Human Zwint-1 Specifies Localization of Zeste White 10 to Kinetochores and Is Essential for Mitotic Checkpoint Signaling

Abstract

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization. In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation. These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore. Finally, our studies show that Zwint-1 is a new component of the mitotic checkpoint, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei. As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.Chromosome movements during mitosis are governed by the interaction of spindle microtubules with a specialized chromosome domain located within the centromere. This specialized region, called the kinetochore (1, 2), is the site for spindle microtubule-centromere association. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore has an active function in chromosomal segregation through microtubule motors and spindle checkpoint sensors located at or near it (3-5).Eukaryotic organisms require extraordinary fidelity in chromosome segregation during meiosis and mitosis as aberrant chromosome segregation can be catastrophic to an organism or its progeny. One of the evolutionarily conserved multiprotein complexes essential for the fidelity of chromosome segregation contains several proteins, including ZW10 1 (Zeste White 10) and ROD (Rough Deal) (6 -8). Mutations in the Drosophila ZW10 or ROD genes cause similar defects, most noticeably in lagging chromatids that remain at the metaphase plate late in anaphase, leading to high levels of aneuploidy among daughter cells.ZW10 and ROD proteins display remarkable dynamics in their intracellular location during cell division (7, 9, 10). Both proteins accumulate strongly at the outer kinetochore plates during prometaphase. At metaphase, ZW10 and ROD depart from the kinetochores and relocate onto spindle microtubules. During anaphase, the proteins are no longer found on kinetochore microtubules and instead localize exclusively to the kinetochores of the separating chromosomes.Besides binding to ROD, ZW10 is responsible for localization of cytoplasmic dynein to kinetochores (10 -12) via a direct contact with dynamitin, a...

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…It remains unclear whether the SAC component, Mps1p, also plays a role in centrosome duplication in higher eukaryotes [Fischer et al, 2004;Fisk et al, 2003;Liu et al, 2003;Stucke et al, 2004;Stucke et al, 2002] as it does in budding [Winey et al, 1991] but not in fission yeast [He et al, 1998]. The mammalian Ndc80p complex [McCleland et al, 2003], the ROD and ZW10 kinetochore proteins [Basto et al, 2000;Chan et al, 2000], the outer kinetochore protein Zwint-1 [Obuse et al, 2004;Wang et al, 2004a], and the kinetochore-associated NEK2A protein [Lou et al, 2004] are also required for the spindle checkpoint, indicating that the kinetochore's role in SAC signaling is evolutionarily conserved, although no ROD, ZW10, or Zwint-1 homologues have been identified in yeast. The mammalian survivin/Aurora B complex regulates BubR1p and Mad2p localization to kinetochores [Lens et al, 2003] and, like the budding yeast Ipl1p/Aurora, is required for SAC function when insufficient tension is applied across sister kinetochores [Carvalho et al, 2003;Hauf et al, 2003;Lens et al, 2003].…”

The Microtubule Plus End Tracking Protein TIP150 Interacts with Cortactin to Steer Directional Cell Migration

“…EB1 siRNA (5Ј-AAGUGAAAUU-CCAAGCUAAGC-3Ј) (39) and CTN siRNA (5Ј-CAAGCTT-CGAGAGAATGTCTT-3Ј) were synthesized by Qiagen. As a control, a duplex-composing scrambled sequence was used (40,41). The 21-mer oligonucleotide RNA duplexes were synthesized from Dharmacon Research, Inc.…”

Mitotic Regulator SKAP Forms a Link between Kinetochore Core Complex KMN and Dynamic Spindle Microtubules

“…Hec1, Mis12, Mis13, and CENP-E siRNA were reported previously (15,19,20). As a control, either a duplex targeting cyclophilin or scramble sequence was used (17,21).…”

“…Antibodies-Both rabbit and mouse antibodies against SKAP were generated using full-length recombinant proteins from bacteria using a standard protocol as described previously (17). Antibodies against Mis13 were generated as described previously (18).…”