A pinboard by
Amanda Broad

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.


Hec1 Tail Phosphorylation Differentially Regulates Mammalian Kinetochore Coupling to Polymerizing and Depolymerizing Microtubules.

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

Premature Silencing of the Spindle Assembly Checkpoint Is Prevented by the Bub1-H2A-Sgo1-PP2A Axis in Saccharomyces cerevisiae.

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