Yinghui Mao

Centromeres and Kinetochores: From Epigenetics to Mitotic Checkpoint Signaling

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).

Activating and Silencing the Mitotic Checkpoint through CENP-E-Dependent Activation/Inactivation of BubR1

The mitotic checkpoint prevents advance to anaphase prior to successful attachment of every centromere/kinetochore to mitotic spindle microtubules. Using purified components and Xenopus egg extracts, the kinetochore-associated microtubule motor CENP-E is now shown to be the activator of the essential checkpoint kinase BubR1. Since kinase activity and the checkpoint are silenced following CENP-E-dependent microtubule attachment in extracts or binding of CENP-E antibodies that do not disrupt CENP-E association with BubR1, CENP-E mediates silencing of BubR1 signaling. Checkpoint signaling requires the normal level of BubR1 containing a functional Mad3 domain implicated in Cdc20 binding, but only a small fraction need be kinase competent. This supports bifunctional roles for BubR1 in the checkpoint: an enzymatic one requiring CENP-E-dependent activation of its kinase activity at kinetochores and a stoichiometric one as a direct inhibitor of Cdc20.

ZW10 links mitotic checkpoint signaling to the structural kinetochore

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80–Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1–Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived “stop anaphase” inhibitor.

Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling

The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible “stop anaphase” inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1–CENP-E–microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.

The mitotic kinesin CENP-E is a processive transport motor

In vivo studies suggest that centromeric protein E (CENP-E), a kinesin-7 family member, plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. How CENP-E accomplishes this crucial task, however, is not clear. Here we present single-molecule measurements of CENP-E that demonstrate that this motor moves processively toward the plus end of microtubules, with an average run length of 2.6 ± 0.2 μm, in a hand-over-hand fashion, taking 8-nm steps with a stall force of 6 ± 0.1 pN. The ATP dependence of motor velocity obeys Michaelis–Menten kinetics with KM,ATP = 35 ± 5 μM. All of these features are remarkably similar to those for kinesin-1—a highly processive transport motor. We, therefore, propose that CENP-E transports chromosomes in a manner analogous to how kinesin-1 transports cytoplasmic vesicles.

Subnuclear distribution of topoisomerase I is linked to ongoing transcription and p53 status

The nonconserved, hydrophilic N-terminal domain of eukaryotic DNA topoisomerase I (topo I) is dispensable for catalytic activity in vitro but essential in vivo. There are at least five putative nuclear localization signals and a nucleolin-binding signal within the first 215 residues of the topo I N-terminal domain. We have investigated physiological functions of the topo I N-terminal domain by fusing it to an enhanced green fluorescent protein (EGFP). The first 170 residues of the N-terminal domain allow efficient import of chimeric proteins into nuclei and nucleoli. The nucleolar localization of this protein does not depend on its interaction with nucleolin, whereas ongoing rDNA transcription clearly is crucial. Immunoprecipitation experiments reveal that the topo I N terminus (topoIN)-EGFP fusion protein associates with the TATA-binding protein in cells. Furthermore, DNA damage results in extensive nuclear redistribution of the topoIN-EGFP chimeric product. The redistribution is also p53-dependent and the N terminus of topo I appears to interact with p53 in vivo. These results show that the topo I localization to the nucleolus is related to the p53 and DNA damage, as well as changes in transcriptional status. Nucleolar release of topo I under conditions of cellular duress may represent an important, antecedent step in tumor cell killing by topoisomerase active agents.

CENP-E–dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

The state of CENP-E–dependent BubR1 autophosphorylation in response to spindle microtubule capture regulates kinetochore function and accurate chromosome segregation.

FORMIN a link between kinetochores and microtubule ends

The mammalian diaphanous-related (mDia) formin proteins are well known for their actin-nucleation and filament-elongation activities in mediating actin dynamics. They also directly bind to microtubules and regulate microtubule stabilization at the leading edge of the cell during cell migration. Recently, the formin mDia3 was shown to associate with the kinetochore and to contribute to metaphase chromosome alignment, a process in which kinetochores form stable attachments with growing and shrinking microtubules. We suggest that the formin mDia3 could contribute to the regulation of kinetochore-bound microtubule dynamics, in coordination with attachment via its own microtubule-binding activity, as well as via its interaction with the tip-tracker EB1 (end-binding protein 1).

Emerging functions of force-producing kinetochore motors

More than two decades of research has resulted in the identification of some 60 microtubule motor proteins, several of which have been implicated in mitosis. Although some kinesin superfamily proteins function as microtubule depolymerases at kinetochores, such as Kinesin-8 and -13, it is now appreciated that there are only two force-producing kinetochore associated motors, the plus end-directed microtubule motor CENP-E and the minus end-directed microtubule motor cytoplasmic dynein. Defining their roles at kinetochores has been hampered by the complexity of mitosis itself, and a multiplicity of mitotic roles, at least for cytoplasmic dynein. Nonetheless, recent advances have served to define the primary roles of the two kinetochore motors in detail.

Catalytic inhibition of DNA topoisomerase IIα by sodium azide

It has been demonstrated previously that sodium azide reduces the clastogenicity of several DNA topoisomerase II (topo II) poisons in cultured mammalian cells. These studies suggested that azide may be a catalytic topo II inhibitor. Azide interferes with mitochondrial production of ATP and is also known to inhibit cellular ATPases. Since topo II requires ATP for catalytic activity (enzyme turnover), it seemed likely that interference with ATP levels or ATP catabolism was the underlying mechanism of topo II inactivation; however, this has not been examined in living cells under conditions where the endogenous topo II is active on genomic DNA. The present studies were carried out to verify that azide inhibits endogenous topo II in cells. We show that azide blocks both decatenation and relaxation activity of purified topo II in a concentration dependent manner and reduces topoII/DNA covalent complex formation in cells. From these studies, it is concluded that sodium azide catalytically inactivates topo II via an ATP-sensitive process.