PhD student , Colorado State University
We are interested in understanding how cell division is regulated by the essential kinase Aurora B.
Mitotic cell division is a fundamental biological process that is essential for all eukaryotes to divide the replicated genome with high fidelity into individual daughter cells. Improper segregation of replicated DNA typically results in chromosome instability, a characteristic that is deleterious to most cells. Critical to the proper segregation of mitotic chromosomes is attachment to spindle microtubules, which are dynamic cytoskeleton filaments that drive the movement of chromosomes during mitosis. A complex network of proteins, collectively called the kinetochore, mediates microtubule attachments to chromosomes. Kinetochores are recruited to individual chromosomes through a specialized heterochromatin domain known as the centromere. Heterochromatin contains tightly packed DNA that is organized into structures called nucleosomes. Each nucleosome contains 147 bp of DNA wrapped around a histone octamer that is typically composed of two histone H2A-H2B dimers and two histone H3-H4 dimers. Centromeric heterochromatin is comprised of both canonical, H3-containing nucleosomes as well as nucleosomes that contain the histone H3 variant CENP-A. Centromeres serve as a central point of organization in mitotic cells, recruiting both structural and regulatory kinetochore proteins to chromosomes. This extensive protein/DNA network ensures the accurate segregation of chromosomes by regulation of proper kinetochore-microtubule (KMT) attachments in mitosis. KMT interactions are regulated by Aurora B kinase (ABK), which phosphorylates outer kinetochore substrates to promote release of erroneous attachments. Although ABK substrates at the kinetochore are defined, little is known about how ABK is recruited to and evicted from kinetochores in early and late mitosis, respectively, to regulate these essential interactions. The current model describing the recruitment of ABK to centromeres proposes that two histone modifications are required. Specifically, phosphorylation of histone H3 (H3-pT3) and histone H2A (H2A-pT120) is suggested to localize ABK to the inner centromere. Contrary to the proposed model, however, immunostaining experiments reveal that H3-pT3 localizes to the inner centromere, while H2A-pT120 distinctly localizes to kinetochores. Thus, major questions remain, including how histone modifications affect the binding of ABK to regulate KMT attachments and whether or not these modifications exist on the same nucleosomes in vivo.
Abstract: The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1, 2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore moves along microtubules [3, 4]. Despite a growing understanding of the molecular components that mediate kinetochore binding [5-7], we do not know how kinetochores physically interact with polymerizing versus depolymerizing microtubule bundles, and whether they use the same mechanisms and regulation to do so. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8-11] in mammalian cells. Hec1's affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12-15], but its role at the interface with polymerizing versus depolymerizing microtubules remains unclear. Here we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1's role on polymerizing versus depolymerizing microtubules. We show that Hec1 tail phosphorylation tunes friction along polymerizing microtubules and yet does not compromise the kinetochore's ability to grip depolymerizing microtubules. Together, the data suggest that kinetochore regulation has differential effects on engagement with growing and shrinking microtubules. Through this mechanism, the kinetochore can modulate its grip on microtubules over mitosis and yet retain its ability to couple to microtubules powering chromosome movement.
Pub.: 30 May '17, Pinned: 29 Jun '17
Abstract: Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation.
Pub.: 01 Jun '17, Pinned: 29 Jun '17
Abstract: During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.
Pub.: 01 Dec '15, Pinned: 29 Jun '17
Abstract: The spindle assembly checkpoint (SAC) monitors mistakes in kinetochore-microtubule interaction and its activation prevents anaphase entry. The SAC remains active until all chromosomes have achieved bipolar attachment that applies tension on kinetochores. Our previous data in budding yeast Saccharomyces cerevisiae show that Ipl1/Aurora B kinase and a centromere-associated protein Sgo1 are required to prevent SAC silencing prior to tension generation, but we believe that this regulatory network is incomplete. Bub1 kinase is one of the SAC components, and Bub1-dependent H2A phosphorylation triggers centromere recruitment of Sgo1 by H2A in yeast and human cells. Although yeast cells lacking the kinase domain of Bub1 show competent SAC activation, we found that the mutant cells fail to maintain a prolonged checkpoint arrest in the presence of tensionless attachment. Mutation of the Bub1 phosphorylation site in H2A also results in premature SAC silencing in yeast cells. Previous data indicate that Sgo1 protein binds to PP2A(Rts1), and we found that rts1Δ mutants exhibited premature SAC silencing as well. We further revealed that sgo1 mutants with abolished binding to H2A or PP2A(Rts1) displayed premature SAC silencing. Together, our results suggest that, in budding yeast S. cerevisiae, the Bub1-H2A-Sgo1-PP2A(Rts1) axis prevents SAC silencing and helps prolonged checkpoint arrest prior to tension establishment at kinetochores.
Pub.: 04 Jan '17, Pinned: 29 Jun '17
Abstract: The four-subunit chromosomal passenger complex (CPC), whose enzymatic subunit is Aurora B kinase, promotes chromosome biorientation by detaching incorrect kinetochore-microtubule attachments. In this study, we use a combination of truncations and artificial dimerization in budding yeast to define the minimal CPC elements essential for its biorientation function. We engineered a minimal CPC comprised of the dimerized last third of the kinase-activating Sli15/INCENP scaffold and the catalytic subunit Ipl1/Aurora B. Although native Sli15 is not oligomeric, artificial dimerization suppressed the biorientation defect and lethality associated with deletion of a majority of its microtubule-binding domain. Dimerization did not act through a physical clustering-based kinase activation mechanism but instead promoted spindle association, likely via a putative helical domain in Sli15 that is essential even when dimerized and is required to target kinetochore substrates. Based on the engineering and characterization of a minimal CPC, we suggest that spindle association is important for active Ipl1/Aurora B complexes to preferentially destabilize misattached kinetochores.
Pub.: 21 Mar '17, Pinned: 29 Jun '17
Abstract: Chromosome alignment on the mitotic spindle is monitored by the spindle checkpoint. We identify Sgo1, a protein involved in meiotic chromosome cohesion, as a spindle checkpoint component. Budding yeast cells with mutations in SGO1 respond normally to microtubule depolymerization but not to lack of tension at the kinetochore, and they have difficulty attaching sister chromatids to opposite poles of the spindle. Sgo1 is thus required for sensing tension between sister chromatids during mitosis, and its degradation when they separate may prevent cell cycle arrest and chromosome loss in anaphase, a time when sister chromatids are no longer under tension.
Pub.: 08 Jan '05, Pinned: 29 Jun '17
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