Specialist α-tubulins for pluralist microtubules

α- and β-tubulins are encoded by multigene families, but the role of tubulin diversity for microtubule function has been a longstanding mystery. A new study (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010155) shows that the two budding yeast α-tubulins have distinct roles during mitotic spindle positioning.

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

The Journal of Cell Biology ◽

10.1083/jcb.145.5.979 ◽

1999 ◽

Vol 145 (5) ◽

pp. 979-991 ◽

Cited By ~ 118

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.

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Cohesin-independent segregation of sister chromatids in budding yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e11-08-0696 ◽

2012 ◽

Vol 23 (4) ◽

pp. 729-739 ◽

Cited By ~ 48

Author(s):

Vincent Guacci ◽

Douglas Koshland

Keyword(s):

Mitotic Spindle ◽

Budding Yeast ◽

Alternative Mechanism ◽

Yeast Viability ◽

Sister Chromatids ◽

Damage Repair ◽

Independent Segregation ◽

Independent Mechanism ◽

Cohesin Subunit

Cohesin generates cohesion between sister chromatids, which enables chromosomes to form bipolar attachments to the mitotic spindle and segregate. Cohesin also functions in chromosome condensation, transcriptional regulation, and DNA damage repair. Here we analyze the role of acetylation in modulating cohesin functions and how it affects budding yeast viability. Previous studies show that cohesion establishment requires Eco1p-mediated acetylation of the cohesin subunit Smc3p at residue K113. Smc3p acetylation was proposed to promote establishment by merely relieving Wpl1p inhibition because deletion of WPL1 bypasses the lethality of an ECO1 deletion (eco1Δ wpl1Δ). We find that little, if any, cohesion is established in eco1Δ wpl1Δ cells, indicating that Eco1p performs a function beyond antagonizing Wpl1p. Cohesion also fails to be established when SMC3 acetyl-mimics (K113Q or K112R,K113Q) are the sole functional SMC3s in cells. These results suggest that Smc3p acetylation levels affect establishment. It is remarkable that, despite their severe cohesion defect, eco1Δ wpl1Δ and smc3-K112R,K113Q strains are viable because a cohesin-independent mechanism enables bipolar attachment and segregation. This alternative mechanism is insufficient for smc3-K113Q strain viability. Smc3-K113Q is defective for condensation, whereas eco1Δ wpl1Δ and smc3-K112R,K113Q strains are competent for condensation. We suggest that Smc3p acetylation and Wpl1p antagonistically regulate cohesin's essential role in condensation.

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The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast

Current Biology ◽

10.1016/s0960-9822(00)00837-x ◽

2000 ◽

Vol 10 (23) ◽

pp. 1497-1506 ◽

Cited By ~ 129

Author(s):

Dale L. Beach ◽

Julie Thibodeaux ◽

Paul Maddox ◽

Elaine Yeh ◽

Kerry Bloom

Keyword(s):

Mitotic Spindle ◽

Budding Yeast

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Control of nuclear centration in the C. elegans zygote by receptor-independent Gα signaling and myosin II

The Journal of Cell Biology ◽

10.1083/jcb.200703159 ◽

2007 ◽

Vol 178 (7) ◽

pp. 1177-1191 ◽

Cited By ~ 29

Author(s):

Morgan B. Goulding ◽

Julie C. Canman ◽

Eric N. Senning ◽

Andrew H. Marcus ◽

Bruce Bowerman

Keyword(s):

Mitotic Spindle ◽

Myosin Ii ◽

Nonmuscle Myosin ◽

Wild Type ◽

Spindle Positioning ◽

C Elegans ◽

Nonmuscle Myosin Ii ◽

Pulling Forces ◽

Actomyosin Contraction

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction.

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Time-Lapse Microscopy Reveals Unique Roles for Kinesins during Anaphase in Budding Yeast

The Journal of Cell Biology ◽

10.1083/jcb.143.3.687 ◽

1998 ◽

Vol 143 (3) ◽

pp. 687-694 ◽

Cited By ~ 181

Author(s):

Aaron F. Straight ◽

John W. Sedat ◽

Andrew W. Murray

Keyword(s):

Mitotic Spindle ◽

Fluorescent Protein ◽

Budding Yeast ◽

Dynamic Structure ◽

Time Lapse ◽

Slow Phase ◽

Rapid Phase ◽

Green Fluorescent ◽

Anaphase B

The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Δ mutants lack the rapid phase of anaphase B, kip1Δ mutants show defects in the slow phase of anaphase B, and kip3Δ mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Δ and kip1Δ mutants affect the duration of anaphase, but cin8Δ does not.

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Mechanisms of MTOC clustering and spindle positioning in the budding yeast Cryptococcus neoformans: a microtubule grow-and-catch model

10.1101/690651 ◽

2019 ◽

Author(s):

Saptarshi Chatterjee ◽

Subhendu Som ◽

Neha Varshney ◽

Kaustuv Sanyal ◽

Raja Paul

Keyword(s):

Nuclear Envelope ◽

Cryptococcus Neoformans ◽

Mitotic Spindle ◽

Computational Models ◽

Budding Yeast ◽

Cell Cortex ◽

Critical Determinant ◽

Spindle Formation ◽

Spindle Positioning ◽

Septin Ring

AbstractMitotic spindle formation in the pathogenic budding yeast, Cryptococcus neoformans, depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before the formation of the mitotic spindle, multiple visible microtubule organizing centers (MTOCs), coalesce into a single focus to serve as an SPB. We propose a ‘grow-and-catch’ model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and catch each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screened various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning - a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintain spatiotemporal spindle localization.Author summaryCells actively self-assemble a bipolar spindle to facilitate chromosomal segregation. Multiple MTOCs, on the outer nuclear envelope, cluster into a single SPB before spindle formation during semi-open mitosis of the budding yeast Cryptococcus neoformans. Eventually, the SPB duplicates and organizes the spindle to position it within the daughter bud near the septin ring during anaphase. In this work, we tested various computational models to match physiological phenomena in an attempt to find plausible mechanisms of MTOC clustering and spindle positioning in C. neoformans. Notably, we propose an MT ‘grow-and-catch’ model that relies on possible redundant mechanisms for timely MTOC clustering mediated by (a) minus end-directed motors that crosslink and slide anti-parallel cMTs from different MTOCs on the nuclear envelope and (b) a Bim1 mediated biased sliding of cMTs along the cell cortex toward the septin ring that pulls MTOCs in the presence of suppressed dynein activity. By combining an analytical model and stochastic MT dynamics simulations, we screened various MT-based forces to detect steady spindle positioning. By screening the outputs of various models, it is revealed that proper spindle positioning near the septin ring requires MT buckling from the cell cortex.

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