Marisa Segal

Control of spindle polarity and orientation in Saccharomyces cerevisiae

Control of mitotic spindle orientation represents a major strategy for the generation of cell diversity during development of metazoans. Studies in the budding yeast Saccharomyces cerevisiae have contributed towards our present understanding of the general principles underlying the regulation of spindle positioning in an asymmetrically dividing cell. In S. cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud emergence, to ensure correct nuclear division between the mother and daughter cells. Establishment of spindle polarity dictates this process and relies on the concerted control of spindle pole function and a precise program of cues originating from the cell cortex that directs cytoplasmic microtubule attachments during spindle morphogenesis. These cues cross talk with the machinery responsible for bud-site selection, indicating that orientation of the spindle in yeast cells is mechanistically coupled to the definition of a polarity axis and the division plane. Here, we propose a model integrating the inherently asymmetric properties of the spindle pathway with the program of positional information contributing towards orienting the spindle in budding yeast. Because the basic machinery orienting the spindle in higher-eukaryotic cells appears to be conserved, it might be expected that similar principles govern centrosome asymmetry in the course of metazoan development.

Dosage Suppressors of pds1 Implicate Ubiquitin-Associated Domains in Checkpoint Control

ABSTRACT In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact withPDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 andDDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae.

In budding yeast, activation of the small Ras-like GTPase Tem1 triggers exit from mitosis and cytokinesis. Tem1 is regulated by Bub2/Bfa1, a two-component GTPase-activating protein (GAP), and by Lte1, a putative guanine nucleotide exchange factor. Lte1 is confined to the bud cortex, and its spatial separation from Tem1 at the spindle pole body (SPB) is important to prevent untimely exit from mitosis. The pathways contributing to Lte1 asymmetry have not been elucidated. Here we show that establishment of Lte1 at the cortex occurs by an actin-independent mechanism, which requires activation of Cdc28/Cln kinase at START and Cdc42, a key regulator of cell polarity and cytoskeletal organisation. This defines a novel role for Cdc42 in late mitotic events. In turn, dissociation of Lte1 from the cortex in telophase depends on activation of the Cdc14 phosphatase. Ectopic expression of Cdc14 at metaphase results in premature dephosphorylation of Lte1 coincident with its release from the cortex. In vitro phosphatase assays confirm that Lte1 is a direct substrate for Cdc14. Our results suggest that the asymmetry in Lte1 localisation is imposed by Cdc28-dependent phosphorylation. Finally, we report a mutational analysis undertaken to investigate intrinsic Lte1 determinants for localisation. Our data suggest that an intrameric interaction between the N-and C-terminal regions of Lte1 is important for cortex association.

Cortical capture of microtubules and spindle polarity in budding yeast - where's the catch?

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.

Mec1p regulates Pds1p levels in S phase: complex coordination of DNA replication and mitosis.

Genetic evidence suggests that the securin Pds1p is the target of a late-S-phase checkpoint control. Here we show that Pds1p becomes essential once two-thirds of the genome has been replicated and that the coupling of the completion of genome replication with mitosis relies on the regulation of Pds1p levels. Mec1p is needed to maintain Pds1p levels under S-phase checkpoint conditions. In contrast, Rad53p and Chk1p, needed for the stabilization of Pds1p in the context of the G2 DNA-damage checkpoint pathway, are dispensable. Thus, the Pds1p-dependent late-S-phase checkpoint pathway couples replication with mitosis but is mechanistically distinct from the G2 DNA-damage checkpoint. Finally, we show that the inhibition of spindle elongation in early S phase, controlled by the Mec1p/Rad53p branch, is not regulated via Pds1p/Esp1p. This can mechanistically explain the need for branched S-phase checkpoint controls.

The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast

In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle [1]. During this interval, S-phase checkpoint controls restrain mitosis until replication is complete [2]. In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly [3] [4], but not when DNA replication is blocked with hydroxyurea (HU). Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control [5]. Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked. This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block. Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis. Further, the S-phase checkpoint comprises at least two distinct pathways. A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase.

Differential contribution of Bud6p and Kar9p to microtubule capture and spindle orientation in S. cerevisiae

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.

S-phase checkpoint controls mitosis via an APC-independent Cdc20p function

Cells divide with remarkable fidelity, allowing complex organisms to develop and possess longevity. Checkpoint controls contribute by ensuring that genome duplication and segregation occur without error so that genomic instability, associated with developmental abnormalities and a hallmark of most human cancers, is avoided. S-phase checkpoints prevent cell division while DNA is replicating. Budding yeast Mec1p and Rad53p, homologues of human checkpoint kinases ATM/ATR and Chk2, are needed for this control system. How Mec1p and Rad53p prevent mitosis in S phase is not known. Here we provide evidence that budding yeasts avoid mitosis during S phase by regulating the anaphase-promoting complex (APC) specificity factor Cdc20p: Mec1p and Rad53p repress the accumulation of Cdc20p in S phase. Because precocious Cdc20p accumulation causes anaphase onset and aneuploidy, Cdc20p concentrations must be precisely regulated during each and every cell cycle. Catastrophic mitosis induced by Cdc20p in S phase occurs even in the absence of core APC components. Thus, Cdc20p can function independently of the APC.

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.

Mechanism for Astral Microtubule Capture by Cortical Bud6p Priming Spindle Polarity in S. cerevisiae

BACKGROUND Budding yeast is a unique model to dissect spindle orientation in a cell dividing asymmetrically. In yeast, this process begins with the capture of pole-derived astral microtubules (MTs) by the polarity determinant Bud6p at the cortex of the bud in G(1). Bud6p couples MT growth and shrinkage with spindle pole movement relative to the contact site. This activity resides in N-terminal sequences away from a domain linked to actin organization. Kip3p (kinesin-8), a MT depolymerase, may be implicated, but other molecular details are essentially unknown. RESULTS We show that Bud6p and Kip3p play antagonistic roles in controlling the length of MTs contacting the bud. The stabilizing role of Bud6p required the plus-end-tracking protein Bim1p (yeast EB1). Bim1p bound Bud6p N terminus, an interaction that proved essential for cortical capture of MTs in vivo. Moreover, Bud6p influenced Kip3p dynamic distribution through its effect on MT stability during cortical contacts via Bim1p. Coupling between Kip3p-driven depolymerization and shrinkage at the cell cortex required Bud6p, Bim1p, and dynein, a minus-end-directed motor helping tether the receding plus ends to the cell cortex. Validating these findings, live imaging of the interplay between dynein and Kip3p demonstrated that both motors decorated single astral MTs with dynein persisting at the plus end in association with the site of cortical contact during shrinkage at the cell cortex. CONCLUSIONS Astral MT shrinkage linked to Bud6p involves its direct interaction with Bim1p and the concerted action of two MT motors-Kip3p and dynein.