scholarly journals Yeast Kinetochores Do Not Stabilize Stu2p-dependent Spindle Microtubule Dynamics

2003 ◽  
Vol 14 (10) ◽  
pp. 4181-4195 ◽  
Author(s):  
Chad G. Pearson ◽  
Paul S. Maddox ◽  
Ted R. Zarzar ◽  
E.D. Salmon ◽  
Kerry Bloom
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Regulatory Mechanism ◽  
Metaphase Plate ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Binding ◽  
Microtubule Stabilization ◽  
Kinetochore Function

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule–kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

scholarly journals Mitotic Centromere–associated Kinesin Is Important for Anaphase Chromosome Segregation

1998 ◽  
Vol 142 (3) ◽  
pp. 787-801 ◽  
Author(s):  
Todd Maney ◽  
Andrew W. Hunter ◽  
Mike Wagenbach ◽  
Linda Wordeman
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Single Gene ◽  
Live Cell ◽  
Spindle Microtubule ◽  
Eventual Loss ◽  
Sister Centromere

Mitotic centromere–associated kinesin (MCAK) is recruited to the centromere at prophase and remains centromere associated until after telophase. MCAK is a homodimer that is encoded by a single gene and has no associated subunits. A motorless version of MCAK that binds centromeres but not microtubules disrupts chromosome segregation during anaphase. Antisense-induced depletion of MCAK results in the same defect. MCAK overexpression induces centromere-independent bundling and eventual loss of spindle microtubule polymer suggesting that centromere-associated bundling and/or depolymerization activity is required for anaphase. Live cell imaging indicates that MCAK may be required to coordinate the onset of sister centromere separation.

scholarly journals The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

2015 ◽  
Vol 211 (2) ◽  
pp. 309-322 ◽  
Author(s):  
Lindsay G. Lammers ◽  
Steven M. Markus
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Coiled Coil ◽  
Binding Domain ◽  
Cell Cortex ◽  
Wild Type ◽  
Microtubule Binding ◽  
Coiled Coil Domain ◽  
Microtubule Binding Domain

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal.

scholarly journals Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1

2002 ◽  
Vol 157 (7) ◽  
pp. 1125-1137 ◽  
Author(s):  
Anja Hagting ◽  
Nicole den Elzen ◽  
Hartmut C. Vodermaier ◽  
Irene C. Waizenegger ◽  
Jan-Michael Peters ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spindle Checkpoint ◽  
Cyclin B1 ◽  
Mutant Form ◽  
Exact Time ◽  
Sister Chromatids ◽  
Sister Chromatid Separation

Progress through mitosis is controlled by the sequential destruction of key regulators including the mitotic cyclins and securin, an inhibitor of anaphase whose destruction is required for sister chromatid separation. Here we have used live cell imaging to determine the exact time when human securin is degraded in mitosis. We show that the timing of securin destruction is set by the spindle checkpoint; securin destruction begins at metaphase once the checkpoint is satisfied. Furthermore, reimposing the checkpoint rapidly inactivates securin destruction. Thus, securin and cyclin B1 destruction have very similar properties. Moreover, we find that both cyclin B1 and securin have to be degraded before sister chromatids can separate. A mutant form of securin that lacks its destruction box (D-box) is still degraded in mitosis, but now this is in anaphase. This destruction requires a KEN box in the NH2 terminus of securin and may indicate the time in mitosis when ubiquitination switches from APCCdc20 to APCCdh1. Lastly, a D-box mutant of securin that cannot be degraded in metaphase inhibits sister chromatid separation, generating a cut phenotype where one cell can inherit both copies of the genome. Thus, defects in securin destruction alter chromosome segregation and may be relevant to the development of aneuploidy in cancer.

scholarly journals Visualization of Mad2 Dynamics at Kinetochores, along Spindle Fibers, and at Spindle Poles in Living Cells

2000 ◽  
Vol 150 (6) ◽  
pp. 1233-1250 ◽  
Author(s):  
B.J. Howell ◽  
D.B. Hoffman ◽  
G. Fang ◽  
A.W. Murray ◽  
E.D. Salmon
Keyword(s):  
Chromosome Segregation ◽  
Binding Sites ◽  
Metaphase Plate ◽  
Atp Depletion ◽  
Anaphase Promoting Complex ◽  
Spindle Fiber ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Kinetochore Function ◽  
Spindle Fibers

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest–deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2–Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t1/2 of ∼24–28 s. Cells entered anaphase ∼10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

scholarly journals Live Cell Imaging of Chromosome Segregation During Mitosis

10.3791/57389 ◽  
2018 ◽  
Author(s):  
Prajakta Varadkar ◽  
Kazuyo Takeda ◽  
Brenton McCright
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell

scholarly journals Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

2002 ◽  
Vol 13 (10) ◽  
pp. 3706-3719 ◽  
Author(s):  
Katie B. Shannon ◽  
Julie C. Canman ◽  
E. D. Salmon
Keyword(s):  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Spindle Checkpoint ◽  
Normal Numbers ◽  
Microtubule Attachment ◽  
Checkpoint Pathway ◽  
Chromosome Congression ◽  
Full Complement ◽  
Checkpoint Genes

The spindle checkpoint monitors microtubule attachment and tension at kinetochores to ensure proper chromosome segregation. Previously, PtK1 cells in hypothermic conditions (23°C) were shown to have a pronounced mitotic delay, despite having normal numbers of kinetochore microtubules. At 23°C, we found that PtK1 cells remained in metaphase for an average of 101 min, compared with 21 min for cells at 37°C. The metaphase delay at 23°C was abrogated by injection of Mad2 inhibitors, showing that Mad2 and the spindle checkpoint were responsible for the prolonged metaphase. Live cell imaging showed that kinetochore Mad2 became undetectable soon after chromosome congression. Measurements of the stretch between sister kinetochores at metaphase found a 24% decrease in tension at 23°C, and metaphase kinetochores at 23°C exhibited higher levels of 3F3/2, Bub1, and BubR1 compared with 37°C. Microinjection of anti-BubR1 antibody abolished the metaphase delay at 23°C, indicating that the higher kinetochore levels of BubR1 may contribute to the delay. Disrupting both Mad2 and BubR1 function induced anaphase with the same timing as single inhibitions, suggesting that these checkpoint genes function in the same pathway. We conclude that reduced tension at kinetochores with a full complement of kinetochore microtubules induces a checkpoint dependent metaphase delay associated with elevated amounts of kinetochore 3F3/2, Bub1, and BubR1 labeling.

scholarly journals Differentiation of Cytoplasmic and Meiotic Spindle Assembly MCAK Functions by Aurora B-dependent Phosphorylation

2004 ◽  
Vol 15 (6) ◽  
pp. 2895-2906 ◽  
Author(s):  
Ryoma Ohi ◽  
Tanuj Sapra ◽  
Jonathan Howard ◽  
Timothy J. Mitchison
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Meiotic Spindle ◽  
Aurora B ◽  
Dependent Manner ◽  
Dynamic Nature ◽  
Spindle Microtubule ◽  
Bipolar Spindles

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylation-dependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly.

scholarly journals CENP-F stabilizes kinetochore-microtubule attachments and limits dynein stripping of corona cargoes

2020 ◽  
Vol 219 (5) ◽  
Author(s):  
Philip Auckland ◽  
Emanuele Roscioli ◽  
Helena Louise Elvidge Coker ◽  
Andrew D. McAinsh
Keyword(s):  
Chromosome Segregation ◽  
Direct Interaction ◽  
Antagonistic Activity ◽  
Early Event ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Microtubule Attachment ◽  
Dynein Motor ◽  
Chromosome Congression ◽  
Kinetochore Function

Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.

114 LIVE-CELL IMAGING FOR THE QUALITY ASSESSMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO IN CYNOMOLGUS MONKEY

2012 ◽  
Vol 24 (1) ◽  
pp. 169
Author(s):  
J. Morichika ◽  
K. Yamagata ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
A. Kusanagi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Intracytoplasmic Sperm Injection ◽  
Cynomolgus Monkey ◽  
Live Cell Imaging ◽  
Embryo Quality ◽  
Cell Imaging ◽  
Imaging Technique ◽  
Live Cell ◽  
The Status ◽  
Normal Offspring

We have developed a live-cell imaging technique to assess measures of embryo quality such as epigenetic status and chromosome integrity during the early cleavage stages of pre-implantation development in the mouse. The advantages of this method are that the procedure is safe for the embryo and pups are not transgenic even after the imaging (Yamagata et al. 2009 Hum. Reprod. 24, 2490–2499). One of the valuable indexes in using this imaging technique is chromosome segregation (CS) during first mitosis; the embryos showing normal CS (NCS) result in normal offspring, whereas abnormal CS (ACS) embryos do not. In this study, we established a live-cell imaging technique for cynomolgus monkey intracytoplasmic sperm injection (ICSI) embryos and we succeeded in obtaining a normal offspring from NCS embryos after the assessment of live-cell imaging. Ovarian stimulation was carried out as previously described by Torii et al. (2001 Exp. Anim. 50, 259). Oocytes were collected by follicular aspiration using laparoscopy and ICSI was performed to metaphase II oocytes. After the ICSI, a mixture of mRNA encoding fluorescent labelled tubulin and histone was injected into ICSI embryos for the evaluation. Live-cell imaging was initiated 4 h after injection by laser confocal microscopy and 2-cell embryos were classified as NCS or ACS the next day. After embryo culture, embryo transfer (ET) was carried out to recipient donors (NCS embryos: 13, ACS embryos: 2) and pregnancy was diagnosed by ultrasonography at 4 weeks after ET. In another experiment, we tried to assess the 2-cell embryos with a snapshot image taken by a conventional fluorescent microscope as a simplified method. A total 121 embryos from 15 monkeys were analysed and embryos were classified as NCS or ACS. Live-cell imaging revealed that the NCS rate was 43.3% and the ACS rate was 56.7%. Pregnancy was confirmed in 2 NCS embryos from 13 ET (15.4%; 2/13); however, no pregnancy was observed in the ACS group (0%, 0/2). Furthermore, one normal offspring was achieved from ET of 2 NCS embryos that were diagnosed by live-cell imaging. In addition, we could also assess the status of chromosome and nuclei in the 2-cell embryos even by fluorescent microscopy and in this case, the NCS rate was 69.2% and the ACS rate was 30.8%. In conclusion, live-cell imaging can be used to evaluate the status of chromosome segregation of ICSI embryos in the cynomolgus monkey under laser confocal and fluorescent microcopy. The results indicate that ACS would be a detrimental factor in the embryonic development in the monkey, similar to in the mouse. Moreover, a normal offspring was born after the imaging and therefore this new technique could be applicable to assessment of embryo quality in human assisted reproductive technology.

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