New aspects of septin assembly and cell cycle control in multinucleated "A. gossypii"

Chapter I Nuclei in the filamentous ascomycete Ashbya gossypii divide asynchronously and most nuclei have the potential to divide (Alberti-Segui et al., 2001; Gladfelter et al., 2006). Although cytoplasmic extension is restricted to growing tips and emerging branches, the distances between nuclei are uniform along the entire hyphal length. This implies active control of nuclear distribution and division to maintain an ideal nuclear to cytoplasmic ratio, potentially depending on environmental conditions. The question of nuclear distribution has already been addressed earlier (Alberti-Segui et al., 2001). We have investigated how the rate of mitosis is regulated in response to intra- and extracellular signals. Here we show that homologues of S. cerevisiae morphogenesis checkpoint components are involved in starvation response in A. gossypii: Phosphorylation of the cyclin dependent kinase AgCdc28p at tyrosine 18 by the protein kinase AgSwe1p is used to delay mitosis under low-nutrient conditions, leading to an increase in the average distance between nuclei. This effect is markedly reduced in Agswe1Δ or Agcdc28Y18F mutants where the CDK cannot be phosphorylated. Overexpression of AgSWE1 leads to decreased nuclear density even under non-starving conditions. Addition of rapamycin mimics starvation response, suggesting that AgSwe1p may be under control of AgTor1/2p. In unperturbed budding yeast cells, ScSwe1p is recruited to the septin ring at the mother-bud neck where it is phosphorylated and subsequently degraded. We have speculated that the septins in A. gossypii could serve as spatial markers to locally inactivate AgSwe1p and increase nuclear division rate in areas of growth. Time-lapse analysis has revealed that mitoses in wild type are most common near branching points. Interestingly, AgSep7p-GFP localizes to branching points and septin deletion mutants show random distribution of mitoses. We propose a model in which AgSwe1p may regulate mitosis in response to cell intrinsic morphogenesis cues and external nutrient availability in multinucleated cells. Chapter II Septins are evolutionary conserved proteins with essential functions in cytokinesis, and more subtle roles throughout the cell cycle. Much of our knowledge about septins originates from studies with S. cerevisiae, where they form a ring-like protein scaffold at the mother-bud neck. We have asked what functions the septins may hold in an organism that does not complete cytokinesis prior to sporulation. Interestingly, all budding yeast septins are conserved in A. gossypii and one is even duplicated (S. Brachat, personal communication; Dietrich et al., 2004). In vivo studies of AgSep7p-GFP have revealed that septins assemble into discontinuous hyphal rings close to growing tips and sites of branch formation and into asymmetric structures at the base of branching points. Rings are made of filaments which are long and diffuse close to growing tips and short and compact further away from the tip. During septum formation, the septin ring splits into two to form a double ring. Agcdc3Δ, Agcdc10Δ, and Agcdc12Δ mutants display aberrant morphology and are defective for actinring formation, chitin-ring formation, and sporulation. Due to the lack of septa, septin deletion mutants are highly sensitive, and lesion of a single hypha can have catastrophic consequences for a young mycelium. Strains lacking AgCDC11A show morphological defects comparable with other septin deletion mutants, but actin- and chitin-ring formation are not disabled. Deletion of AgCDC11B results in no detectable phenotype under standard laboratory conditions.