Role of Tem1 in Signalling Mitotic Exit in the Human Fungal Pathogen Candida albicans
Milne, Stephen William
Date: 9 May 2011
Thesis or dissertation
Publisher
University of Exeter
Degree Title
PhD in Biological Sciences
Abstract
The human pathogen Candida albicans is polymorphic, and its ability to switch growth forms is
thought to play an important role in virulence. The primary research aim of this thesis was to
understand the role the mitotic exit network plays in C. albicans with particular focus on the
Tem1 GTPase protein. This aim was split into three ...
The human pathogen Candida albicans is polymorphic, and its ability to switch growth forms is
thought to play an important role in virulence. The primary research aim of this thesis was to
understand the role the mitotic exit network plays in C. albicans with particular focus on the
Tem1 GTPase protein. This aim was split into three specific goals; to study the role of Tem1
through the construction of a regulatable tem1 mutant, to understand the regulation of Tem1
through localisation and protein interaction studies, and to construct new molecular tools
utilising the NAT1 positive selection marker in order to achieve two previous goals.
In this thesis we demonstrated that TEM1 is an essential gene in C. albicans, and its essential
function is signalled through the Cdc15 protein. Surprisingly, Tem1p depleted cells arrested as
hyper-polarised filaments containing one or two nuclei and ultimately lost viability. These
filaments formed from budding yeast cells, suggestive of a blockage late in the cell cycle.
Ultimately the failure of these filaments to undergo cytokinesis was linked to a defect in septin
ring dynamics and the formation of actomyosin ring.
To understand the regulation of Tem1 we localised both the Tem1 and Lte1 proteins and found
that Tem1 localised to spindle pole bodies in a cell-cycle dependent fashion by recruited at the
onset of S phase. In contrast, the Lte1 homolog localised to the daughter cell cortex prior to
release into the cytoplasm at the end of the cell cycle. A yeast 2-hybrid analysis of the MEN
components demonstrated the potential of Bfa1/Bub2 and Tem1 to form a complex and the
ability of Tem1 to homodimerise which may play a role in its self-activation.
In order to carry out various aspects of this work we constructed a fully functional set of
cassettes, including the constitutively active ENO1 promoter, V5-6xHIS epitope tag and various
fluorescent protein genes fused to the NAT1 positive selection marker.
When considered together, these results indicate that Tem1 is required for timely mitotic exit
and cytokinesis in C. albicans, similar to S. cerevisiae, but the final output of the pathway must
have diverged.
Doctoral Theses
Doctoral College
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