Sharpening the anaphase switch

2015 ◽  
Vol 43 (1) ◽  
pp. 19-22 ◽  
Author(s):  
John C. Meadows ◽  
Jonathan B.A. Millar
Keyword(s):  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Mitotic Chromosomes ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Substantial Progress ◽  
A Cell ◽  
Spindle Elongation ◽  
Anaphase B

The segregation of sister chromatids during mitosis is one of the most easily visualized, yet most remarkable, events during the life cycle of a cell. The accuracy of this process is essential to maintain ploidy during cell duplication. Over the past 20 years, substantial progress has been made in identifying components of both the kinetochore and the mitotic spindle that generate the force to move mitotic chromosomes. Additionally, we now have a reasonable, albeit incomplete, understanding of the molecular and biochemical events that are involved in establishing and dissolving sister-chromatid cohesion. However, it is less well-understood how this dissolution of cohesion occurs synchronously on all chromosomes at the onset of anaphase. At the centre of the action is the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that, in association with its activator cell-division cycle protein 20 homologue (Cdc20), is responsible for the destruction of securin. This leads to the activation of separase, a specialized protease that cleaves the kleisin-subunit of the cohesin complex, to relieve cohesion between sister chromatids. APC/C–Cdc20 is also responsible for the destruction of cyclin B and therefore inactivation of the cyclin B–cyclin-dependent kinase 1 (Cdk1). This latter event induces a change in the microtubule dynamics that results in the movement of sister chromatids to spindle poles (anaphase A), spindle elongation (anaphase B) and the onset of cytokinesis. In the present paper, we review the emerging evidence that multiple, spatially and temporally regulated feedback loops ensure anaphase onset is rapid, co-ordinated and irreversible.

scholarly journals Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast

2001 ◽  
Vol 155 (5) ◽  
pp. 711-718 ◽  
Author(s):  
Fedor Severin ◽  
Anthony A. Hyman ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Budding Yeast ◽  
Sister Chromatid Cohesion ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Spindle Elongation

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

scholarly journals Cdc14-regulated midzone assembly controls anaphase B

2007 ◽  
Vol 177 (6) ◽  
pp. 981-993 ◽  
Author(s):  
Anton Khmelinskii ◽  
Clare Lawrence ◽  
Johanna Roostalu ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Protein Phosphatase ◽  
Cross Linking ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Spindle Midzone ◽  
A Cell ◽  
Spindle Elongation ◽  
And Function ◽  
Anaphase B

Spindle elongation in anaphase of mitosis is a cell cycle–regulated process that requires coordination between polymerization, cross-linking, and sliding of microtubules (MTs). Proteins that assemble at the spindle midzone may be important for this process. In this study, we show that Ase1 and the separase–Slk19 complex drive midzone assembly in yeast. Whereas the conserved MT-bundling protein Ase1 establishes a midzone, separase–Slk19 is required to focus and center midzone components. An important step leading to spindle midzone assembly is the dephosphorylation of Ase1 by the protein phosphatase Cdc14 at the beginning of anaphase. Failure to dephosphorylate Ase1 delocalizes midzone proteins and delays the second, slower phase of anaphase B. In contrast, in cells expressing nonphosphorylated Ase1, anaphase spindle extension is faster, and spindles frequently break. Cdc14 also controls the separase–Slk19 complex indirectly via the Aurora B kinase. Thus, Cdc14 regulates spindle midzone assembly and function directly through Ase1 and indirectly via the separase–Slk19 complex.

scholarly journals The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

2008 ◽  
Vol 183 (2) ◽  
pp. 267-277 ◽  
Author(s):  
Evan C. Osmundson ◽  
Dipankar Ray ◽  
Finola E. Moore ◽  
Qingshen Gao ◽  
Gerald H. Thomsen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
E3 Ligase ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Carboxyl Terminus ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Anaphase Onset

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

Mitotic motors and chromosome segregation: the mechanism of anaphase B

2011 ◽  
Vol 39 (5) ◽  
pp. 1149-1153 ◽  
Author(s):  
Ingrid Brust-Mascher ◽  
Jonathan M. Scholey
Keyword(s):  
Chromosome Segregation ◽  
Cyclin B ◽  
Spindle Poles ◽  
Mitotic Motors ◽  
Balance Of Forces ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Relationship Of ◽  
The Relationship ◽  
Acting In Concert

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an ‘on/off’ switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned ‘off’, tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.

scholarly journals Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

2017 ◽  
Author(s):  
Charles L. Asbury
Keyword(s):  
Fluorescence Microscopy ◽  
Mechanistic Models ◽  
Chromosome Movement ◽  
Load Bearing ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Chromosome Movements ◽  
Cellular Movement

The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly, or ‘melting’ of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through a ‘pac-man’ mechanism, where tubulin subunits are lost from kinetochore-attached plus ends and the kinetochore appears to consume its microtubule track as it moves poleward. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive pac-man and flux-based movements, are discussed.

The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised

2002 ◽  
Vol 115 (14) ◽  
pp. 2847-2856 ◽  
Author(s):  
Jun-yong Huang ◽  
Jordan W. Raff
Keyword(s):  
Fusion Proteins ◽  
Cyclin B ◽  
Subcellular Localisation ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Mitotic Chromosomes ◽  
Nuclear Envelope Breakdown ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Drosophila Cells

In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularised embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos,only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction,but its subcellular localisation remains controversial. We constructed GFP fusions of two core APC/C subunits, Cdc16 and Cdc27. These fusion proteins were incorporated into the endogenous APC/C and were largely localised in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulated in the nucleus prior to nuclear envelope breakdown but only weakly associated with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly,we noticed that GFP-Cdc27 associated with mitotic chromosomes, whereas GFP-Cdc16 did not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90%in tissue culture cells led to a transient mitotic arrest that was both biochemically and morphologically distinct. Taken together, our results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localised and perform distinct functions.

scholarly journals A Subunit of the Anaphase-Promoting Complex Is a Centromere-Associated Protein in Mammalian Cells

1998 ◽  
Vol 18 (1) ◽  
pp. 468-476 ◽  
Author(s):  
Pia-Marie Jörgensen ◽  
Eva Brundell ◽  
Maria Starborg ◽  
Christer Höög
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Separation Process ◽  
Soluble Form ◽  
Anaphase Promoting Complex ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Ubiquitin Ligase Complex ◽  
A Subunit

ABSTRACT Sister chromatids in early mitotic cells are held together mainly by interactions between centromeres. The separation of sister chromatids at the transition between the metaphase and the anaphase stages of mitosis depends on the anaphase-promoting complex (APC), a 20S ubiquitin-ligase complex that targets proteins for destruction. A subunit of the APC, called APC-α in Xenopus (and whose homologs are APC-1, Cut4, BIME, and Tsg24), has recently been identified and shown to be required for entry into anaphase. We now show that the mammalian APC-α homolog, Tsg24, is a centromere-associated protein. While this protein is detected only during the prophase to the anaphase stages of mitosis in Chinese hamster cells, it is constitutively associated with the centromeres in murine cells. We show that there are two forms of this protein in mammalian cells, a soluble form associated with other components of the APC and a centromere-bound form. We also show that both the Tsg24 protein and the Cdc27 protein, another APC component, are bound to isolated mitotic chromosomes. These results therefore support a model in which the APC by ubiquitination of a centromere protein regulates the sister chromatid separation process.

scholarly journals The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects

2007 ◽  
Vol 177 (4) ◽  
pp. 599-611 ◽  
Author(s):  
Elena Chiroli ◽  
Valentina Rossio ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Protein Phosphatase ◽  
Budding Yeast ◽  
S Phase ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
Chromosome Transmission ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Noncanonical Pathway

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2ACdc55 in controlling a noncanonical pathway of chromatid cohesion removal.

scholarly journals Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient

2007 ◽  
Vol 177 (6) ◽  
pp. 995-1004 ◽  
Author(s):  
Dhanya K. Cheerambathur ◽  
Gul Civelekoglu-Scholey ◽  
Ingrid Brust-Mascher ◽  
Patrizia Sommi ◽  
Alex Mogilner ◽  
...  
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Complete Recovery ◽  
Cyclin B ◽  
Spatial Gradient ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Mechanical Switch ◽  
Lower Recovery ◽  
Drosophila Embryos

Anaphase B in Drosophila embryos is initiated by the inhibition of microtubule (MT) depolymerization at spindle poles, which allows outwardly sliding interpolar (ip) MTs to drive pole–pole separation. Using fluorescence recovery after photobleaching, we observed that MTs throughout the preanaphase B spindle are very dynamic and display complete recovery of fluorescence, but during anaphase B, MTs proximal to the poles stabilize and therefore display lower recovery than those elsewhere. Fluorescence microscopy of the MT tip tracker EB1 revealed that growing MT plus ends localize throughout the preanaphase B spindle but concentrate in the overlap region of interpolar MTs (ipMTs) at anaphase B onset. None of these changes occurred in the presence of nondegradable cyclin B. Modeling suggests that they depend on the establishment of a spatial gradient of MT plus-end catastrophe frequencies, decreasing toward the equator. The resulting redistribution of ipMT plus ends to the overlap zone, together with the suppression of minus-end depolymerization at the poles, could constitute a mechanical switch that initiates spindle elongation.

The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A

1996 ◽  
Vol 109 (5) ◽  
pp. 961-969 ◽  
Author(s):  
K.D. Brown ◽  
K.W. Wood ◽  
D.W. Cleveland
Keyword(s):  
Chromosome Segregation ◽  
Chromosome Movement ◽  
Initial Alignment ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Elongation ◽  
Anaphase B

The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

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