Function and organisation of actin and septins in Neurospora crassa
This thesis deals with the organisation and function of actin and septins in the model filamentous fungus, Neurospora crassa. Firstly, study demonstrates the utility of the Lifeact peptide probe for the investigation of actin dynamics in N. crassa. Lifeact fused to fluorescent proteins allowed live-cell imaging of actin patches, cables and rings without interfering with cellular functions. Actin cables and patches localised to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organisation was markedly different between the tip regions of mature hyphae and germ tubes. Only mature hyphae displayed a sub-apical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Proper organisation of actin cables required the class-V myosin, MYO-5, and the frequency of rapid transport of actin patches was reduced in its absence, suggesting that MYO-5 participates in actin patch translocation. Deletion of myo-5 caused gross morphological and polarity defects, demonstrating the importance of this motor for normal cell function. GFP-tagged MYO-5 localised as a crescent at germ tube tips and to the core of the Spitzenkörper in mature hyphae. Secondly, analysis of septin null mutants demonstrated that septins limit the emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins showed different patterns of localisation at hyphal tips, with GFP-CDC-10 and CDC- 11-GFP organised as a collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP constituted as a cap at the tip apex and GFP-SPN-1 forming an extended collar. Septins formed a range of different higher-order structures in N. crassa – rings, loops, fibres, bar-like structures, and caps – which can co-exist within the same cell. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of SPN-1 revealed that this septin interacts with the core septin complex.
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