Dynein collective behavior in mitotic nuclear positioning of Saccharomyces cerevisiae

1AbstractPositioning the nucleus at the bud-neck prior during Saccharomyces cerevisiae mitosis during anaphase involves pulling forces of cytoplasmic dynein localized in the daughter cell. While genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and dyneins numbers driving the process has remained difficult. In order to better understand the role of motor-microtubule mechanics during nuclear positioning and the role of dynein, we have used a computational model of nuclear mobility in S. cerevisiae and reconciled it to the mobility of labelled spindle pole bodies (SPBs) measured by quantifying fluorescence microscopy time-series. We model the apparent random-walk mobility of SPBs by combining diffusion of the nucleus and active pushing of MTs at the cell membrane. By minimizing the deviation between tracks of fluorescently tagged SPBs and simulations, we estimate the effective cytoplasmic viscosity to be 0.5 Pa s. The directed transport of nuclei during the budding process is similarly quantified by tracking the daughter SPB (SPB-D) in experiment. Using force-balance, we find 2 to 8 motors are required to pull the nucleus to the bud-neck. Simulations of the cytoplasmic MT (cMT) ‘search and capture’ by dynein suggest single motor binding is followed by a rapid saturation of number of bound motors. The short time and length of MT interactions with the cortex and minimal collective dynein force required, predict a functional role for dynein clustering in nuclear positioning.

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Saccharomyces cerevisiae Mob1p Is Required for Cytokinesis and Mitotic Exit

Molecular and Cellular Biology ◽

10.1128/mcb.21.20.6972-6983.2001 ◽

2001 ◽

Vol 21 (20) ◽

pp. 6972-6983 ◽

Cited By ~ 92

Author(s):

Francis C. Luca ◽

Manali Mody ◽

Cornelia Kurischko ◽

David M. Roof ◽

Thomas H. Giddings ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Spindle Pole Body ◽

Spindle Pole ◽

Mitotic Exit ◽

Live Cells ◽

Ring Assembly ◽

Spindle Poles ◽

Spindle Pole Bodies ◽

Bud Neck ◽

Mitotic Cyclin

ABSTRACT The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize inmob1 mutants, suggesting that MOB1functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck requiredCDC3, MEN genes CDC5,CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent ofMYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis.

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Structural role of Sfi1p–centrin filaments in budding yeast spindle pole body duplication

The Journal of Cell Biology ◽

10.1083/jcb.200603153 ◽

2006 ◽

Vol 173 (6) ◽

pp. 867-877 ◽

Cited By ~ 94

Author(s):

Sam Li ◽

Alan M. Sandercock ◽

Paul Conduit ◽

Carol V. Robinson ◽

Roger L. Williams ◽

...

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Crystal Structures ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pole Body ◽

Spindle Pole Bodies ◽

N Terminus ◽

Α Helix

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p–centrin complexes containing several repeats show Sfi1p as an α helix with centrins wrapped around each repeat and similar centrin–centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p–centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous α helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p–centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200405018 ◽

2004 ◽

Vol 167 (1) ◽

pp. 75-85 ◽

Cited By ~ 79

Author(s):

Brenton L. Scott ◽

Jeffrey S. Van Komen ◽

Hassan Irshad ◽

Song Liu ◽

Kirilee A. Wilson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bud Neck ◽

Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Spatial signals link exit from mitosis to spindle position

eLife ◽

10.7554/elife.14036 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 18

Author(s):

Jill Elaine Falk ◽

Dai Tsuchiya ◽

Jolien Verdaasdonk ◽

Soni Lacefield ◽

Kerry Bloom ◽

...

Keyword(s):

Spindle Pole ◽

Mitotic Exit ◽

Zone Model ◽

Cytoplasmic Microtubule ◽

Binucleate Cells ◽

Spindle Pole Bodies ◽

Mitotic Exit Network ◽

Bud Neck ◽

Competing Models ◽

Spindle Position

In budding yeast, if the spindle becomes mispositioned, cells prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN is a signaling cascade that localizes to spindle pole bodies (SPBs) and activates the phosphatase Cdc14. There are two competing models that explain MEN regulation by spindle position. In the 'zone model', exit from mitosis occurs when a MEN-bearing SPB enters the bud. The 'cMT-bud neck model' posits that cytoplasmic microtubule (cMT)-bud neck interactions prevent MEN activity. Here we find that 1) eliminating cMT– bud neck interactions does not trigger exit from mitosis and 2) loss of these interactions does not precede Cdc14 activation. Furthermore, using binucleate cells, we show that exit from mitosis occurs when one SPB enters the bud despite the presence of a mispositioned spindle. We conclude that exit from mitosis is triggered by a correctly positioned spindle rather than inhibited by improper spindle position.

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Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae.

Molecular Biology of the Cell ◽

10.1091/mbc.3.12.1443 ◽

1992 ◽

Vol 3 (12) ◽

pp. 1443-1454 ◽

Cited By ~ 65

Author(s):

J T McGrew ◽

L Goetsch ◽

B Byers ◽

P Baum

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Normal Cell ◽

Cell Cycle Progression ◽

Nuclear Division ◽

Flow Cytometric Analysis ◽

Spindle Pole ◽

Spindle Pole Bodies ◽

Mutant Cells ◽

Normal Cell Cycle

Mutations in the ESP1 gene of Saccharomyces cerevisiae disrupt normal cell-cycle control and cause many cells in a mutant population to accumulate extra spindle pole bodies. To determine the stage at which the esp1 gene product becomes essential for normal cell-cycle progression, synchronous cultures of ESP1 mutant cells were exposed to the nonpermissive temperature for various periods of time. The mutant cells retained viability until the onset of mitosis, when their viability dropped markedly. Examination of these cells by fluorescence and electron microscopy showed the first detectable defect to be a structural failure in the spindle. Additionally, flow cytometric analysis of DNA content demonstrated that massive chromosome missegregation accompanied this failure of spindle function. Cytokinesis occurred despite the aberrant nuclear division, which often resulted in segregation of both spindle poles to the same cell. At later times, the missegregated spindle pole bodies entered a new cycle of duplication, thereby leading to the accumulation of extra spindle pole bodies within a single nucleus. The DNA sequence predicts a protein product similar to those of two other genes that are also required for nuclear division: the cut1 gene of Schizosaccharomyces pombe and the bimB gene of Aspergillus nidulans.

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Early Spindle Assembly in Drosophila Embryos: Role of a Force Balance Involving Cytoskeletal Dynamics and Nuclear Mechanics

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0154 ◽

2005 ◽

Vol 16 (10) ◽

pp. 4967-4981 ◽

Cited By ~ 51

Author(s):

E. N. Cytrynbaum ◽

P. Sommi ◽

I. Brust-Mascher ◽

J. M. Scholey ◽

A. Mogilner

Keyword(s):

Spindle Assembly ◽

Spindle Pole ◽

Cortical Reorganization ◽

Force Balance ◽

Cell Cortex ◽

Nuclear Dynamics ◽

Nuclear Mechanics ◽

Cytoskeletal Dynamics ◽

Drosophila Embryos

Mitotic spindle morphogenesis depends upon the action of microtubules (MTs), motors and the cell cortex. Previously, we proposed that cortical- and MT-based motors acting alone can coordinate early spindle assembly in Drosophila embryos. Here, we tested this model using microscopy of living embryos to analyze spindle pole separation, cortical reorganization, and nuclear dynamics in interphase-prophase of cycles 11-13. We observe that actin caps remain flat as they expand and that furrows do not ingress. As centrosomes separate, they follow a linear trajectory, maintaining a constant pole-to-furrow distance while the nucleus progressively deforms along the elongating pole-pole axis. These observations are incorporated into a model in which outward forces generated by zones of active cortical dynein are balanced by inward forces produced by nuclear elasticity and during cycle 13, by Ncd, which localizes to interpolar MTs. Thus, the force-balance driving early spindle morphogenesis depends upon MT-based motors acting in concert with the cortex and nucleus.

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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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Conservative Duplication of Spindle Poles during Meiosis in Saccharomyces cerevisiae

Journal of Bacteriology ◽

10.1128/jb.183.7.2372-2375.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2372-2375 ◽

Cited By ~ 10

Author(s):

Andreas Wesp ◽

Susanne Prinz ◽

Gerald R. Fink

Keyword(s):

Saccharomyces Cerevisiae ◽

Spindle Pole Body ◽

Spindle Pole ◽

Spore Formation ◽

Wild Type ◽

Body Distribution ◽

Spindle Poles ◽

Pole Body ◽

Spindle Pole Bodies ◽

Type Strains

ABSTRACT During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

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Progression Into the First Meiotic Division Is Sensitive to Histone HN-HZB Dimer Concentration in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/145.3.647 ◽

1997 ◽

Vol 145 (3) ◽

pp. 647-659

Author(s):

Kochung Tsui ◽

Lee Simon ◽

David Norris

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Division ◽

Expression Patterns ◽

Spindle Pole ◽

Chain Elongation ◽

Histone H2a ◽

Yeast Saccharomyces Cerevisiae ◽

Spindle Pole Bodies ◽

Dna Chain ◽

Mitosis And Meiosis

The yeast Saccharomyces cerevisiae contains two genes for histone H2A and two for histone H2B located in two divergently transcribed gene pairs: HTA1-HTB1 and HTA2-HTB2. Diploid strains lacking HTA1-HTB1 (hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2) grow vegetatively, but will not sporulate. This sporulation phenotype results from a partial depletion of H2A-H2B dimers. Since the expression patterns of HTA1-HTB1 and HTA2-HTB2 are similar in mitosis and meiosis, the sporulation pathway is therefore more sensitive than the mitotic cycle to depletion of H2A-H2B dimers. After completing premeiotic DNA replication, commitment to meiotic recombination, and chiasma resolution, the hta1-htb1Δ/hta1-htb1Δ, HTA2-HTB2/HTA2-HTB2 mutant arrests before the first meiotic division. The arrest is not due to any obvious disruptions in spindle pole bodies or microtubules. The meiotic block is not bypassed in backgrounds homozygous for spo13, rad50Δ, or rad9Δ mutations, but is bypassed in the presence of hydroxyurea, a drug known to inhibit DNA chain elongation. We hypothesize that the deposition of H2A-H2B dimers in the mutant is unable to keep pace with the replication fork, thereby leading to a disruption in chromosome structure that interferes with the meiotic divisions.

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Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1

eLife ◽

10.7554/elife.36745 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 11

Author(s):

Safia Omer ◽

Samuel R Greenberg ◽

Wei-Lih Lee

Keyword(s):

Saccharomyces Cerevisiae ◽

Spatial Distribution ◽

Direct Evidence ◽

Critical Factor ◽

Spindle Positioning ◽

Dynein Motor ◽

Independent Population ◽

Bud Neck ◽

Pulling Forces ◽

Pulling Force

Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

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