Septins are conserved GTP-binding proteins that play essential roles in morphogenesis in fungi and higher eukaryotic cells. In budding yeast, septins form a series of filaments that assemble as a ring at the bud neck during vegetative growth. Septin rings are thought to function as a scaffold to recruit proteins to the bud neck and as a boundary domain to restrict diffusion during budding and cytokinesis (reviewed in [10–12]). Previous reports have shown that in S. cerevisiae septins localize to the PSM during sporogenesis, although it is not known what their function is in this process. It has been proposed that septins could be required for the correct assembly of PSMs, although mutants lacking several septins have no defects in spore formation, which suggests that septins could act redundantly during this process . In the course of the analysis of the role of the Cdc15 kinase during sporulation , we used Cdc11 as a marker for analysis of PSM morphology by indirect immunofluorescence and noticed that Cdc11 had a localization pattern similar to that of the microtubule cytoskeleton at early time-points of the sporulation time-course. In addition, previous reports had already observed the localization of several septins, such as Cdc11 or Spr3, at the microtubules although it was assumed that it was a cross of fluorescence channels [17, 28].
Dynamic behavior of septins during sporulation
In S. cerevisiae, septin localization at the bud neck during vegetative growth is relatively constant [9, 29]. By contrast, our results show here that septins localization and behavior is much more variable through the process of sporulation. Analysis of septin localization during sporulation has been addressed by using two different techniques, in vivo analysis using GFP fusions and immunofluorescence in fixed cells. Microtubule association of Cdc11 and other septins was mainly detected by indirect immunofluorescence, while it was not detected using GFP fusions. Conversely, prospore membrane localization was clearly seen with both techniques. One possible reason to explain these differences is the relative amounts of septins associated with these two structures. It could be that the fraction of septins that interact with the microtubule cytoskeleton is low and that its interaction is very dynamic, making difficult its detection in vivo. On the other hand, fixation of the cells for immunofluorescence could stabilize the fraction of septins associated to microtubules or modify the proteins, facilitating its detection with specific antibodies.
During sporulation, Cdc11 localizes to different cell compartments; the SPBs, astral microtubules (cytoplasm), the meiotic spindle (nucleus) and the PSM. The association of Cdc11 with microtubules was confirmed by immunoprecipitation experiments, although our results do not indicate whether the interaction between tubulin and septins is direct or mediated by other proteins. Furthermore, analysis of Cdc10 and Spr28 localization indicated that septin-microtubules interaction during sporulation is not exclusive to Cdc11, but it is a characteristic of other septins as well. Spr28 and Cdc10 localization to the microtubules was weak but both proteins strongly accumulated at the PSMs later in sporogenesis, whereas the Cdc11 signal was more intense in the microtubule cytoskeleton and declined during spore wall formation. Colocalization of Spr28-GFP and Cdc11 (data not shown) supported the idea that affinity for microtubules or the PSM is different. Although this observation should be verified in greater detail, it is possible that these differences only reflect the different temporal pattern of expression of the genes during sporulation, since SPR28 and CDC10 are induced as middle sporulation genes while CDC11 is constitutively expressed, but at a lower level .
Our experiments show that tubulin-Cdc11 interaction and Cdc11-PSM interaction during sporulation are independent. This is supported by several experiments: First, depolymerization of the microtubule cytoskeleton using benomyl resulted in delocalization of the fraction of Cdc11 that was associated to tubulin, but it had no effect on those molecules that were localized to the PSM. In contrast, the absence of GIP1 is sufficient to disorganize septins at the PSM , but has no effects on the septins localizing to the microtubule cytoskeleton. Second, nutritional experiments and the fact that Spo20 vesicles arrived earlier than Cdc11 to the meiotic outer plate of the SPB suggests that Cdc11 localization to microtubules and PSM are independently regulated and respond to different signals. Thus, our results clearly indicate that Cdc11-microtubule association is triggered by a nutritional signal and also occurs during vegetative growth, while septin-PSM association is part of the developmental program that leads to the production of spores [17, 19, 23].
The genetic requirements for these different localizations are also distinct during sporulation. During vegetative growth, absence of an essential septin results in defects in the localization of the other septins and septin ring disassembly. In contrast, during sporulation, Cdc11 can localize to microtubules in the absence of SPR3 and SPR28 or GIP1, but its subsequent localization on the prospore membrane is altered. In the absence of SPR28, the remaining septins cannot form discrete bars but are localized uniformly around the prospore membrane throughout meiosis II. Mutation of SPR3 is distinct from spr28 Δ in that not just the sheets, but the association of the remaining septins with the prospore membrane is greatly reduced. In this respect, mutation of SPR3 resembles a gip1 mutant . Thus, though the function of the septins at the prospore membrane remains obscure, the sporulation-specific septins play direct and distinct roles in the organization of septin complexes during sporulation. The differential genetic requirements for septin localization at different stages of sporulation suggest that the organization of the septins themselves is changing during this process. It is noteworthy that the ability of Spr3 to associate with other septins depends on the phase of the life cycle. When ectopically expressed in vegetative cells, Spr3 does not localize with other septins at the bud neck . Taken together, these observations suggest that both the localization and the organization of the septins are changing dynamically throughout sporulation.
One possible explanation for these observations is that the stoichiometry of the septin subunits could be different during the successive phases of the sporulation process, and these differences could be important for their association to microtubules or PSM. Structural studies of mammalian septins suggest that the basic organization of a septin filament is a heterotrimer . It may be that different combinations of septins produce filaments with different properties. If so, understanding what these changes are and how they are regulated in yeast could prove insightful in other organisms which, like S. cerevisiae, express more than three septin proteins.
Septin-tubulin interactions had been previously observed in budding yeast , but this interaction was not analyzed in this organism. However, in mammalian cells, septins localize not only to the plasma membrane but also throughout the cytoplasm, interacting with the microtubule and actin cytoskeletons, where they act as a scaffold for cytoskeleton-binding proteins (reviewed in ). For example, the mammalian septin Sept9 (formerly known as MSF) colocalizes during interphase with actin, microtubules and another mammalian septin, Sept2 (Nedd5) during mitosis . Sept2 filaments are associated with actin fibers, and play a role in stabilization of actin stress fibers preventing actin turnover [32–34]. It has also been shown that Sept2 and Sept9 can align along microtubules during interphase in PC12 and HeLa cells [35, 36], and Sept2 has been reported to associate with spindle microtubules during mitosis . Furthermore, in mammalian cells septins not only interact with microtubules or actin cytoskeleton, but also control their organization and stability. This is the case of the mammalian septin complex composed of Sept2/6/7, which regulates microtubule stability through an interaction with the microtubule-binding protein MAP4  or actin organization through nuclear accumulation of the adaptor protein NCK . Moreover, it has also been suggested that Sept9 might have a function in microtubule organization during cytokinesis . In yeast, it is unlikely that septins may play a similar role in microtubule cytoskeleton organization, although it has been reported that microtubules interact with the septin ring to align the mitotic spindle along the mother-bud axis during vegetative growth .
Septin localization to microtubules and nutritional stimuli
In unicellular organisms, sporulation is a process necessary for cells to survive when environmental conditions change. In S. cerevisiae, sporulation is triggered in response to nitrogen deprivation and, this process is induced when cells are grown in a non-fermentable carbon source such as acetate. Analysis of Cdc11 localization in sporulating cells indicated that Cdc11 localized to the microtubules shortly after transfer to sporulation medium, and the same pattern was observed also in haploid cells, suggesting that the nutritional stimulus is the main signal responsible for the change in septin distribution. A deeper analysis of Cdc11 localization to microtubules in haploid cells has resulted in the following conclusions: 1) Cdc11 associates with microtubules during vegetative growth under certain nutritional stimuli, so Cdc11 can interact with tubulin independently of the developmental stage; 2) Non-fermentable carbon sources, such as acetate or glycerol, were the signal triggering Cdc11 association with microtubules; 3) the presence of non-fermentable carbon sources per se and not the transition to the respiratory metabolism is the origin of the stimulus that promotes Cdc11 localization to the tubulin cytoskeleton; and 4) though nitrogen starvation cannot trigger septin relocalization to microtubules, the absence of nitrogen enhances the effect of a non-fermentable carbon source. Thus, these results suggest that Cdc11 has the ability to interact to tubulin at different moments of the yeast life cycle.
At the moment, we do not know the exact nature of the signal that triggers Cdc11 association to microtubules. One possibility is that it involves changes in the intracellular concentration of a secondary messenger, such as cAMP or intracellular pH, allowing the interaction of tubulin and septins, two families of structural proteins, to maintain cytoskeleton polymerization under these conditions. Whether these complexes constitute only a response to intracellular changes or they have an active role has yet to be elucidated. The first possibility in good agreement with results from mammalian cells, in which Sept2 polymerizes forming structures inside the cytoplasm without any defined function when actin cytoskeleton is perturbed . In budding yeast, release of septins from the neck and polymerization of proteins in the cytoplasm could be a mechanism to store proteins temporarily until intracellular conditions would be reestablished to promote an active role of these proteins.