Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes

Cytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis. Keywords:dynein, ubiquitin, NSC697923, PYR-41, mitosis, microtubule

Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program

The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA–microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (Grant GM088313)American Cancer Society (Research Scholar Grant 121776)National Science Foundation (U.S.). Graduate Research Fellowshi

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Functional analyses of mitotic microtubule-binding complexes

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.Mitosis is the process by which a single cell divides to form two identical daughter cells. Each daughter cell must inherit a full complement of the genetic material. Thus, a critical aspect of mitosis is the faithful segregation of each duplicated chromosome. Chromosome segregation is achieved through the attachment of a chromosome-localized macromolecular complex, termed the kinetochore, to microtubules. Microtubules are dynamic polymers comprised of tubulin heterodimers. The successful execution of mitosis additionally depends on the organization of the microtubules into a bipolar array, termed the mitotic spindle. The depolymerization of kinetochore-bound microtubules generates the force required to properly segregate the chromosomes. The work in this thesis analyzes the molecular basis for the function and activity of two key players in microtubule function. First, I investigate the mechanisms by which the Ska1 complex facilitates the continued association of the kinetochore with microtubules, even as the microtubules grow and shrink. I show that Ska1 uses multiple surfaces to interact with diverse tubulin substrates, and each of these surfaces are required for microtubule tip tracking and optimal mitotic progression. Second, I analyze cytoplasmic dynein, a microtubule-based motor that is critically required to maintain spindle bipolarity and execute numerous other cellular processes throughout the cell cycle. The execution of these diverse functions of dynein relies on precise temporal and spatial regulation of dynein activity. Dynein regulation is accomplished in part by the association of adaptor proteins with the dynein complex, including Nde1. Here, I show that Nde1 utilizes distinct intermolecular interactions to regulate different dynein functions. I also identify a previously uncharacterized interaction between Nde1 and the 26S proteasome. Finally, I explore a potential role for post-translational modifications in regulating dynein function. I find that the localization of dynein during mitosis is rapidly altered following the addition of small molecule inhibitors of ubiquitination enzymes. Together, these findings provide new insights into the function and regulation of diverse components of the mitotic machinery.by Julie Kathryn Monda.Ph. D

Nde1 promotes diverse dynein functions through differential interactions and exhibits an isoform-specific proteasome association

Nde1 is a key regulator of cytoplasmic dynein, binding directly to both dynein itself and the dynein adaptor, Lis1. Nde1 and Lis1 are thought to function together to promote dynein function, yet mutations in each result in distinct neurodevelopment phenotypes. To reconcile these phenotypic differences, we sought to dissect the contribution of Nde1 to dynein regulation and explore the cellular functions of Nde1. Here we show that an Nde1- Lis1 interaction is required for spindle pole focusing and Golgi organization but is largely dispensable for centrosome placement, despite Lis1 itself being required. Thus, diverse functions of dynein rely on distinct Nde1- and Lis1-mediated regulatory mechanisms. Additionally, we discovered a robust, isoform-specific interaction between human Nde1 and the 26S proteasome and identify precise mutations in Nde1 that disrupt the proteasome interaction. Together, our work suggests that Nde1 makes unique contributions to human neurodevelopment through its regulation of both dynein and proteasome function.National Institute of General Medical Sciences (Grant GM088313)National Institute of General Medical Sciences (Grant GM108718)National Science Foundation (Grant 1122374

Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.National Institute of General Medical Sciences (U.S.) (Grant GM088313

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces

The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation. Monda and Whitney et al. demonstrate that the kinetochore-localized Ska1 complex autonomously tracks with both growing and depolymerizing microtubule ends in vitro and that this activity requires multiple microtubule binding surfaces. Ska1 mutants that bind microtubules, but are unable to tip track, result in defective mitotic progression in cells. Keywords: mitosis; kinetochore; microtubule; tubulin; chromosome segregationNational Science Foundation (U.S.) (Grant 1122374