Theses and Dissertations

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Open Access
Discovery and characterization of the SLAC complex and its role in actin polymerization during clathrin-mediated endocytosis
(Colorado State University. Libraries, 2013) Feliciano, Daniel, author; Di Pietro, Santiago, advisor; Bamburg, James, committee member; Curthoys, Norman, committee member; Chen, Chaoping, committee member; Reist, Noreen, committee member
Endocytosis is the process by which cells control the lipid and transmembrane protein compositions in order to comply with certain requirements essential for cellular function. The different forms of endocytosis provide the cell with a discriminatory system where specific cargoes are selected, packed and internalized when there is a particular physiological demand. Given the importance of endocytic internalization routes for a variety of cellular processes, it is not surprising that defects in the protein machinery involved in these pathways leads to pathologies. Examples of some metabolic disorders associated to defects in adaptor or receptor function include autosomal recessive and familial hypercholesterolemia. In other cases, mutations in actin regulatory proteins, such as WASp, can cause many blood disorders that include primary immunodeficiency and thrombocytopenia. Clathrin-mediated endocytosis (CME) is a fundamental pathway conserved from yeast to humans that proceeds by forming a clathrin coat at the plasma membrane followed by the recruitment of proteins that promote membrane curvature, actin polymerization, and scission. CME is the mayor route for nutrient uptake, distribution of membrane components, and receptor internalization. During CME, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (WASp) is the strongest activator of the Arp2/3 complex in yeast cells, it is not autoinhibited, and arrives to endocytic sites 20 seconds before actin polymerization begins. One of the most outstanding questions in the field has been how Las17 is inhibited during the initial 20 seconds after its arrival to sites of endocytosis. In this dissertation, the discovery and characterization of a stable complex between Las17 and the clathrin adaptor Sla1 is described, in which Las17 is inhibited. This interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I (RxxPxxP) and class II (PxxPxR). In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on a new class I/II Las17 polyproline motifs. The inhibition is based on competition between Sla1 and monomeric actin for binding to sequences comprising a novel G-actin binding site in Las17 that is also characterized. The Las17 novel G-actin binding module 1 (LGM1) requires two sets of arginine-rich sites for normal Las17 function in vitro and in vivo. Furthermore, live cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, its inhibition during the initial 20 seconds, and for efficient endocytosis. Within this complex, Las17 requires full length Bzz1, a membrane tubulation protein, for its activation in vitro through a mechanism that does not depend on complex dissociation. Since Sla1 and Las17 regulate actin polymerization during clathrin-mediated endocytosis, this complex has been named SLAC. The discovery and characterization of the SLAC complex help to define the negative and positive mechanisms regulating Las17 activity and answer one of the most outstanding questions in the field. This work also sets the stage to decipher the roles of other WASp homologues in mammalian cells. Overall, findings reported here advance our understanding of the regulation of actin polymerization by Las17 during clathrin-mediated endocytosis.
Open Access
Investigation of the molecular mechanisms and therapeutic potential of oncogene-induced kinetochore-microtubule defects
(Colorado State University. Libraries, 2015) Herman, Jacob A., author; DeLuca, Jenniver G., advisor; Bamburg, James, committee member; Stargell, Laurie, committee member; Nickoloff, Jac, committee member
Kinetochores, large protein structures assembled on centromeric DNA during mitosis, bind to microtubules of the mitotic spindle to orchestrate and power chromosome movements. Deregulation of kinetochore-microtubule (kinetochore-MT) attachments has been implicated in driving chromosome instability and cancer evolution; however, the nature and source of kinetochore-MT attachment defects in cancer cells remain largely unknown. Here, we identify kinetochore-MT attachments, and their regulation by Aurora B kinase (ABK) as key targets for selective therapeutic intervention in glioblastoma and other cancers. We observe that accessory regulators of ABK and kinetochore-microtubule attachment stability are compromised in some cancers and fundamentally alter kinetochore signaling. First we identify RAS/MAPK oncogenic transformation as sufficient to induce these defects through an enzymatic cascade targeting the kinetochore. We then identify BUBR1 kinetochore recruitment and kinetochore-associated PP2A activity as cancer-essential activities, which are required for some cancers to form robust physical interactions between kinetochores and MTs. We also verify previous findings that many cancers are characterized by chromosome segregation errors arising from merotelic kinetochore-microtubule attachments (a single kinetochore bound to microtubules emanating from both spindle poles). We attribute the cause of these errors to be a decrease in MT dynamics independent of the physical attachments status. Finally we characterize a novel kinetochore component, BUGZ, which serves as a molecular chaperone for BUB3 and thus indirectly stimulates ABK activity. We find that BUGZ binds to BUB3 through a conserved GLEBS domain, and this interaction is required for BUB3 kinetochore localization. BUGZ depletion decreases ABK activity resulting in lethal chromosome alignment defects in glioblastoma cells and genetically transformed cells. Together these findings further elucidate the molecular mechanism by which kinetochore-MT attachments are regulated and importantly, how this mechanism is perturbed upon transformation. These results will make the design and application of novel anti-cancer drugs with reduced side effects possible because the specifically target cancer populations.
Open Access
Mediation of kinetochore-microtubule interactions through the Ndc80 complex component Hec1
(Colorado State University. Libraries, 2011) Guimaraes, Geoffry J., author; DeLuca, Jennifer, advisor; Bamburg, James, committee member; Peersen, Olve, committee member; Reddy, A. S. N., committee member
To view the abstract, please see the full text of the document.
Open Access
Multiple domains in the NDC80 complex are required for generating and regulating kinetochore-microtubule attachments in mitosis
(Colorado State University. Libraries, 2012) Sundin, Lynsie, author; DeLuca, Jennifer G., advisor; Bamburg, James, committee member; Curthoys, Norman, committee member; Bailey, Susan, committee member
The goal of mitosis is to accurately segregate chromosomes into two new daughter cells. It is critical that this process occurs appropriately because the consequences of chromosome nondisjunction or missegregation are severe, most notably birth defects and cancer. Kinetochores are built at the centromeric region of mitotic chromosomes and serve several functions during mitosis. First, the kinetochore is the physical scaffold at which microtubule binding sites are built. Second, kinetochores regulate the strength of the attachments to microtubules to ensure proper chromosome movements. Finally, the kinetochore is the origin of a soluble 'wait anaphase' signal that prevents premature entry into anaphase. Together these functions culminate with chromosome alignment at the spindle equator of a cell, ultimately resulting in accurate chromosome segregation in anaphase. While the kinetochore can be considered the director of kinetochore-microtubule attachment, microtubules drive the process of cell division by providing the force behind chromosome movements. The mechanism of kinetochore-microtubule attachment remains elusive as kinetochores must generate and maintain connections to microtubules that are constantly polymerizing and depolymerzing. Extensive studies into this process have revealed that the KMN (KNL1 complex, MIS12 complex, and NDC80 complex) network, a supercomplex of proteins at the outer kinetochore, comprises the core microtubule binding site in cells. As part of this network the NDC80 complex has been an attractive candidate as an essential part of the microtubule binding machinery. Here we have used a combination of in vivo, in vitro, and in silico methods to characterize three discrete domains of the NDC80 complex that each contribute to the process of kinetochore-microtubule attachment in distinct ways. Our data have elucidated some of the molecular details of how kinetochore-microtubule attachments are both generated and regulated. We show that the Hec1 CH domain is absolutely required for kinetochore-microtubule attachment. Our data suggest that the Hec1 CH domain makes direct contacts with microtubules, while the CH domain of Nuf2 does not, indicating functionally distinct roles for these protein domains in mitosis. We characterize the Hec1 loop domain, demonstrating that it is required for stable kinetochore-microtubule attachments and mitotic progression. Our data suggest that the Hec1 loop domain is required to recruit accessory proteins to the kinetochore during mitosis. Furthermore, we show that kinetochore-microtubule attachment strength is highly sensitive to small changes in Hec1 tail phosphorylation. Finally we also demonstrate that incremental phosphorylation of the Hec1 tail domain is a primary mechanism of regulating kinetochore-microtubule attachment strength. Together our data highlight the diverse functions of a single kinetochore component and implicate the NDC80 complex as the principle site for direct binding to microtubules and as a site of regulation for these attachments.
Open Access
Solving the crystallographic structure of the Cl2J construct and occupancy titration trials to quantitatively determine isomer ratio
(Colorado State University. Libraries, 2011) Rummel, Brittany L., author; Ho, P. Shing, advisor; Curthoys, Norman, committee member; Bamburg, James, committee member; Kennan, Alan, committee member
Halogen atoms are commonly found in biological organic compounds such as plastic polymers, flame retardants, coolant fluids, insecticides and herbicides. Halogens are known to mediate neurotransmitters in the brain and are required for the production of many hormones (i.e. thyroxine). Because halogen atoms are frequently incorporated in pharmaceuticals and antibiotics (i.e. clindamycin and chloramphenicol), it is important to characterize the interactions that those atoms participate in. Currently, there is little information known about halogen bonds and these interactions are not modeled accurately by molecular simulations. The long-term objective of Dr. Shing Ho's laboratory has been to characterize halogen bonds through structural and energetic determinations. As part of that larger goal, the studies in this thesis aim to address the structure-energy relationship of chlorinated halogen bonds or X-bonds. The experimental assay that allowed the study of halogen bonds is the 4-stranded DNA Holliday junction. Incorporating engineered halogen bonds into the structure results in halogen bonds competing energetically against hydrogen bonds for stabilization of the junction. The structure that was refined in order to analyze chlorinated halogen bonds is referred to as the Cl2J construct. The Cl2J construct is a crystallized Holliday junction crystal in which 2 chlorine atoms are incorporated into the structure as chlorinated uracil nucleotides, and thus, sets chlorine halogen bonding energies and hydrogen bonding energies in opposition. Occupancy titrations were conducted to quantify isomeric ratios of halogen bond versus hydrogen bond stabilized junctions (X- and H-isomers, respectively) within these crystals. The initial estimate of the isomer ratios of the Cl2J construct was 50/50 X-to-H-isomer from the initial electron density maps. The crystallographic model and subsequent occupancy titration trials actually indicate a higher ratio of approximately 3/1 X-to-H-isomer ratio, respectively. The occupancy titrations and crystallographic models of other constructs, F2J, Br2J and I2J, were analyzed in comparison to the Cl2J construct in order to define a protocol that accurately quantifies these isomeric ratios. Differential scanning calorimetry (DSC) studies are also presented to corroborate in solution any conclusions drawn from occupancy titrations in the crystals.
Open Access
Understanding regulation of HIV-1 protease precursor autoprocessing
(Colorado State University. Libraries, 2019) Tien, Chih-Feng, author; Chen, Chaoping, advisor; Bamburg, James, committee member; Peersen, Olve, committee member; Quackenbush, Sandra, committee member
The HIV-1 protease (PR) is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. Protease autoprocessing is generally referred to proteolytic reactions catalyzed by the precursor itself leading to liberation of free, mature PR in a highly regulated manner. We study the precursor autoprocessing mechanism using engineered fusion precursors carrying the p6*-PR miniprecursor sandwiched between various proteins and/or epitope peptides expressed in transfected mammalian cells. The studies reported here examined and identified factors involved in regulation of precursor autoprocessing. Modulation of precursor autoprocessing activity and outcomes by the 26 amino acid maltose binding protein signal peptide (SigP) mimicking the proviral constructs. A H69D mutation in PR abolished autoprocessing of SigP-containing fusion precursors or Gag processing in viral particles whereas it only partially suppressed autoprocessing of fusion precursors lacking SigP. The mature PRs released from SigP-carrying precursors or associated with the viral particles are both resistant to self-degradation whereas those released from SigP-lacking fusion precursors are prone to self-degradation. Furthermore, the PR-containing autoprocessing intermediate fragments released from a SigP fusion precursor or a proviral constructs showed protease inhibitor response profiles distinct to those released from the corresponding fusion precursor lacking SigP. These findings of context-dependent modulation reveals the complexity of precursor autoprocessing regulation that most likely accompanies sequence variation imposed by the evolution of the upstream Gag moiety. We also examined trans proteolysis for its functional correlation with precursor dimerization. Fusion enzymes carrying GST, a well-known dimer forming protein, processed the GST-fused substrate in trans as expected. Interestingly, positive trans processing was also detected between enzyme and substrate precursors carrying maltose binding protein (MBP), a known monomeric tag, or lacking any dimer-inducing tag, suggesting that a dimer-inducing flanking tag is not required for trans proteolysis in the transfected cells. Sucrose gradient sedimentation analysis detected dimeric substrates, with or without dimer-inducing GST, as the major complexes in transfected cell lysates. In the presence of a protease inhibitor (PI) at high enough concentrations, dimeric enzymes were predominantly detected. Without PI treatment, fusion enzymes with different tags and varied p6* sequences showed monomers or dimers or mixtures, suggesting modulation of enzyme dimerization by p6* peptides and flanking tags. Precursors carrying two PRs in tandem tethered by a GGS linker demonstrated higher propensities of forming inter-molecular dimers than intra-molecular dimers, indicating a role of p6* peptide in regulating precursor dimerization. Collectively, our results decoupled the requirement of a dimer-inducing tag upstream of the p6*-PR miniprecursor for precursor trans proteolysis and demonstrated elements within and beyond p6*-PR miniprecursor that collectively influence precursor dimerization, which revealed additional complexity involved in precursor autoprocessing regulation. In summary, this dissertation highlights complicated regulations and more than one productive pathway involved in HIV-1 protease precursor autoprocessing.

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