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Title: Imaging vesicle trafficking and organelle dynamics in living fungal hyphae
Author: Hickey, Patrick C.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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The aims of the research were to develop and apply live cell imaging techniques using confocal microscopy to image and analyse vesicle trafficking, organelle dynamics and molecular localization at high spatial and temporal resolution in filamentous fungi. The dyes FM4-64, and to a lesser extent, FM1-43, were used to analyse vesicle trafficking, and as general probes to image hyphal tip growth, branching, septum formation, hyphal fusion, conidiophore development and the early stages of protoperithecium development. Neurospora crassa was the main system studied, although a taxonomically diverse range of other species were also analysed. Uptake of FM4-64 into hyphae was shown to be time- and energy-dependent, consistent with internalization being mediated by endocytosis. FM4-64 proved to be an excellent vital stain for different cell components including the apical vesicle cluster (AVC), vacuolar network and mitochondria. The dye was used to compare the morphology of the AVC in 15 different species. The green fluorescent protein (GFP) is a recombinant fluorescent probe that can be targeted to organelles and used to label specific proteins within living cells. Confocal microscopy was used to image transformants of Aspergillus nidulans expressing GFP targeted to the vacuolar network, mitochondria, nuclei, spindle pole bodies, endoplasmic reticulum, and Golgi cistemae. In addition, a range of vital fluorescent dyes were used to image organelles, and results from these studies were compared to those obtained with GFP transformants. Vacuolar staining revealed an extensive tubular network in actively growing regions of hyphae, whereas in sub-apical regions large spherical vacuoles were observed. Division of a single mitochondrion was imaged in Aspergillus nidulans expressing GFP targeted to mitochondria. Potentiometric dyes (especially rhodamine-123) indicated that the membrane potential of mitochondria in growing hyphal tips was higher than that in sub-apical regions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available