Browsing by Author "Bowerman, Bruce"
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Item Restricted The Construction and Deconstruction of Signaling Systems that Regulate Mitotic Spindle Positioning(University of Oregon, 2013-07-11) Lu, Michelle; Bowerman, BruceSignaling systems regulate the flow of cellular information by organizing proteins in space and time to coordinate a variety of cellular activities that are critical for the proper development, function, and maintenance of cells. Signaling molecules can exhibit several levels of complexity through the utilization of modular protein interactions, which can generate simple linear behaviors or complex behaviors such as ultrasensitivity. Protein modularity also serves as the basis for the vast protein networks that form the regulatory networks that govern several biological activities. My work focuses on the importance of protein modularity in complex biological systems, in particular the regulatory pathways of spindle positioning. The first part of my work involves the construction of a synthetic regulatory network using modular protein interactions in an effort to understand the complex behavior of the natural spindle orientation regulator Pins. Utilizing well-characterized protein domains and their binding partners, I built an autoinhibited protein switch that can be activated by a small protein domain. We found that the input-output relationship of the synthetic protein switch could be tuned by the simple addition of "decoy" domains, domains that bind and sequester input signal, thereby impeding the onset of the output response to generate an input threshold. By varying the number and affinities of the decoy domains, we found that we could transform a simple linear response into a complex, ultrasensitive one. Thus, modular protein interactions can serve as a source of complex behaviors. The second part of my work focuses on elucidating the molecular mechanisms underlying spindle positioning in the Drosophila neuroblast. I found that Pins orients the mitotic spindle by coordinating two opposite-polarity microtubule motors Dynein and Kinesin-73 through its multiple domains. Kinesin-73 also relies on its modular domain architecture to perform its duties in Pins-mediated spindle positioning, where its N-terminal half functions in coordinating cortical-microtubule capture while its C-terminal half functions as a region necessary for the activation of Dynein. Thus, modular protein design allows for the organization of spindle orientation regulators in space to achieve the complex biological activity that is spindle positioning. This dissertation includes previously published and unpublished coauthored material.Item Open Access Control of Histone H3 Lysine 27 Trimethylation in Neurospora crassa(University of Oregon, 2015-01-14) Jamieson, Kirsty; Bowerman, BruceTrimethylation of histone H3 lysine 27 (H3K27me3) marks facultative heterochromatin, containing silent genes. My research investigated factors that influence the distribution of H3K27me3 in the filamentous fungus Neurospora crassa. The H3K27 methyltransferase complex, PRC2, is well conserved in eukaryotes and consists of four core members: E(Z), EED, SUZ12 and P55. I showed that three of the PRC2 subunits (SET-7, the homolog of E(Z), EED and SUZ12) are required for H3K27me3 in Neurospora, while NPF, the homolog of P55, is only required for a subset of H3K27me3 domains. H3K27me3 is organized into large, gene-rich domains in Neurospora and normally does not overlap with constitutive heterochromatin, which is marked by both H3K9me3 and DNA methylation and bound by heterochromatin protein 1 (HP1). I discovered that loss of HP1 binding results in a genome-wide relocalization of H3K27me3. Specifically, it is lost from many of its normal domains while it becomes associated with much of the genome that is constitutive heterochromatin. This contrasts plant and mouse studies in which the loss of DNA methylation relocalizes H3K27me3. The DCDC complex is the H3K9-specific methyltransferase consisting of DIM-5, DIM-7, DIM-9, CUL4 and DIM-8. Separate deletions of DCDC subunits, with the exception of dim-7, relocalized H3K27me3 to constitutive heterochromatin, presumably due to the loss of HP1 binding. The deletion of dim-7 resulted in the loss of all H3K27me3, suggesting a novel role for dim-7. To look for a recruitment signal for PRC2, I moved large fragments contained within an H3K27me3 domain to loci devoid of H3K27me3, his-3 and csr-1. None of the fragments induced H3K27me3, demonstrating that a recruitment signal is not present within every fragment of H3K27me3-marked DNA. Large chromosomal rearrangements had profound effects on H3K27me3 domains, resulting in the loss of some H3K27me3 domains and the formation of others. In Drosophila and mammals, a subset of PRC2 complexes contains the histone deacetylase, Rpd3. A close homolog of Rpd3 in Neurospora, HDA-3, did not appear to be a member of PRC2 in Neurospora. This dissertation includes both previously published and unpublished co-authored material.Item Open Access Drosophila Embryonic Type II Neuroblasts: Origin, Temporal Patterning and Contribution to the Adult Central Complex(University of Oregon, 2018-04-10) Walsh, Kathleen; Bowerman, BruceThe large numbers of neurons that comprise the adult brain display an immense diversity. Repeated divisions of a relatively small pool of neural stem cells generate this neuronal diversity during development. To increase progress towards medical treatments for neurodegenerative diseases, it is of interest to understand both how neural stem cells generate the assortment of neurons and how these neurons come together to form a functional brain. Brain assembly occurs sequentially across time with early events laying the foundation for later events. Drosophila neural stem cells, neuroblasts (NBs), are an excellent model for investigating how neural diversity is generated and what roles early and late born neurons have in shaping the stereotypical adult brain structure. Generation of neural diversity, begins with specifying the diverse population of stem cells, called spatial patterning, and continues with diversifying neurons made from the diverse stem cells, called temporal patterning. Drosophila NBs exhibit both spatial and temporal patterning. Drosophila NBs have three types of division modes: type 0, type I and type II. Type II NBs expand the number of neurons made with progeny that exhibit a transit-amplifying division pattern, similar to that of mammalian outer subventricular zone (OSVZ) progenitors. Additionally, type II NBs exhibit temporal patterning across both the NB and their progeny to generate a large diversity of neurons that populate a conserved region of the brain responsible for many sensory and motor functions, called the central complex. Type II NBs have only been identified and studied during later stages in development, with nothing known about their origin or early divisions. In this dissertation, I describe the early lineages of the type II NBs within the Drosophila embryo. I show that type II NBs and lineages originate early in development, exhibit temporal patterning across both the NB and transit-amplifying progeny, and produce neurons that survive into the adult brain to innervate and potentially serve as a foundation within the adult central complex. Additionally, I explain how live imaging of the developing Drosophila brain can answer questions not easily addressed through other methods.Item Open Access Excess crossovers impede faithful meiotic chromosome segregation in C. elegans(Public Library of Science, 2020-09-04) Hollis, Jeremy A.; Glover, Marissa L.; Schlientz, Aleesa J.; Cahoon, Cori K.; Bowerman, Bruce; Wignall, Sarah M.; Libuda, Diana E.During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis.Item Open Access Massively Parallel Sequencing-Based Analyses of Genome and Protein Function(University of Oregon, 2015-08-18) Kamps-Hughes, Nicholas; Bowerman, BruceThe advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory. Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures. Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila. This dissertation includes previously published, co-authored materialItem Open Access Mechanisms of Branched Actin Network Formation through Coordinate Activation of Arp2/3 Complex(University of Oregon, 2015-01-14) Helgeson, Luke; Bowerman, BruceFundamental cellular processes such as motility and endocytosis rely on the actin cytoskeleton to translate biochemical protein interactions into mechanical forces. Cells utilize an extensive collection of actin binding proteins to comprehensively regulate actin networks during these dynamic cell operations. Branched actin networks, which are geometrically and functionally disparate from linear networks, are required for numerous cellular actions. Actin-related protein 2/3 complex (Arp2/3 complex) nucleates branched actin filaments upon activation by regulatory proteins known as nucleation promoting factors (NPFs). Often, several biochemically distinct NPFs are required for the same cellular structure, leading us to hypothesize that multiple NPFs can coordinately activate Arp2/3 complex to regulate the nucleation, architecture and assembly of branched networks. We identified and dissected the mechanisms of two sets of NPFs which coordinately activate Arp2/3 complex. Overall, these findings provide a better understanding of how Arp2/3 complex is activated and how cells control branched actin networks. In chapters II and III, we investigated the mechanism of synergistic activation of Arp2/3 complex by the NPFs cortactin and WASP family proteins. We found that cortactin accelerates the release of WASP family proteins from a branching intermediate, a previously unknown rate limiting step. Further dissection of the mechanism revealed that cortactin is specifically suited to displace WASP family proteins through a unique Arp2/3 complex binding region and target stalled branching intermediates with high affinity. Three different WASP family members were tested for their capacity to synergize with cortactin in Arp2/3 complex activation, establishing a list of cellular structures where cortactin-mediated synergistic activation is likely occurring. In chapter IV, we investigated the ability of Dip1 and Wsp1 to coordinately activate Arp2/3 complex during branched network formation. We established that Dip1 activation of Arp2/3 complex results in the formation of linear filaments which can template Wsp1 mediated branching. Subsequent kinetic data and modeling revealed that Dip1 and Wsp1 likely increase the rate of network formation by simultaneously binding to and co-activating Arp2/3 complex. These findings suggest that, together, Dip1 and Wsp1 regulate the initiation and rate of branched network assembly. This dissertation includes previously published and unpublished co-authored material and videos files.Item Open Access Oocyte Meiotic Cell Division: Spindle Assembly, Chromosome Segregation, and Cytokinesis.(University of Oregon, 2020-09-24) Schlientz, Aleesa; Bowerman, BruceMeiosis, the specialized cell division process that results in haploid gamete formation, is characterized by a single round of genome duplication followed by two successive divisions. During meiosis, replicated homologous chromosomes must pair and recombine or cross-over to allow for efficient chromosome segregation and formation of daughter cells with the correct chromosomal content. Defects in the meiotic division process, including the failure or mis-segregation of chromosomes or failed cytokinesis, can lead to complications such as aneuploid disorders (ex. trisomy 21), miscarriages, or infertility. Female gamete precursors, called oocytes, are remarkably error-prone during the meiotic division process, with approximately 10-30% of human oocytes having an incorrect number of chromosomes. Interestingly, the process of both spindle assembly and cytokinesis are unique in oocytes and differ greatly from the mitotic and sperm meiotic processes, with oocytes forming spindles in the absence of centrosomes and the cytokinetic apparatus, the polar body contractile ring, forming distal to both spindle poles rather than between them. These aspects of the meiotic division process lead to a number of questions about the requirements for building a meiotic spindle in the absence of centrosomes, segregating chromosomes, assembling a contractile ring, and the potential relationships between these processes. To better understand the oocyte meiotic division process, we used a combination of Caenorhabditis elegans genetics and spinning-disk confocal time-lapse microscopy to live-image the meiotic divisions in oocytes. We examined the requirements for the earliest stages of meiotic spindle assembly, focusing on microtubule nucleation and spindle formation dynamics, the impact of supernumerary crossovers on the process of chromosome segregation, and the relationship between oocyte meiotic spindle assembly/chromosome segregation on the assembly and dynamics of the cytokinetic contractile ring. These analyses allow for a better understanding of the functional requirements of the oocyte meiotic division process This dissertation includes unpublished co-authored material.Item Open Access Spatial Regulation of the Polarity Protein aPKC During Asymmetric Cell Division of Drosophila Neuroblasts(University of Oregon, 2015-08-18) Drummond, Mike; Bowerman, BruceThe Par complex protein, atypical protein kinase C (aPKC), plays an instrumental role in diverse cell polarities. aPKC is able to restrict substrate localization through a phosphorylation-induced cortical exclusion mechanism, allowing for the generation of molecularly distinct cortical domains. Thus, controlling the localization of aPKC is central to Par-mediated polarity but the mechanism by which aPKC is polarized remains poorly understood. In this dissertation I investigated the restriction of aPKC to the apical cortex of Drosophila neural stem cells, neuroblasts, as these cells dynamically polarize aPKC through repeated asymmetric cell divisions. The polarity created through aPKC phosphorylation must be tightly regulated in order to ensure proper balance between self-renewal and differentiation. To begin, I investigated whether or not aPKC’s so called ‘maturation’ by PDK1 phosphorylation is required for aPKC activity and localization. We found that aPKC’s phosphorylation by PDK1 is required for both polarity and full activity. An aPKC containing an unphosphorylatable activation loop mutation localizes symmetrically around the cortex in a manner independent of its binding partner, Par-6, suggesting that aPKC could interact with the cortex by an unknown mechanism. To investigate how aPKC is able to localize to the cortex independent of Par-6, I used an in vivo structure function analysis of domains within aPKC, accompanied by biochemical approaches. I identified a necessity for the aPKC C1 domain for binding to the neuroblast cortex. This interaction is mediated by negatively charged phospholipids. Neither aPKC interaction, with phospholipids or Par-6, is sufficient to restrict aPKC to the apical cortex. Thus, aPKC polarization utilizes a dual interaction mechanism that takes advantage of both protein-lipid and protein-protein interactions, and proper control of each of these signals is required to prevent neuroblast division defects. One interaction, mediated by the C1, is a general cortical targeting mechanism, whereas the other specifies polarization mediated by Par complex interactions. We conclude that a conformational change induced by these interactions activates aPKC’s catalytic activity, thereby coupling localization and activity. This dissertation includes unpublished co-authored material.Item Open Access Temporal Patterning and Generation of Neural Diversity in Drosophila Type II Neuroblast Lineages(University of Oregon, 2013-10-03) Bayraktar, Omer; Bowerman, BruceThe central nervous system (CNS) has an astonishing diversity of neurons and glia. The diversity of cell types in the CNS has greatly increased throughout evolution and underlies our unique cognitive abilities. The diverse neurons and glia in the CNS are made from a relatively small pool of neural stem cells and progenitors. Understanding the developmental mechanisms that generate diverse cell types from neural progenitors will provide insight into the complexity of the mammalian CNS and guide stem cell based therapies for brain repair. Temporal patterning, during which individual neural progenitors change over time to make different neurons and a glia, is essential for the generation of neural diversity. However, the regulation of temporal patterning is poorly understood. Human outer subventricular zone (OSVZ) neural stem cells and Drosophila type II neural stem cells (called neuroblasts) both generate transit-amplifying intermediate neural progenitors (INPs). INPs undergo additional rounds of cell division to increase the number of neurons and glia generated in neural stem cell lineages. However, it is unknown whether INPs simply expand the numbers of a particular cell type or make diverse neural progeny. In this dissertation, I show that type II neuroblast lineages give rise to extraordinary neural diversity in the Drosophila adult brain and contribute diverse neurons to a major brain structure, the central complex. I find that INPs undergo temporal patterning to expand neural diversity in type II lineages. I show that INPs sequentially generate distinct neural subtypes; that INPs sequentially express Dichaete, Grainyhead, and Eyeless transcription factors; and that these transcription factors are required for the production of distinct neural subtypes. Moreover, I find that parental type II neuroblasts also sequentially express transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. I conclude that neuroblast and INP temporal patterning axes act combinatorially to specify diverse neural cell types within adult central complex; OSVZ neural stem cells may use similar mechanisms to increase neural diversity in the human brain. This dissertation includes previously published co-authored material.Item Open Access XMAP215/ZYG-9 and TACC/TAC-1 Promote Bipolar Spindle Assembly and Stability during C. elegans Oocyte Meiotic Cell Division(University of Oregon, 2022-10-26) Harvey, Austin; Bowerman, BruceDespite lacking canonical, centriole-containing microtubule organizing centers, oocytes can still organize microtubules into a bipolar spindle and accurately separate chromosomes. How oocyte acentrosomal spindles achieve bipolarity and separate chromosomes remains unclear, yet accuracy is critical as defects can lead to severe health consequences such as aneuploidy. The conserved two-component modulator of microtubule stability, comprised of XMAP215/ZYG-9 and TACC/TAC-1 in Caenorhabditis elegans, is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division, with C. elegans zyg-9 and tac-1 mutant oocytes exhibiting multiple and indistinguishable defects beginning early in meiosis I. To better understand the role of XMAP215/ZYG-9 and TACC/TAC-1 during acentrosomal spindle assembly, we aimed to determine if these defects represent one early requirement, with additional later and indirect consequences, or multiple temporally distinct and more direct requirements. To accomplish this, we used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect at high resolution their meiotic spindle assembly requirements in C. elegans oocytes. Our results from temperature upshift and downshift experiments indicate that the XMAP215/ZYG-9 and TAC-1/TACC complex has multiple and temporally distinct requirements throughout oocyte meiotic cell division. First, we show that ZYG-9 and TAC-1 appear to promote the coalescence of early pole foci into a bipolar structure both by promoting pole stability and by limiting pole growth during meiosis I, with these requirements being independent of earlier defects in both microtubule organization and levels. Second, during metaphase I, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation, and this pole stability is important for maintaining chromosome congression at the metaphase plate. Finally, we show that ZYG-9/TAC-1 also are required for the proper coalescence of pole foci during meiosis II, independently of their requirements during meiosis I. Together, these analyses reveal that the ZYG-9 and TAC-1 complex has separable, stage-specific requirements throughout meiosis I and II, and we also discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.