Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpoint

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
The EMBO Journal (Impact Factor: 10.43). 03/2009; 28(7):902-14. DOI: 10.1038/emboj.2009.38
Source: PubMed


The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re-expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1-phosphorylation-deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.

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    • "Loss of any component impairs dynein/ dynactin binding (Starr et al, 1998; Griffis et al, 2007; Yang et al, 2007; Chan et al, 2009) and leads to improper kinetochore–MT attachments in prometaphase (Sharp et al, 2000; Gassmann et al, 2008; Varma et al, 2008) and loss of Mad1 and Mad2 (Buffin et al, 2005; Kops et al, 2005). In metaphase, impairment of dynein/dynactin function leads to prolonged checkpoint activation and retention of kinetochore-associated checkpoint proteins, which has led to the hypothesis that dynein-based 'stripping' of key regulators from kinetochores drives checkpoint silencing (Howell et al, 2000; Sivaram et al, 2009). Dynein/dynactin perturbation also typically results in significant spindle derangement owing to the focusing activities they provide at spindle poles, which makes it all the more difficult to tease out their roles at kinetochores. "
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    ABSTRACT: Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150Glued subunit, but little is known about the 'pointed-end complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed β-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.
    Full-text · Article · Mar 2013 · The EMBO Journal
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    • "For instance, unattached kinetochores activate the SAC by recruiting the Mad2 component of the SAC to the kinetochores first (Waters et al. 1998; Essex et al. 2009). Once all of the kinetochores have achieved the proper attachment , the SAC is silenced by the minus-end–directed protein dynein, which " walks " away the Mad2 and other SAC components from kinetochores along mictotubules to centrosomes (Griffis et al. 2007; Howell et al. 2001; Schmidt et al. 2005; Sivaram et al. 2009). If the removal of the SAC components by dynein is compromised, the SAC remains activated even when the proper attachment is achieved, leading to unnecessary delay in anaphase onset due to the inhibition of APC/C activity. "
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    ABSTRACT: Cyclin-dependent kinases (CDK) and their compulsory cofactors, the cyclins, are the two key classes of regulatory molecules that determine the eukaryotic cell's progress through the cell cycle by substrate phosphorylation. Cdk1 forms complexes with B-type cyclins and phosphorylates a number of substrates as cells prepare to enter mitosis. CYB-3 (Cyclin B3) is a B-type cyclin that has been recently shown to be required for the timely metaphase-to-anaphase transition, presumably by alleviating a spindle assembly checkpoint (SAC) block. Previously, we have shown that doubling the CYB-3 dosage suppresses sterility in the absence of the essential SAC component MDF-1/Mad1. Here we demonstrate the importance of the Mos1-mediated single-copy insertion method for understanding the effects of gene dosage by generating strains that have more (two or three) copies of the cyb-3 in wild-type and mdf-1(gk2) backgrounds to investigate dosage effect of CYB-3 on mitotic progression as well as development and fertility in the absence and the presence of the MDF-1 checkpoint component. We show that tripling the dosage of CYB-3 results in a significantly variable metaphase-to-anaphase transition, both in wild-type and mdf-1(gk2) mutant backgrounds. Although a majority of embryos initiate anaphase onset normally, a significant number of embryos initiate anaphase with a delay. We also show that tripling the dosage of CYB-3 has no effect on viability in the wild-type background; however, it does reduce the sterility caused by the absence of MDF-1. Together, these data reveal that proper dosage of CYB-3 is important for precision of timely execution of anaphase onset regardless of the presence of the MDF-1 checkpoint component.
    Full-text · Article · Aug 2012 · G3-Genes Genomes Genetics
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    • "This conclusion is supported by the fact that dynein localizes to multiple subcellular structures in mitosis where it fulfills diverse functions. It is involved in centrosome separation during prophase (Splinter et al., 2010; Tanenbaum et al., 2010); connects astral microtubules to the cell cortex, possibly pulling spindle poles apart (Busson et al., 1998; Gö nczy et al., 1999; Grill and Hyman, 2005; Laan et al., 2012); transports microtubule nucleation factors and spindle assembly checkpoint factors along kinetochore fibers from kinetochores to spindle poles (Ma et al., 2010; Sivaram et al., 2009; Chan et al., 2009 and references therein); and is involved in pole focusing (Gaglio et al., 1996; Shimamoto et al., 2011). Taking these diverse functions into consideration, it is not surprising that depletion of a multifunctional protein such as dynein results in unexpected and highly complex patterns of spindle formation. "
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    ABSTRACT: During cell division, the molecular motor Eg5 crosslinks overlapping antiparallel microtubules and pushes them apart to separate mitotic spindle poles. Dynein has been proposed as a direct antagonist of Eg5 at the spindle equator, pulling on antiparallel microtubules and favoring spindle collapse. Some of the experiments supporting this hypothesis relied on endpoint quantifications of spindle phenotypes rather than following individual cell fates over time. Here, we present a mathematical model and proof-of-principle experiments to demonstrate that endpoint quantifications can be fundamentally misleading because they overestimate defective phenotypes. Indeed, live-cell imaging reveals that, while depletion of dynein or the dynein binding protein Lis1 enables spindle formation in presence of an Eg5 inhibitor, the activities of dynein and Eg5 cannot be titrated against each other. Thus, dynein most likely antagonizes Eg5 indirectly by exerting force at different spindle locations rather than through a simple push-pull mechanism at the spindle equator.
    Full-text · Article · May 2012 · Cell Reports
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