Publications by authors named "Marisa Segal"

19 Publications

  • Page 1 of 1

Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase.

Elife 2020 08 27;9. Epub 2020 Aug 27.

Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

Asymmetric astral microtubule organization drives the polarized orientation of the mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.
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http://dx.doi.org/10.7554/eLife.59222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470843PMC
August 2020

Intrinsic and Extrinsic Determinants Linking Spindle Pole Fate, Spindle Polarity, and Asymmetric Cell Division in the Budding Yeast S. cerevisiae.

Results Probl Cell Differ 2017 ;61:49-82

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK.

The budding yeast S. cerevisiae is a powerful model to understand the multiple layers of control driving an asymmetric cell division. In budding yeast, asymmetric targeting of the spindle poles to the mother and bud cell compartments respectively orients the mitotic spindle along the mother-bud axis. This program exploits an intrinsic functional asymmetry arising from the age distinction between the spindle poles-one inherited from the preceding division and the other newly assembled. Extrinsic mechanisms convert this age distinction into differential fate. Execution of this program couples spindle orientation with the segregation of the older spindle pole to the bud. Remarkably, similar stereotyped patterns of inheritance occur in self-renewing stem cell divisions underscoring the general importance of studying spindle polarity and differential fate in yeast. Here, we review the mechanisms accounting for this pivotal interplay between intrinsic and extrinsic asymmetries that translate spindle pole age into differential fate.
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http://dx.doi.org/10.1007/978-3-319-53150-2_3DOI Listing
July 2017

Analysis of the Localization of MEN Components by Live Cell Imaging Microscopy.

Methods Mol Biol 2017 ;1505:151-166

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK.

Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem.
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http://dx.doi.org/10.1007/978-1-4939-6502-1_12DOI Listing
January 2018

Spindle pole body history intrinsically links pole identity with asymmetric fate in budding yeast.

Curr Biol 2013 Jul 27;23(14):1310-9. Epub 2013 Jun 27.

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.

Background: Budding yeast is a unique model for exploring differential fate in a cell dividing asymmetrically. In yeast, spindle orientation begins with the old spindle pole body (SPB) (from the preceding cell cycle) contacting the bud by its existing astral microtubules (aMTs) while the new pole delays astral microtubule organization. This appears to prime the inheritance of the old pole by the bud. The basis for this asymmetry and the discrimination of the poles by virtue of their history remain a mystery.

Results: Here, we report that asymmetric aMT organization stems from an outstanding structural asymmetry linked to the SPB cycle. We show that the γ-tubulin nucleation complex (γTC) favors the old spindle pole, an asymmetry inherent to the outer plaque (the cytoplasmic face of the SPB). Indeed, Spc72 (the receptor for the γTC) is acquired by the new SPB outer plaque partway through spindle assembly. The significance of this asymmetry was explored in cells expressing an Spc72(1-276)-Cnm67 fusion that forced symmetric nucleation at the SPB outer plaques. This manipulation triggered simultaneous aMT organization by both spindle poles from the outset and led to symmetric contacts between poles and the bud, effectively disrupting the program for spindle polarity. Temporally symmetric aMT organization perturbed Kar9 polarization by randomizing the choice of the pole to be guided toward the bud. Accordingly, the pattern of SPB inheritance was also randomized.

Conclusions: Spc72 differential recruitment imparting asymmetric aMT organization represents the most upstream determinant linking SPB historical identity and fate.
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http://dx.doi.org/10.1016/j.cub.2013.05.057DOI Listing
July 2013

Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae.

Curr Biol 2012 Jun 17;22(12):1075-83. Epub 2012 May 17.

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.

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.
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http://dx.doi.org/10.1016/j.cub.2012.04.059DOI Listing
June 2012

Mitotic exit control: a space and time odyssey.

Authors:
Marisa Segal

Curr Biol 2011 Oct;21(20):R857-9

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

The mitotic exit network (MEN), a protein kinase cascade under the switch-like control of the small GTPase Tem1, triggers exit from mitosis in budding yeast. Now it emerges that signals from both Tem1 and the yeast Polo kinase Cdc5 converge onto the MEN kinase Cdc15 to accurately restrict MEN activation to late mitosis.
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http://dx.doi.org/10.1016/j.cub.2011.09.023DOI Listing
October 2011

Ase1p phosphorylation by cyclin-dependent kinase promotes correct spindle assembly in S. cerevisiae.

Cell Cycle 2011 Jun 15;10(12):1988-97. Epub 2011 Jun 15.

Department of Genetics, University of Cambridge, Cambridge, UK.

Spindle morphogenesis and dynamics follow an orderly sequence of events coupled to the oscillatory activation of cyclin-dependent kinase (CDK). Using S. cerevisiae, we have addressed the requirement of CDK for phosphorylation of the spindle midzone component Ase1p and its significance to spindle assembly. Ase1p is related to human PRC1, a protein negatively regulated by CDK until late mitosis, when it is required for central spindle organization and cytokinesis. By contrast, we show here that Ase1p phosphorylation by CDK promotes correct spindle assembly. Indeed, Ase1p phosphorylation coincident with spindle assembly requires Clb5p, Clb3p and Clb4p. Moreover, in clb5Δ cells, Ase1p recruitment and the kinetics of spindle formation were perturbed. These phenotypes were enhanced in a cdc28-4 clb5Δ mutant to the extent that midzone disruption resulted in transient breaks of the short spindle. By contrast, clb3Δ clb4Δ cells delayed spindle assembly downstream to Ase1p recruitment. Expression of Ase1(7D) p that mimics the phosphorylated state restored timely recruitment in clb5Δ cells and fully rescued the corresponding spindle phenotypes. Finally, Ase1(7D) p partially suppressed the spindle assembly delay in clb3Δ clb4Δ cells. Thus, Ase1p phosphorylation by CDK promotes the assembly and stability of the mitotic spindle. It follows that CDK may differentially alter the functionality of members of the Ase1p/PRC1 family to place their distinct roles in their respective stage-specific contexts, a further factor of complexity in the organization of pathways promoting spindle assembly and dynamics.
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http://dx.doi.org/10.4161/cc.10.12.15864DOI Listing
June 2011

Actin-mediated delivery of astral microtubules instructs Kar9p asymmetric loading to the bud-ward spindle pole.

Mol Biol Cell 2010 Aug 9;21(15):2685-95. Epub 2010 Jun 9.

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

In Saccharomyces cerevisiae, Kar9p, one player in spindle alignment, guides the bud-ward spindle pole by linking astral microtubule plus ends to Myo2p-based transport along actin cables generated by the formins Bni1p and Bnr1p and the polarity determinant Bud6p. Initially, Kar9p labels both poles but progressively singles out the bud-ward pole. Here, we show that this polarization requires cell polarity determinants, actin cables, and microtubules. Indeed, in a bud6 Delta bni1 Delta mutant or upon direct depolymerization of actin cables Kar9p symmetry increased. Furthermore, symmetry was selectively induced by myo2 alleles, preventing Kar9p binding to the Myo2p cargo domain. Kar9p polarity was rebuilt after transient disruption of microtubules, dependent on cell polarity and actin cables. Symmetry breaking also occurred after transient depolymerization of actin cables, with Kar9p increasing at the spindle pole engaging in repeated cycles of Kar9p-mediated transport. Kar9p returning to the spindle pole on shrinking astral microtubules may contribute toward this bias. Thus, Myo2p transport along actin cables may support a feedback loop by which delivery of astral microtubule plus ends sustains Kar9p polarized recruitment to the bud-ward spindle pole. Our findings also explain the link between Kar9p polarity and the choice setting aside the old spindle pole for daughter-bound fate.
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http://dx.doi.org/10.1091/mbc.e10-03-0197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912354PMC
August 2010

Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae.

J Cell Sci 2008 Nov 28;121(Pt 22):3803-14. Epub 2008 Oct 28.

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.

In S. cerevisiae, spindle orientation is linked to the inheritance of the ;old' spindle pole by the bud. A player in this asymmetric commitment, Bud6p, promotes cortical capture of astral microtubules. Additionally, Bud6p stimulates actin cable formation though the formin Bni1p. A relationship with the second formin, Bnr1p, is unclear. Another player is Kar9p, a protein that guides microtubules along actin cables organised by formins. Here, we ask whether formins mediate Bud6p-dependent microtubule capture beyond any links to Kar9p and actin. We found that both formins control Bud6p localisation. bni1 mutations advanced recruitment of Bud6p at the bud neck, ahead of spindle assembly, whereas bnr1Delta reduced Bud6p association with the bud neck. Accordingly, bni1 or bnr1 mutations redirected microtubule capture to or away from the bud neck, respectively. Furthermore, a Bni1p truncation that can form actin cables independently of Bud6p could not bypass a bud6Delta for microtubule capture. Conversely, Bud6(1-565)p, a truncation insufficient for correct actin organisation via formins, supported microtubule capture. Finally, Bud6p or Bud6(1-565)p associated with microtubules in vitro. Thus, surprisingly, Bud6p may promote microtubule capture independently of its links to actin organisation, whereas formins would contribute to the program of Bud6p-dependent microtubule-cortex interactions by controlling Bud6p localisation.
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http://dx.doi.org/10.1242/jcs.036269DOI Listing
November 2008

The protease activity of yeast separase (esp1) is required for anaphase spindle elongation independently of its role in cleavage of cohesin.

Genetics 2008 Apr;178(4):2361-72

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Separase is a caspase-family protease required for the metaphase-anaphase transition in eukaryotes. In budding yeast, the separase ortholog, Esp1, has been shown to cleave a subunit of cohesin, Mcd1 (Scc1), thereby releasing sister chromatids from cohesion and allowing anaphase. However, whether Esp1 has other substrates required for anaphase has been controversial. Whereas it has been reported that cleavage of Mcd1 is sufficient to trigger anaphase in the absence of Esp1 activation, another study using a temperature-sensitive esp1 mutant concluded that depletion of Mcd1 was not sufficient for anaphase in the absence of Esp1 function. Here we revisit the issue and demonstrate that neither depletion of Mcd1 nor ectopic cleavage of Mcd1 by Tev1 protease is sufficient to support anaphase in an esp1 temperature-sensitive mutant. Furthermore, we demonstrate that the catalytic activity of the Esp1 protease is required for this Mcd1-independent anaphase function. These data suggest that another protein, possibly a spindle-associated protein, is cleaved by Esp1 to allow anaphase. Such a function is consistent with the previous observation that Esp1 localizes to the mitotic spindle during anaphase.
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http://dx.doi.org/10.1534/genetics.107.085308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323821PMC
April 2008

Phosphorylation of Spc110p by Cdc28p-Clb5p kinase contributes to correct spindle morphogenesis in S. cerevisiae.

J Cell Sci 2007 Feb 9;120(Pt 3):435-46. Epub 2007 Jan 9.

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK.

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 spc110(Delta)(13), encoding a truncation of the spindle pole body component Spc110p (the intranuclear receptor for the gamma-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 spc110(Delta)(13) 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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http://dx.doi.org/10.1242/jcs.03342DOI Listing
February 2007

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

J Cell Sci 2005 Feb;118(Pt 3):463-71

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK.

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.
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http://dx.doi.org/10.1242/jcs.01650DOI Listing
February 2005

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

J Cell Biol 2004 Oct 18;167(2):231-44. Epub 2004 Oct 18.

Department of Genetics, University of Cambridge, Cambridge, CB2 3EH UK.

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, kar9Delta cells maintained Bud6p-dependent MT capture. Conversely, bud6Delta cells supported Kar9p-associated MT delivery to the bud. Moreover, additive phenotypes in bud6Delta kar9Delta or bud6Delta dyn1Delta mutants underscored the separate contributions of Bud6p, Kar9p, and dynein to spindle positioning. Finally, tub2C354S, a mutation decreasing MT dynamics, suppressed a kar9Delta 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.
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http://dx.doi.org/10.1083/jcb.200407167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172562PMC
October 2004

Temporal coupling of spindle disassembly and cytokinesis is disrupted by deletion of LTE1 in budding yeast.

Cell Cycle 2004 Jun 28;3(6):817-22. Epub 2004 Jun 28.

Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, London, UK.

The mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis in budding yeast by triggering the nucleolar release and hence activation of the Cdc14 phosphatase. Activation of the MEN is tightly coordinated with spindle position in such a way that Cdc14 is only fully released upon spindle pole body (SPB) migration into the daughter cell. This temporal regulation of the MEN has been proposed to rely in part on the spatial separation of the G-protein Tem1 at the SPB and its nucleotide exchange factor Lte1 confined to the daughter cell cortex. However, the dispensability of LTE1 for survival has raised questions regarding this model. Here using real-time microscopy we show that lte1Delta mutants not only delay exit from mitosis but also uncouple the normal coordination between spindle disassembly and contraction of the actomyosin ring at cell division. These mitotic defects can be suppressed by a bub2Delta mutation or by Cdc14 over-expression suggesting that they are caused by compromised MEN activity. Thus Lte1 function is important to fine-tune the timing of mitotic exit and to couple this event with cytokinesis in budding yeast.
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June 2004

Cdc20 in S-phase: the Banquo at replication's banquet.

Cell Cycle 2004 Mar 1;3(3):276-9. Epub 2004 Mar 1.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that covalently attaches ubiquitins onto proteins to target them for proteolysis by the 26S proteasome. During mitosis, the APC/C is instrumental in allowing the cell to enter and exit from mitosis. The APC/C accomplishes this by using different specificity factors to recognize, interact with, and ubiquitylate key proteins that block cell cycle progression. The specificity factors, Cdc20p and Cdh1p, are not always associated with the APC/C and indeed they have the ability to interact with substrates in isolation. The molecular events that take place in order for Cdc20p and Cdh1p to couple substrates and APC/C are currently being resolved. Meanwhile, evidence has emerged suggesting that at least one of the specificity factors, Cdc20p, might be capable of functioning independently of the APC/C.
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March 2004

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

Nat Cell Biol 2003 Oct 21;5(10):928-35. Epub 2003 Sep 21.

The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

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.
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http://dx.doi.org/10.1038/ncb1046DOI Listing
October 2003

Kar9p-independent microtubule capture at Bud6p cortical sites primes spindle polarity before bud emergence in Saccharomyces cerevisiae.

Mol Biol Cell 2002 Dec;13(12):4141-55

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Spindle orientation is critical for accurate chromosomal segregation in eukaryotic cells. In the yeast Saccharomyces cerevisiae, orientation of the mitotic spindle is achieved by a program of microtubule-cortex interactions coupled to spindle morphogenesis. We previously implicated Bud6p in directing microtubule capture throughout this program. Herein, we have analyzed cells coexpressing GFP:Bud6 and GFP:Tub1 fusions, providing a kinetic view of Bud6p-microtubule interactions in live cells. Surprisingly, even during the G1 phase, microtubule capture at the recent division site and the incipient bud is dictated by Bud6p. These contacts are eliminated in bud6 delta cells but are proficient in kar9 delta cells. Thus, Bud6p cues microtubule capture, as soon as a new cell polarity axis is established independent of Kar9p. Bud6p increases the duration of interactions and promotes distinct modes of cortical association within the bud and neck regions. In particular, microtubule shrinkage and growth at the cortex rarely occur away from Bud6p sites. These are the interactions selectively impaired at the bud cortex in bud6 delta cells. Finally, interactions away from Bud6p sites within the bud differ from those occurring at the mother cell cortex, pointing to the existence of an independent factor controlling cortical contacts in mother cells after bud emergence.
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http://dx.doi.org/10.1091/mbc.02-05-0067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC138622PMC
December 2002

Spindle polarity in S. cerevisiae: MEN can tell.

Cell Cycle 2002 Sep-Oct;1(5):308-11

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.

Spatial coordination between the axis of the spindle and the division plane is critical in asymmetric cell divisions. In the budding yeast S. cerevisiae, orientation of the mitotic spindle responds to two intertwined programs dictating the position of the spindle poles: one providing the blueprint for built-in pole asymmetry, the other sequentially confining microtubule-cortex interactions to the bud and the bud neck. The first program sets a temporal asymmetry to limit astral microtubules to a single pole prior to spindle pole separation. The second enforces this polarity by allowing these early formed microtubules to undergo capture at the bud cell cortex while stopping newly formed microtubules once cortical capture shifts to the bud neck. The remarkable precision of this integrated program results in an invariant pattern of spindle pole inheritance in which the "old" spindle pole is destined to the bud. An additional layer of asymmetry is superimposed to couple successful chromosomal segregation between the mother and the bud with mitotic exit. This is based on the asymmetric localization to the committed daughter-bound pole of signaling components of the mitotic exit network. This system operates irrespective of intrinsic spindle polarity to ensure that it is always the pole translocating into the bud that carries the signal to regulate mitotic exit.
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http://dx.doi.org/10.4161/cc.1.5.143DOI Listing
June 2003

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

J Cell Sci 2002 Dec;115(Pt 24):4977-91

Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

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.
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http://dx.doi.org/10.1242/jcs.00189DOI Listing
December 2002