiOC6 did not label a putative apical vesicle cluster. Time lapse images with both dyes showed endomembrane movements. Although theoretically possible, we were not able to effectively unmix signals from DiOC6 and GFP-tagged cytoplasmic microtubule arrays due to spatial rather than spectral constraints. FM4-64 arrays co-localized well with GFP-ER, but generally not with GFP-Golgi. Comparing endomembrane and cytoplasmic microtubule arrays in developing branches in wildtype and hypA1 mutants, we find that Golgi cluster at branch initiation sites, whereas both Golgi and cytoplasmic microtubules have roles in branch extension
2005

abstract No: 

27.

Full conference title: 

23rd Fungal Genetics Conference
    • Fungal Genetics Conference 23rd (2002)