Endocytosis in filamentous fungi
dc.contributor.author
Kalkman, Edward R I C
en
dc.date.accessioned
2007-08-31T13:08:55Z
dc.date.available
2007-08-31T13:08:55Z
dc.date.issued
2007
dc.description.abstract
Endocytosis is little understood in filamentous fungi. For some time it has been
controversial as to whether endocytosis occurs in filamentous fungi. A comparative
genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous
fungal species showed that filamentous fungi possess complex endocytic machineries.
The use of the endocytic marker dye FM4-64, and various vesicle trafficking
inhibitors revealed many similarities between endocytosis in the filamentous fungus
Neurospora crassa, and endocytosis in budding yeast and mammalian cells. Actin
polymerization was found to be crucial for endocytosis in N. crassa, and the microtubule
cytoskeleton seemed to be necessary for long distance movement of putative early
endosomes. Brefeldin A (BFA) blocked vesicular transport to the Spitzenkörper.
Three putative endocytic proteins (WASP, clathrin light chain and Rab5) were
labelled with fluorescent proteins in N. crassa. WASP-GFP was found to localise to
motile, punctate structures in the plasma membrane just behind the hyphal apex in
growing hyphae. This localisation changed to the hyphal apex when growth was
temporarily arrested, indicating a possible role in endocytosis and polarized growth.
Clathrin light chain-GFP was found to be concentrated in a region just behind the
Spitzenkörper, which is consistent with there being a high concentration of clathrinmediated
endocytosis in this region. Clathrin light chain-GFP also labelled putative
Golgi and this labelling was found to be BFA sensitive, whereas BFA did not have a
detectable effect on FM4-64 internalisation and organelle staining. GFP-Rab5 labelled
putative early endosomes and decorated microtubules.
Knock-outs of putative endocytic proteins in N. crassa, generated as part of the
Neurospora genome consortium gene knock-out project, were analysed for defects in
endocytosis. 14 out of 17 gene knock-outs were found to be ascospore lethal. The Rab5
knock-out was viable, but did not show a detectable effect on the endocytic
internalisation of FM4-64 or its pattern of staining. However, it did exhibit a defect in
sexual crossing.
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26607347 bytes
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31693143 bytes
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dc.format.mimetype
application/pdf
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application/octet-stream
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dc.identifier.uri
http://hdl.handle.net/1842/1970
dc.language.iso
en
dc.subject
biological sciences
en
dc.subject
cell biology
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dc.title
Endocytosis in filamentous fungi
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dc.type
Thesis or Dissertation
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dc.type.qualificationlevel
Doctoral
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dc.type.qualificationname
PhD Doctor of Philosophy
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