In Vivo Dynamics of the Rough Deal Checkpoint Protein during Drosophila Mitosis

Basto, Renata; Scaerou, Frederic; Mische, Sarah; Wojcik, Edward; Lefebvre, Christophe; Hays, Thomas S.; Karess, Roger E.

doi:10.1016/j.cub.2003.12.025

Cited by 105 publications

(120 citation statements)

References 22 publications

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“…SAC proteins are highly colocalized with the bipolar spindle. They originally accumulate on unattached kinetochores 30, 31, 32, 33, 34 ; but upon stabilization of a proper kinetochore-spindle attachment, they undergo dynein-mediated poleward streaming along the spindles toward the associated spindle pole 11, 35, 36, 37, 38 Furthermore, such poleward streaming is continuous until anaphase onset 37, 39, 40 . Inhibiting the poleward streaming causes mitotic arrest 35, 36, 41, 42 .…”

mentioning

confidence: 99%

Spatial-temporal model for silencing of the mitotic spindle assembly checkpoint

Chen

Liu

2014

Nat Commun

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The spindle assembly checkpoint arrests mitotic progression until each kinetochore secures a stable attachment to the spindle. Despite fluctuating noise, this checkpoint remains robust and remarkably sensitive to even a single unattached kinetochore among many attached kinetochores; moreover, the checkpoint is silenced only after the final kinetochore-spindle attachment. Experimental observations have shown that checkpoint components stream from attached kinetochores along microtubules toward spindle poles. Here, we incorporate this streaming behavior into a theoretical model that accounts for the robustness of checkpoint silencing. Poleward streams are integrated at spindle poles, but are diverted by any unattached kinetochore; consequently, accumulation of checkpoint components at spindle poles increases markedly only when every kinetochore is properly attached. This step-change robustly triggers checkpoint silencing after, and only after, the final kinetochore-spindle attachment. Our model offers a conceptual framework that highlights the role of spatiotemporal regulation in mitotic spindle checkpoint signaling and fidelity of chromosome segregation.

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mentioning

confidence: 99%

Spatial-temporal model for silencing of the mitotic spindle assembly checkpoint

Chen

Liu

2014

Nat Commun

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“…Also, RNA interference reduction of Bub3 levels leads to the accumulation of cells in prophase (24). Cytoplasmic dynein is thought to contribute to the inactivation of the spindle checkpoint by transporting checkpoint proteins from kinetochores to the spindle poles along the newly attached kinetochore microtubules (27)(28)(29)(30)(31)(32). However, the molecular basis underlying the association of checkpoint proteins with dynein was unknown.…”

mentioning

confidence: 99%

The DYNLT3 Light Chain Directly Links Cytoplasmic Dynein to a Spindle Checkpoint Protein, Bub3

Kogoy

Pfister

2007

Journal of Biological Chemistry

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Cytoplasmic dynein is the motor protein responsible for the intracellular transport of various organelles and other cargoes toward microtubule minus ends. However, it remains to be determined how dynein is regulated to accomplish its varied roles. The dynein complex contains six subunits, including three classes of light chains. The two isoforms of the DYNLT (Tctex1) family of light chains, DYNLT1 and DYNLT3, have been proposed to link dynein to specific cargoes. However, no specific binding partner had been found for the DYNLT3 light chain. We find that DYNLT3 binds to Bub3, a spindle checkpoint protein.Bub3 binds exclusively to DYNLT3 and not to the other dynein light chains. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that Bub3 interacts with the cytoplasmic dynein complex. DYNLT3 is present on kinetochores at prometaphase, but not later mitotic stages, demonstrating that this dynein light chain, like Bub3 and other checkpoint proteins, is depleted from the kinetochore during chromosome alignment. Knockdown of DYNLT3 with small interference RNA increases the mitotic index, in particular, the number of cells in prophase/prometaphase. These results demonstrate that dynein binds directly to a component of the spindle checkpoint complex through the DYNLT3 light chain. Thus, DYNLT3 contributes to dynein cargo binding specificity. These data also suggest that the subpopulation of dynein, containing the DYNLT3 light chain, may be important for chromosome congression, in addition to having a role in the transport of checkpoint proteins from the kinetochore to the spindle pole.Cytoplasmic dynein 1 is a large multisubunit ATPase that moves toward the minus ends of microtubules. It is involved in multiple important cellular processes, including mitosis, nuclear migration, Golgi and centrosome localization, organelle transport, and viral transport (1, 2). This dynein complex has a molecular mass greater than 1 MDa and contains two identical heavy chains, each containing motor domains that generate the force necessary to move along the microtubule (3-5). The cytoplasmic dynein cargo binding domain consists of five subunits that assemble into the complex as dimers: the intermediate chain, light intermediate chain, and three classes of light chains; DYNLL, DYNLT, and DYNLRB.2 There are at least two isoforms of each of these five subunits (6). The DYNLT light chain family has two members, DYNLT1 (previously called Tctex1) and DYNLT3 (previously called rp3) (2, 7-9). The two family members share ϳ55% amino acid identity. Both light chains are expressed in all the cells and tissues so far examined, and they incorporate into dynein complexes as homodimers (7,10).3 Thus, a dynein complex contains either DYNLT1 or DYNLT3 but not both.While the dynactin complex is responsible for binding dynein to many cargoes, there is increasing evidence that the isoforms of cargo binding subunits are important in binding dynein to specific cargoes (reviewed in Ref. 1). DYNLT1 has been shown to specifically ...

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“…These results suggest that the Rod/Zw10 complex has a critical role in the centromeric recruitment of PIASy and, thus, in the SUMOylation of chromosomal substrates near the centromere. Previous studies in Drosophila embryos revealed that GFP-Rod enters the nucleus by the time that nuclear envelope breaks down, accumulates on kinetochores, and disperses as sister chromatids are separated (24). This aspect of Rod and Zw10 distribution during mitosis highly resembles PIASy distribution, leading to an explanation for how PIASy executes centromeric SUMOylation during mitosis.…”

Section: Discussionmentioning

confidence: 86%

“…The RZZ complex is localized at kinetochores from pro/metaphase to metaphase in somatic cells (24). PIASy was also observed near centromeres in XEE assays (Fig.…”

Section: The N Terminus Of Piasy Is Sufficient For Localization To Thmentioning

confidence: 85%

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Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

Ryu

Azuma

2010

Journal of Biological Chemistry

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SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.SUMOylation is a protein modification process conserved from yeast to vertebrates (1). The consequences of SUMO 2 (small ubiquitin-like modifier) modification that have been elucidated over the past decade include modulation of gene transcription, DNA repair, protein translocation, protein/protein interaction, chromosomal organization, and sister chromatid segregation (2-4). Vertebrates have three SUMO isoforms and all three display roughly 50% identity with the single SUMO found in yeast (1). SUMO is conjugated to cellular substrates by an analogous pathway to that of ubiquitin. It has been reported that SUMOylation is mediated without E3 ligases in vitro (5, 6), but under physiological conditions, SUMO E3 ligases are essential to execute SUMOylation of cellular substrates (7-10). There are mainly two types of SUMO E3 ligases in vertebrates, RanBP2 (Nup358), and Siz/PIAS. RanBP2 has no homolog in yeast, and its ligase function is independent of either HECT or Ring finger-type ubiquitin E3 ligases (11). Siz/PIAS, on the other hand, initially identified in budding yeast, functions similar to Ring finger ubiquitin ligases (8). Vertebrates have four PIAS proteins (PIAS1, PIAS3, PIASx, and PIASy) that share important conserved functional domains (12). The SAP (scaffold attachment factor-A/B, acinus and PIAS) domain is positioned at the N terminus, and directly binds AT-rich regions of DNA (13-15). The SP-Ring domain is related to that of ubiquitin E3 ligase and is responsible for Ubc9 recruitment (16). The SUMO-interacting motif (SIM) is situated after the SP-Ring and redirects the Ubc9ϳSUMO complex on substrate proteins, potentially contributing to SUMO paralogue specificity (17,18). Doma...

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In Vivo Dynamics of the Rough Deal Checkpoint Protein during Drosophila Mitosis

Cited by 105 publications

References 22 publications

Spatial-temporal model for silencing of the mitotic spindle assembly checkpoint

Spatial-temporal model for silencing of the mitotic spindle assembly checkpoint

The DYNLT3 Light Chain Directly Links Cytoplasmic Dynein to a Spindle Checkpoint Protein, Bub3

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

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