Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases.

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (CLE)-like effector proteins. These proteins act as ligand mimics of plant CLE peptides and are required for successful nematode infection. Previously, we showed that the CLV2/CORYNE (CRN) heterodimer receptor complex is required for nematode CLE signaling. However, there was only a partial reduction in nematode infection when this signaling was disrupted, indicating that there might be additional nematode CLE receptors. In this study, we demonstrate that CLV1 and RECEPTOR-LIKE PROTEIN KINASE 2/TOADSTOOL2 (RPK2), two additional receptors that can transmit the CLV3 signal independent of CLV2/CRN for shoot apical meristem maintenance, also play a role in nematode CLE perception. Localization studies showed that both receptors are expressed in nematode-induced syncytia. Infection assays with clv1 and rpk2 single mutants revealed a decrease in both nematode infection and syncytium size. Significantly, further reduction in nematode infection was observed when rpk2 was combined with clv1 and clv2 mutants. Taken together, our results indicate that parallel signaling pathways involving CLV1, CLV2, and RPK2 are important for nematode parasitism.

Summary The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.

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We have identified the cyclin domain-containing proteins encoded by the genomes of 17 species of Aspergillus as well as 15 members of other genera of filamentous ascomycetes. Phylogenetic analyses reveal that the cyclins fall into three groups, as in other eukaryotic phyla, and, more significantly, that they are remarkably conserved in these fungi. All 32 species examined, for example, have three group I cyclins, cyclins that are particularly important because they regulate the cell cycle, and these are highly conserved. Within the group I cyclins there are three distinct clades, and each fungus has a single member of each clade. These findings are in marked contrast to the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, which have more numerous group I cyclins. These results indicate that findings on cyclin function made with a model Aspergillus species, such as A. nidulans, are likely to apply to other Aspergilli and be informative for a broad range of filamentous ascomycetes. In this regard, we note that the functions of only one Aspergillus group I cyclin have been analysed (NimECyclin B of A. nidulans). We have consequently carried out an analysis of the members of the other two clades using A. nidulans as our model. We have found that one of these cyclins, PucA, is essential, but deletion of PucA in a strain carrying a deletion of CdhA, an activator of the anaphase promoting complex/cyclosome (APC/C), is not lethal. These data, coupled with data from heterokaryon rescue experiments, indicate that PucA is an essential G1/S cyclin that is required for the inactivation of the APC/C-CdhA, which, in turn, allows the initiation of the S phase of the cell cycle. Our data also reveal that PucA has additional, non-essential, roles in the cell cycle in interphase. The A. nidulans member of the third clade (AN2137) has not previously been named or analyzed. We designate this gene clbA. ClbA localizes to kinetochores from mid G2 until just prior to chromosomal condensation. Deletion of clbA does not affect viability. However, by using a regulatable promoter system new to Aspergillus, we have found that expression of a version of ClbA in which the destruction box sequences have been removed is lethal and causes a mitotic arrest and a high frequency of non-disjunction. Thus, although ClbA is not essential, its timely destruction is essential for viability, chromosomal disjunction, and successful completion of mitosis.

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