| dc.description.abstract | In addition to its well-established role in nucleating microtubules at microtubule-organizing centers, γ-tubulin has essential, microtubule-independent functions that are incompletely understood [reviewed in (Oakley et al., 2015)]. Experiments in our lab with the cold-sensitive γ-tubulin mutant mipAD159 in the filamentous fungus Aspergillus nidulans revealed that γ-tubulin has a role in inactivating the anaphase promoting complex/cyclosome (APC/C) resulting in continuous destruction of cyclin B and failure of nuclei to progress through the cell cycle (Nayak et al., 2010). Deletion of the APC/C co-activator CdhA (the A. nidulans Cdh1 homolog) restores cyclin B accumulation and these and other data demonstrate that γ-tubulin plays an important role in inactivating APC/C-CdhA at the G1/S boundary. However, cdhAΔ, mipAD159 strains are as cold sensitive as the mipAD159 parent, indicating that the cold sensitivity is not due to continuous APC/C-CdhA activity (Edgerton-Morgan and Oakley, 2012). Although the underlying molecular mechanism(s) by which γ-tubulin regulates CdhA are not yet known, our data do not support a direct interaction between γ-tubulin and CdhA. Instead, we hypothesize that γ-tubulin acts through regulators of CdhA. Proteins involved in Cdh1 inhibition and inactivation have been identified in other organisms but not in A. nidulans prior to my work. Thus, the first part of my main project consisted of identifying and characterizing regulators of CdhA. As filamentous fungi are hugely important medically, agriculturally and commercially [reviewed in (Meyer et al., 2016)], it is vital that we understand the cell biology of filamentous fungi to be able to combat fungal pathogens and to maximize their growth for production of desirable products. The second part of my main project was aimed at determining whether these CdhA regulators are candidates through which γ-tubulin acts to regulate CdhA at the G1/S transition. In many organisms, initial Cdh1 inactivation at G1/S occurs via phosphorylation by cyclin/CDK complexes which then triggers Cdh1 ubiquitination by the Skp1-Cullin1-F-box (SCF) complex. However, cyclins have not been well studied in members of aspergilli, including A. nidulans. In chapter 3 of this work, I report my identification of all cyclin domain-containing proteins in A. nidulans and establish that this cyclin repertoire is well-conserved in closely and distantly related filamentous ascomycetes. This is significant as the complement of cyclins found in model yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans) differs considerably from one another and from the great majority of filamentous ascomycetes. Thus, A. nidulans is a much better model than these yeasts for studying cyclin function and cell cycle regulation in filamentous fungi. My phylogenetic analyses reveal there are three A. nidulans cyclins that likely carry out cell cycle-related functions (NimECyclin B, PucA, and ClbA). In Chapter 4 of this work, I focus on cyclins PucA and ClbA as they had not been characterized previously. My results reveal that ClbA is not essential, but its destruction is required for mitotic exit. ClbA also appears to function at the G2/M transition. My experiments further demonstrate that both NimECyclin B and ClbA play critical roles in chromosomal disjunction. My results also reveal that PucA is the essential cyclin required for CdhA inactivation at the G1/S transition and that there are no other redundant mechanisms for CdhA inactivation in A. nidulans. Finally, my data indicate that PucA function is required for some of the growth limiting effects of two mipA mutants, including mipAD159, although the mechanism of this interaction is not yet understood. I have also determined that the SCF complex plays a role in regulating CdhA in A. nidulans. In Chapter 5 of this work, I focus on two essential components of the SCF complex, Cullin A (CulA) and SkpA. I have determined their terminal phenotypes via the heterokaryon rescue technique, and I have studied their in vivo localization patterns using fluorescent protein fusions I have generated. Interestingly, CulA-GFP strains in a wild-type background are slightly cold sensitive, and CulA-GFP causes strong synthetic growth reduction with mipAD159. The strong, synthetic genetic interaction between mipAD159 and culA-GFP indicates that γ-tubulin and CulA are involved in a common function that is required for growth. Additionally, I have found that the SCF complex in A. nidulans has a crucial role in suppressing septation near the hyphal tip, which is essential for rapid tip extension in filamentous fungi. My study of cell cycle-related cyclins and SCF components in A. nidulans provides new insights into the regulation of the cell cycle and growth of filamentous fungi. My phylogenetic cyclin analyses also indicate that A. nidulans is a well-suited model compared to popular model yeasts for studying cyclin function and cell cycle regulation in aspergilli and other filamentous fungi. Finally, my data also indicate that PucA and CulA are strong candidates through which γ-tubulin acts to control APC/C-CdhA activity and are worthy of follow-up studies. | |
