2,981 research outputs found
Video of SHINE! by Kim Scott feat. Blake Aaron and Philip N. Davis
No full textLive performance from the album release concert for "SHINE!", Kim's 5th album on the Innervision Records label. Kim Scott, flute; Phil Davis, keys; Eric Essix, guitar; Sean Michael Ray, bass; James "PJ" Spraggins, drums; Kelley Oneal, sax and flut
Regulation of Interpolar Microtubules by Microtubule Plus-End Binding Proteins in <italic>Saccharomyces cerevisiae</italic>
No full textThesis (Ph.D.)--University of Washington, 2013Cells carrying out mitosis must ensure that each daughter cell receives a full complement of chromosomes. As part of this process, multiprotein complexes called kinetochores link centromeric DNA to microtubule plus ends. This attachment allows cells to carry out two tasks that are essential for proper chromosome segregation. It couples chromosome position to microtubule plus end dynamics to allow maneuvering of chromosomes. It also senses whether chromosomes have been correctly attached to the spindle, correcting errors and delaying the cell cycle as necessary. That the kinetochore can maintain attachment to a microtubule plus end that is constantly polymerizing and disintegrating under its grip is one of the kinetochore's more extraordinary feats. Why evolution has chosen to utilize the plus-end, one of the cell's most ephemeral structures, as the site of its crucial chromosomal attachments is unknown. My lab previously isolated a Saccharomyces cerevisiae kinetochore mutant, DAM1-765, whose kinetochores retain their initial lateral attachment to the microtubule lattice instead of maturing into end-on attachments. Despite this fundamental defect in spindle organization, DAM1-765 cells proliferate nearly as rapidly as wild-type. To determine how end-on kinetochore-microtubule attachments benefit the cell, I performed a synthetic lethal screen against DAM1-765. This screen produced mutant alleles of the EB1 homolog Bim1, CLIP-170 homolog Bik1, and the kinesin-14 subunits Kar3 and Cik1. Cells possessing these mutations fail to properly bundle spindle microtubules and struggle to biorient chromosomes, phenotypes which are exacerbated in the presence of DAM1-765. The presence of a kinetochore capping the plus end of a kinetochore microtubule appears to assist in the proper development of interpolar microtubules. In the absence of such a cap, spindle microtubules are poorly organized, and spindles are short and subject to collapse. Three proteins identified in my synthetic lethal screen, Bim1 and Kar3/Cik1, are believed to be involved in bundling and regulating interpolar microtubules. It is unknown, however, how these proteins accomplish these tasks when they are only known to be able to bind to a single microtubule. I have identified a potential Bim1 binding motif, SxIP, on Cik1's N-terminus. This motif is highly conserved among kinesin-14s. To determine the role this motif plays in kinesin-14 function on the mitotic spindle, I mutated this motif to alanine residues and assayed the mutation's effect on spindle structure via fluorescence microscopy. The motif is important for recruitment of Cik1 to the spindle and for maintenance of proper spindle length in both metaphase and anaphase. The existence of a greater Bim1/Kar3/Cik1 complex capable of binding to two microtubules (one via Bim1's calponin homology domain, and one via Kar3/Cik1's motor and motor homology domains) could explain how Bim1 and Kar3/Cik1 are able to bundle interpolar microtubules together. This work confirms that Bim1, Kar3, and Cik1 are important bundlers and regulators of interpolar microtubules and raises the possibility that Bik1 may be involved in this process as well. It suggests a mechanism by which Bim1 and Kar3/Cik1 may carry out these tasks. It also demonstrates that regulation of the different cohorts of spindle microtubules is intimately linked; interpolar microtubules rely on the proper maturation of kinetochore microtubules to properly develop themselves
Kinetochore-microtubule coupling and its regulation during mitosis
No full textThesis (Ph.D.)--University of Washington, 2014The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain, which has been the focus of biochemical and structural characterization. However, the function of the remainder of Ndc80 is poorly understood. We coupled high-throughput sequencing to a saturating linker-scanning mutagenesis screen in Saccharomyces cerevisiae and identified essential domains in previously uncharacterized regions of Ndc80. By analyzing mutants from the screen, we found that one domain within the Ndc80 coiled-coil is involved in assembling, but not maintaining, metaphase spindles. This process likely depends on a conformational change in the complex. Furthermore, we found that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex in vitro, and a C-terminal segment of Ndc80 is required for tetramerization of the complex in vivo. In S. cerevisiae, the Ndc80 complex recruits the microtubule-binding Dam1 complex to kinetochores. The Ndc80 and Dam1 complexes are not redundant, but their distinct contributions are unknown. We show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to couple to microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by Aurora B. This provides evidence for a mechanism by which Aurora B resets aberrant kinetochore-microtubule attachments in S. cerevisiae. In higher eukaryotes, recent findings suggest that Aurora B also influences microtubule dynamics as part of its error correction mechanism. We show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). Human Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even while robustly coupled to disassembling microtubule tips. Together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the human Ndc80 complex
Regulation of microtubule nucleation and attachment to spindle pole bodies
No full textThesis (Ph.D.)--University of Washington, 2016-03The precise regulation and coordination of the mitotic spindle is vital for accurate chromosomal segregation within a dividing cell. The centrosome is the microtubule organizing center of the cell, responsible for nucleating and organizing the microtubules of the mitotic spindle. In yeast, the centrosome functional equivalent is called the spindle pole body. The studies presented here aimed to understand how microtubules are nucleated at the spindle pole body and also studied the regulation and mechanical strength of the yeast spindle pole body in vitro. In yeast, the γ-tubulin small complex, a highly conserved heterotetramer essential for microtubule nucleation, is composed of two copies of Tub4, and one copy each of Spc97 and Spc98. In this study, a comprehensive mutagenesis technique coupled with high-throughput sequencing was used to identify regions of Spc97 and Spc98 that were essential for the formation of the γ-tubulin small complex and mutations that influenced the organization of the mitotic spindle. Regions essential for structure and function of the complex were mapped onto the protein sequence for both Spc97 and Spc98 and temperature sensitive mutants identified by the mutagenic screen were isolated and their unique nucleation phenotypes characterized. Improvements were made to historical spindle pole body purification methods to facilitate a variety of in vitro biochemical and biophysical assays that had previously been hindered by technological constraints. Higher spindle pole body yields, purity, and reproducibility enabled a re-evaluation of the yeast spindle pole body cell cycle phosphoproteome. High confidence phosphorylation assignments were collected and compared to previously published data sets to reduce the ambiguity in the phosphoproteome data set while also expanding the data set to analyze the novel phosphorylation profile of the spindle pole body in G1/S. In vivo characterization of the high confidence phosphorylation sites revealed a combination of phosphorylation events in Spc97 that were essential for cell viability. The adaptability of the spindle pole body purification methods also allowed for the purification of genetic mutants of the spindle pole body, which were studied by biophysical assays. The strength of the microtubule attachment to the spindle pole body was probed by a laser trapping technique to determine how mutations in spindle pole body component Spc110 affected the physical integrity of the spindle pole body. It was shown that mutations in Spc110 decreased the force at which the microtubule was pulled from the spindle pole body, providing mechanical evidence for in vivo phenotypes of mitotic spindle failure. Together, these experiments interrogated several aspects of the yeast spindle pole body in vivo and in vitro to better understand the requirements for and regulation of microtubule nucleation
Biophysical mechanisms of the kinetochore-microtubule interface and its regulation during mitosis
No full textThesis (Ph.D.)--University of Washington, 2014Chromosomes carry the genetic information that acts as a "blueprint" for every organism. When cells divide, replicated copies of the chromosomes are segregated into two new cells that are genetic copies of the original. This process is mediated by the mitotic spindle, a bipolar array of dynamic microtubules that connect to chromosomes and drive their segregation. Microtubules link to chromosomes via kinetochores, which assemble on the centromeres and present microtubule attachment sites. These kinetochore-microtubule linkages are tightly regulated to ensure accurate transmission of the genetic material during each division. In the budding yeast <italic>Saccharomyces cerevisiae</italic>, kinetochore-microtubule attachments are mediated by the Ndc80 and Dam1 complexes. Both are essential for viability, though their distinct contributions to kinetochore-microtubule coupling were previously unknown. We showed that these complexes interact directly to form robust linkages to dynamic microtubule ends. Furthermore, the interaction between these complexes can be disrupted by the mitotic regulatory kinase, Aurora B. During error correction, Aurora B detaches aberrant kinetochore-microtubule linkages, providing another chance to form correct attachments. We propose that Aurora B targets the interaction between the Ndc80 and Dam1 complexes during corrective detachment. In higher eukaryotes, error correction appears to depend additionally on modulating microtubule dynamics to promote microtubule disassembly. We showed that this effect is exerted through Aurora B regulation of the human Ndc80 complex. Kinetochore-microtubule linkages require the combined activity of many different kinetochore components. Moreover, these components are present in multiple copies, as ~20 Ndc80 complexes and ~30 Dam1 complexes act collectively at each kinetochore-microtubule interface. <italic>In vitro</italic>, Dam1 complexes associate together to form large oligomeric rings that encircle microtubules; how this oligomerization contributes to kinetochore function has remained unclear. We found that oligomerization of the Dam1 kinetochore complex is required for its ability to form microtubule attachments that are robust against tension <italic>in vitro</italic> and <italic>in vivo</italic>. In higher eukaryotes, the Ndc80 and Ska complexes are both reported to oligomerize on microtubules. Therefore, we propose that oligomerization is an essential and conserved feature of kinetochore components that is required for accurate chromosome segregation during mitosis
Molecular Architecture and Regulation of the Budding Yeast Kinetochore-Microtubule Interface
No full textThesis (Ph.D.)--University of Washington, 2018Equal partitioning of replicated chromosomes during mitosis is vital. The genetic information stored in chromosomes provide the template for cells to produce RNA and protein molecules, which carry out cellular functions. Failure to equally divide chromosomes between two daughter cells means a disturbance in this central dogma of biology. Chromosome division is mediated by a bipolar structure specific to mitosis, called the spindle. Cytoskeletal microtubules anchored at each pole grow towards the other pole, resulting in the separation of two poles. During this process, the sister chromatids (replicated pair of chromosomes connected to each other) bind to spindle microtubules. This is made possible by the kinetochore, which assembles on the centromeric DNA region of each sister chromatid. During metaphase, sister kinetochores establish bioriented attachments on dynamic spindle microtubules. In anaphase, microtubule depolymerization is coupled with the segregation of sister chromatids towards their respective poles. Thus, the main function of the kinetochore is to make strong and processive attachments to microtubules. In the budding yeast S. cerevisiae outer kinetochore, the essential Ndc80 and Dam1 complexes are the main microtubule binding components. I show that these two protein complexes have a tripartite interaction and disrupting any one interaction site results in chromosome segregation error. Through these three interactions, the elongated Ndc80 complex bridges two Dam1 complex rings. In addition, I detail the mechanisms by which the Aurora B kinase and Cyclin dependent kinase 1 (Cdk1) regulate the kinetochore-microtubule interface. The Aurora B kinase phosphorylates the Dam1 complex to disrupt each interaction site between the Dam1 and Ndc80 complexes. Further phosphorylation of the Dam1 complex results in the inhibition of both cooperative and single molecule microtubule-binding ability. In contrast, the Cdk1 phosphorylates the Dam1 complex to enhance the cooperative binding onto microtubules. Together, I describe how the Ndc80 and Dam1 complexes are organized at the kinetochore-microtubule interface and the intricate mechanisms regulating these interactions. The Spc105 and MIND complexes are also essential and conserved components of the outer kinetochore. I show for the first time a recombinant expression and purification method for the Spc105 complex. I detected coiled-coil interactions between the two proteins that compose the Spc105 complex, Spc105p and Kre28p. Purification of the Spc105 complex from budding yeast co-purifies both MIND and Ndc80 complexes. These three protein complexes represent the highly conserved KMN network. Future studies will further utilize these tools for investigating how the Spc105 complex is organized with other outer kinetochore components
The Role of Outer Kinetochore Proteins in Forming Load-Bearing Interactions with the Dynamic Microtubule Tip
No full textThesis (Ph.D.)--University of Washington, 2021Mitosis, a biological process where newly duplicated DNA is condensed and segregated between a dividing cell, is an essential process for all eukaryotic systems. During mitosis, dynamic spindle microtubules, emanated from the spindle pole body, forms an interaction with the kinetochore. The kinetochore, a molecular machine composed of over 50 different proteins in budding yeast, is first assembled on a specific region on the chromosomes, known as the centromere, and connects with the spindle microtubule. The kinetochore plays many roles during chromosome segregation. 1) The kinetochore forms end-on attachments with the dynamic microtubule tip. 2). Once end-on attachments are made, the kinetochore ensures these interactions are load bearing. 3). Lastly, the kinetochore acts as a regulatory hub to ensure proper chromosome segregation. Here I investigate how the kinetochore forms load-bearing interactions with the dynamic microtubule tip. The two essential microtubule binding elements of the budding yeast kinetochore, Ndc80 and Dam1 complexes, interact with one another via three interaction regions. I found that two of these regions form load-bearing interactions between the Ndc80 and Dam1 complexes during microtubule assembling. These two regions are regulated by the Ipl1 kinase. All three interaction regions establish load-bearing interactions while on a disassembling microtubule tip
Building a Functional Kinetochore: From Microtubule to Centromere
No full textThesis (Ph.D.)--University of Washington, 2020Equal partitioning of duplicated chromosomes between daughter cells is a microtubule- mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, tethers chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and initiates error correction mechanisms. But though the field has arrived at a “parts list” of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. I reconstituted functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self- assembling in vitro, tethering centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal that two inner kinetochore protein subcomplexes, Mif2 and OA, are independently capable of transmitting force from MIND to the centromeric nucleosome and suggest that these two pathways of outer kinetochore recruitment may be differentially regulated
The Role of Microtubule-Associated Proteins During Microtubule Nucleation and Organization
No full textThesis (Ph.D.)--University of Washington, 2020Successful mitotic division is fundamental to life. During mitosis, the cytoskeletal network is reorganized to form the bipolar spindle: microtubules extend from microtubule-organizing centers to attach to and separate chromosomes. The mitotic spindle is formed by 1) transport of existing microtubules and 2) targeted nucleation of new microtubules. These processes are governed by the microtubule-organizing center and proteins associated with the microtubule. Here I investigate how the MTOCs and the microtubule associated proteins work together to nucleate microtubules. Using a turbidity assay, I find that XMAP215 is a classically defined microtubule nucleator, as it reduces the nucleation lag seen in bulk tubulin assembly. I further characterize the role of XMAP215 in nucleation, finding its ability to nucleate correlates with its ability to elongate existing microtubules and this is true in both the presence and absence of gamma-tubulin. Then using a novel tool, I define the role of three microtubule associated proteins (Stu2, Bim1 and Bik1) and two motors (Kip3 and Vik1) during assembly of microtubule arrays from an ectopic microtubule nucleation site expressed in vivo. I found that Stu2 and Bim1 are required for microtubule nucleation from the ectopic site, whereas Bik1, Kip3 and Vik1 are required for organization of the microtubule arrays
Using biophysical analysis to study the regulation of the budding yeast kinetochore
No full textThesis (Ph.D.)--University of Washington, 2019Kinetochores are large macromolecular machines that serve to physically segregate the genomic material of a cell during mitosis. To achieve this, the kinetochore must make strong attachments to dynamic microtubules under force for long periods of time, but also be able to correct erroneous attachments. These complicated tasks require exquisite regulation within the kinetochore. The flexible outer kinetochore complex Ndc80 is essential to all of these functions. Previous studies have primarily focused on posttranslational modifications that affect regulation of the complex. I sought to determine how additional kinetochore components might regulate the microtubule binding capacity of the Saccharomyces cerevisiae Ndc80 complex, as well as if the complex’s intrinsic flexibility (conformational changes) affects its function. I and a previous member of the lab initially found that the conserved MIND complex, a central kinetochore component, and the Ndc80 complex are connected by an extensive network of contacts: an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. However, MIND binds Ndc80 complex far from the microtubule-binding domain. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a tightly bent conformation. From these results, I hypothesized a previously unidentified allosteric mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex: the binding of MIND opposes the tightly bent (auto-inhibited) form of the Ndc80 complex, stabilizing the complex in a more open conformation which results in better microtubule binding. To more directly test this hypothesis, I developed a single molecule FRET assay to examine conformational changes of the complex while simultaneously monitoring its microtubule-binding activity. I uncovered a novel mechanism of regulation of the Ndc80 complex that is intrinsic to its structure; tight bending of the complex inhibits its microtubule binding. I showed that the Ndc80 complex can fluctuate between straight and bent forms, and that binding of the complex to microtubules selects for straightened forms. In addition, the kinetochore complex MIND enhances microtubule binding by opposing the auto-inhibited conformation of Ndc80 complex. I suggest that prior to its assembly at the kinetochore, the Ndc80 complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 complex in a straightened form for higher affinity microtubule binding
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