Nicole Duggan

The structure of purified kinetochores reveals multiple microtubule-attachment sites

Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.

The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein.

The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-CMif2 protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

The Experiences and Care Preferences of People With Diabetes at the End of Life: A Qualitative Study

The aims of the study were to explore the past and current diabetes management experiences of people with diabetes at the end of life and identify their preferences for how they want their diabetes managed at the end of life. Fourteen semistructured interviews were conducted with people with diabetes receiving palliative care in a regional city in Victoria, Australia. The interviews were audio recorded, transcribed, and analyzed using framework analysis. Five key themes were identified from the data: living with diabetes, sometimes they “bugger it up,” it is a complicated subject, when I am very ill, and plans for the very end. The findings suggest that health professionals should continue monitoring blood glucose levels when a person has a life-limiting illness, listen to the views of the person with diabetes about his/her diabetes management, and identify and acknowledge the patient’s diabetes management preferences when he/she is very ill.

Kinetochore Function and Chromosome Segregation Rely on Critical Residues in Histones H3 and H4 in Budding Yeast

Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.

Diabetes and end of life: ethical and methodological issues in gathering evidence to guide care.

INTRODUCTION Providing palliative care for people with diabetes at the end of life is part of the chronic disease care trajectory, but end of life care is complex and the presence of diabetes further complicates management. AIM The aim of the paper is to discuss the ethical and methodological issues encountered when undertaking research to develop guidelines for managing diabetes at the end of life and the strategies used to address the issues. METHOD The issues emerged as we developed guidelines for managing diabetes at the end of life, which included conducting individual interviews with 14 people with diabetes requiring palliative care and 10 family members. A reflexive researcher journal was maintained throughout the guideline development process. The interview transcripts and researchers journal were analysed to determine key methodological, ethical and researcher-related issues. FINDINGS Key themes were vulnerability of the sampling population, methodological issues included recruiting participants and ensuring rigor, ethical issues concerned benefit and risk, justice, autonomy, privacy, professional boundaries and informed consent. Researcher-related issues were identified such as managing participant distress and their own emotional distress. People were willing to discuss end of life diabetes management preferences. CONCLUSIONS Undertaking research with people at the end of life is complex because of their vulnerability and the ethical issues involved. However, the ethical principles of autonomy and justice apply and people should be given the relevant information and opportunity to decide whether to participate or not.

Caring for People with Diabetes at the End of Life

End-of-life care planning is assuming global significance. While general end-of-life care guidelines apply to diabetes, there are some diabetes-specific issues that need to be considered. These include the usual long trajectory to end-of-life care that enables clinicians and people with diabetes to proactively discuss when to change the focus of care from preventing diabetes complications (tight control) to a palliative approach. Palliative care aims to promote comfort and quality of life and reduce the unnecessary burden of care on individuals and their families. The aim of this paper is to discuss common disease trajectories and their relationship to diabetes care, outline strategies for proactively discussing these issues and suggest indications that palliative care is warranted.