scholarly journals γ-Tubulin Is Required for Proper Recruitment and Assembly of Kar9–Bim1 Complexes in Budding Yeast

2006 ◽  
Vol 17 (10) ◽  
pp. 4420-4434 ◽  
Author(s):  
Lara Cuschieri ◽  
Rita Miller ◽  
Jackie Vogel
Keyword(s):  
Budding Yeast ◽  
Spindle Pole ◽  
Microtubule Dynamics ◽  
Interacting Proteins ◽  
Cortical Actin ◽  
Dynamic Interactions ◽  
Spindle Positioning ◽  
Astral Microtubule ◽  
Spindle Pole Bodies

Microtubule plus-end–interacting proteins (+TIPs) promote the dynamic interactions between the plus ends (+ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, +TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast γ-tubulin Tub4 (tub4-Δdsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependant spindle positioning. Surprisingly, we find Kar9 localizes to microtubule +ends in tub4-Δdsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-Δdsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-Δdsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2- and F-actin–dependent dwelling of Kar9 in the bud is observed in tub4-Δdsyl cells, suggesting defective Kar9 complexes tether microtubule +ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-Δdsyl mutant, reduces cortical dwelling, and promotes Bim1–Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional +TIP complexes before their deployment to microtubule +ends.

scholarly journals Reorientation of Mispositioned Spindles in Short Astral Microtubule Mutantspc72Δ Is Dependent on Spindle Pole Body Outer Plaque and Kar3 Motor Protein

2002 ◽  
Vol 13 (4) ◽  
pp. 1366-1380 ◽  
Author(s):  
Dominic Hoepfner ◽  
Florian Schaerer ◽  
Arndt Brachat ◽  
Achim Wach ◽  
Peter Philippsen
Keyword(s):  
Spindle Pole Body ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Astral Microtubule ◽  
Spindle Pole Bodies ◽  
Microtubule Motor ◽  
Microtubule Formation ◽  
Mother Cells

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic γ-tubulin complex, can only generate very short (<1 μm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However,SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.

scholarly journals Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

1999 ◽  
Vol 145 (5) ◽  
pp. 979-991 ◽  
Author(s):  
Roberta Fraschini ◽  
Elisa Formenti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cycle Progression ◽  
Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

scholarly journals Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

2020 ◽  
Author(s):  
Divya Singh ◽  
Nadine Schmidt ◽  
Franziska Müller ◽  
Tanja Bange ◽  
Alexander W. Bird
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Microtubule Dynamics ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Tissue Organization ◽  
Microtubule Stability ◽  
Astral Microtubule ◽  
Early Mitosis

AbstractThe precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

scholarly journals A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 46
Author(s):  
Michael Vannini ◽  
Victoria R. Mingione ◽  
Ashleigh Meyer ◽  
Courtney Sniffen ◽  
Jenna Whalen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Signal Transduction Pathway ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Independent Manner ◽  
Spindle Pole Bodies ◽  
Mitotic Exit Network ◽  
Cell Cycle Transition ◽  
Spindle Position

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

scholarly journals Discrete regions of the kinesin-8 Kip3 tail differentially mediate astral microtubule stability and spindle disassembly

2018 ◽  
Vol 29 (15) ◽  
pp. 1866-1877 ◽  
Author(s):  
Sandeep Dave ◽  
Samuel J. Anderson ◽  
Pallavi Sinha Roy ◽  
Emmanuel T. Nsamba ◽  
Angela R. Bunning ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Microtubule Dynamics ◽  
Motor Domain ◽  
Spindle Positioning ◽  
Cellular Processes ◽  
Microtubule Stability ◽  
Astral Microtubule ◽  
Cellular Context ◽  
Spindle Disassembly ◽  
The Stability

To function in diverse cellular processes, the dynamic properties of microtubules must be tightly regulated. Cellular microtubules are influenced by a multitude of regulatory proteins, but how their activities are spatiotemporally coordinated within the cell, or on specific microtubules, remains mostly obscure. The conserved kinesin-8 motor proteins are important microtubule regulators, and family members from diverse species combine directed motility with the ability to modify microtubule dynamics. Yet how kinesin-8 activities are appropriately deployed in the cellular context is largely unknown. Here we reveal the importance of the nonmotor tail in differentially controlling the physiological functions of the budding yeast kinesin-8, Kip3. We demonstrate that the tailless Kip3 motor domain adequately governs microtubule dynamics at the bud tip to allow spindle positioning in early mitosis. Notably, discrete regions of the tail mediate specific functions of Kip3 on astral and spindle microtubules. The region proximal to the motor domain operates to spatially regulate astral microtubule stability, while the distal tail serves a previously unrecognized role to control the timing of mitotic spindle disassembly. These findings provide insights into how nonmotor tail domains differentially control kinesin functions in cells and the mechanisms that spatiotemporally control the stability of cellular microtubules.

scholarly journals Aurora kinase Ipl1 facilitates bilobed distribution of clustered kinetochores to ensure error-free chromosome segregation in Candida albicans

2019 ◽  
Author(s):  
Neha Varshney ◽  
Kaustuv Sanyal
Keyword(s):  
Candida Albicans ◽  
Chromosome Segregation ◽  
Structural Integrity ◽  
Aurora Kinase ◽  
Spindle Pole ◽  
Microtubule Dynamics ◽  
Aurora Kinase B ◽  
Spindle Pole Bodies ◽  
Candida Infections ◽  
Anti Fungal

Candida albicans, an ascomycete, has an ability to switch to diverse morphological forms. While C. albicans is predominatly diploid, it can tolerate aneuploidy as a survival strategy under stress. Aurora kinase B homolog Ipl1 is a critical ploidy regulator that controls microtubule dynamics and chromosome segregation in Saccharomyces cerevisiae. In this study, we show that Ipl1 in C. albicans has a longer activation loop than that of the well-studied ascomycete S. cerevisiae. Ipl1 localizes to the kinetochores during the G1/S phase and associates with the spindle during mitosis. Ipl1 regulates cell morphogenesis and is required for cell viability. Ipl1 monitors microtubule dynamics which is mediated by separation of spindle pole bodies. While Ipl1 is dispensable for maintaining structural integrity and clustering of kinetochores in C. albicans, it is required for the maintenance of kinetochore geometry to form bilobed structures along the mitotic spindle, a feature of Ipl1 that was not observed in other yeasts. Depletion of Ipl1 results in erroneous kinetochore-microtubule attachments leading to aneuploidy-associated resistance to fluconazole, the most common anti-fungal drug used to treat Candida infections. Taking together, we suggest that Ipl1 spatiotemporally ensures kinetochore geometry to facilitate bipolar spindle assembly crucial for ploidy maintenance in C. albicans.

scholarly journals Checkpoint control of mitotic exit—do budding yeast mind the GAP?

2006 ◽  
Vol 172 (3) ◽  
pp. 331-333 ◽  
Author(s):  
John A. Cooper ◽  
Scott A. Nelson
Keyword(s):  
Budding Yeast ◽  
Spindle Pole ◽  
Small Gtpase ◽  
Mitotic Exit ◽  
Cell Cycle Checkpoints ◽  
Nucleotide Exchange ◽  
Checkpoint Control ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Spindle Pole Bodies

Cell cycle checkpoints can delay mitotic exit in budding yeast. The master controller is the small GTPase Tem1, with inputs from a proposed guanine nucleotide exchange factor (GEF), Lte1, and a GTPase-activating protein (GAP), Bub2/Bfa1. In this issue, Fraschini et al. (p. 335) show that GAP activity of Bub2/Bfa1 appears to be dispensable for inactivation of Tem1 in cells. Their results call into question the GTP/GDP switch model for Tem1 activity, as have other results in the past. The paper also focuses attention on the two spindle pole bodies as potential sites for regulation of Tem1.

scholarly journals Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast

2000 ◽  
Vol 148 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Marisa Segal ◽  
Duncan J. Clarke ◽  
Paul Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Dynamic Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

scholarly journals Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28.

1996 ◽  
Vol 16 (11) ◽  
pp. 6385-6397 ◽  
Author(s):  
H H Lim ◽  
P Y Goh ◽  
U Surana
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Pole Bodies ◽  
M Phase ◽  
Yeast Homolog ◽  
Bipolar Spindles

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.

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