In Saccharomyces cerevisiae, spindle orientation is controlled by a temporal and spatial program of microtubule (MT)–cortex interactions. This program requires Bud6p/Aip3p to direct the old pole to the bud and confine the new pole to the mother cell. Bud6p function has been linked to Kar9p, a protein guiding MTs along actin cables. Here, we show that Kar9p does not mediate Bud6p functions in spindle orientation. Based on live microscopy analysis, kar9Δ cells maintained Bud6p-dependent MT capture. Conversely, bud6Δ cells supported Kar9p-associated MT delivery to the bud. Moreover, additive phenotypes in bud6Δ kar9Δ or bud6Δ dyn1Δ mutants underscored the separate contributions of Bud6p, Kar9p, and dynein to spindle positioning. Finally, tub2 C354S, a mutation decreasing MT dynamics, suppressed a kar9Δ mutation in a BUD6-dependent manner. Thus, Kar9p-independent capture at Bud6p sites can effect spindle orientation provided MT turnover is reduced. Together, these results demonstrate Bud6p function in MT capture at the cell cortex, independent of Kar9p-mediated MT delivery along actin cables.

Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.

In asymmetric divisions, the mitotic spindle must align according to the cell polarity axis. This is achieved through targeting astral microtubules emanating from each spindle pole to opposite cell cortex compartments. Saccharomyces cerevisiae is a powerful genetic model for dissection of this complex process. Intense research in this yeast has rendered detailed models for a program linking actin organization and spindle orientation along the mother-bud axis. This program requires the separate contributions of Kar9p, a protein guiding microtubules along polarized actin cables, and the polarity determinant Bud6p/Aip3 that marks sites for cortical capture at the bud tip and bud neck. In an added layer of complexity, cyclin-dependent kinase (Cdk) differentially regulates spindle pole function to dictate asymmetric spindle pole fate. Asymmetric contacts established by the spindle poles impart a further layer of extrinsic asymmetry restricting recruitment of Kar9p to the pole destined for the daughter cell. As a result, astral microtubules from a single pole are guided to the bud compartment after spindle assembly. Finally, Cdk might also translocate along astral microtubules in association with Kar9p to modulate microtubule-cortex interactions following spindle alignment. Insertion of the mitotic spindle into the bud neck is driven by the microtubule motor dynein. This process relies on the combined action of microtubule-plus-end-tracking proteins and kinesins that control the cell-cycle-dependent abundance of dynein at microtubule plus ends. Thus, this actin-independent pathway for spindle orientation might also be influenced by Cdk.

Spindle morphogenesis is regulated by cyclin-dependent kinases and monitored by checkpoint pathways to accurately coordinate chromosomal segregation with other events in the cell cycle. We have previously dissected the contribution of individual B-type cyclins to spindle morphogenesis in Saccharomyces cerevisiae. We showed that the S-phase cyclin Clb5p is required for coupling spindle assembly and orientation. Loss of Clb5p-dependent kinase abolishes intrinsic asymmetry between the spindle poles resulting in lethal translocation of the spindle into the bud with high penetrance in diploid cells. This phenotype was exploited in a screen for high dosage suppressors that yielded spc110Δ13, encoding a truncation of the spindle pole body component Spc110p (the intranuclear receptor for the γ-tubulin complex). We found that Clb5p-GFP was localised to the spindle poles and intranuclear microtubules and that Clb5p-dependent kinase promoted cell cycle dependent phosphorylation of Spc110p contributing to spindle integrity. Two cyclin-dependent kinase consensus sites were required for this phosphorylation and were critical for the activity of spc110Δ13 as a suppressor. Together, our results point to the function of cyclin-dependent kinase phosphorylation of Spc110p and provide, in addition, support to a model for Clb5p control of spindle polarity at the level of astral microtubule organisation.

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