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Browsing by Author "Tsunoda, Susan, committee member"

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    A clonable selenium nanoparticle in action: high resolution localization of FtsZ using electron tomography
    (Colorado State University. Libraries, 2021) Borgognoni, Kanda, author; Ackerson, Christopher J., advisor; Neilson, James, committee member; Kennan, Alan J., committee member; Tsunoda, Susan, committee member
    A meaningful understanding of biochemistry requires that we understand the function of proteins, which is heavily dependent on their structure and location within an organism. As the Resolution Revolution of cryo-electron microscopy gains unprecedented ground largely due to the recent development of commercially available direct electron detectors, energy filters, and high-end computation, thousands of protein structures have been solved at atomic or near-atomic resolution, with the highest resolution structure to date being solved at 1.2 Å. A major challenge that has limited the broad use of cryo-electron tomography (cryo-ET) is locating a protein of interest in an organism, as no commercially available high-contrast markers which can be generated in vivo exist. Herein, we present a breakthrough study which aims to solve this problem by synthesizing high contrast metal nanoparticles labeling desired proteins in situ. We isolated a Glutathione Reductase-like Metalloid Reductase (GRLMR), which can reduce selenite and selenate into selenium nanoparticles (SeNPs), from Pseudomonas moraviensis stanleyae found in the roots of a Se hyperaccumulator Stanleya pinnata, or Desert Princes' Plume. A recombinant variant, denoted as a clonable Selenium NanoParticle (cSeNP), was fused to filamentous temperature sensitive protein Z (FtsZ), and the chimera was expressed in vivo using a T7 expression system in model organism E. coli for a proof-of-concept study. Because the SeNPs biogenically produced are amorphous, they exist in a quasistable state and are composed of polymeric Sen in the form of chains and rings that are constantly breaking and reforming. To stabilize the particles during cellular preservation ex aqua, a disproportionation-like reaction can be done either in vivo or as a post-fixation step to form crystalline metal selenide (MSe) NPs that can withstand the processing liquids used. Thereafter, electron tomography was used to acquire a tilt series that was reconstructed into a tomogram and segmented using IMOD, generating a model representing MSeNPs labeling FtsZ filaments. As such, we have demonstrated the potential of using cSeNP as a high resolution marker for cryo-ET. While our study relied on traditional preservation and embedment techniques, we anticipate that for cells preserved via vitrification, cloned SeNPs can be used without subsequent transformation to MSeNPs, as the amorphous particles are stable in aqueous media. Prospectively, we expect that clonable nanoparticle technology will revolutionize cryo-ET, allowing us to localize proteins in vivo at high resolution while maintaining organism viability through metal immobilization. Furthermore, this technique can be expanded to other imaging modalities, such as light microscopy and X-ray tomography, through the discovery and engineering of other clonable nanoparticles.
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    An investigation of the molecular complexities that regulate molting in decapod crustaceans
    (Colorado State University. Libraries, 2015) Pitts, Natalie Lynn, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Tjalkens, Ronald B., committee member; Tsunoda, Susan, committee member
    Molting in decapod crustaceans is regulated by the interaction of two hormones, molt inhibiting hormone (MIH) and ecdysteroids. Ecdysteroids are steroid hormones secreted from the molting gland or Y-organ (YO) and fluctuations in hemolymph ecdysteroid titers regulate progression through the molt cycle. Secretion of ecdysteroids is controlled by the peptide hormone MIH, which is synthesized and released from the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). The field of crustacean endocrinology has mainly focused on understanding the molecular underpinnings of MIH’s action on ecdysteroid production in the YO. The goal of this dissertation was to examine how MIH synthesis and secretion from the XO/SG complex contributes to molt cycle progression. Blackback land crabs, Gecarcinus lateralis, were induced to molt via autotomy of five or more walking legs (multiple limb autotomy or MLA). ESG were collected from intermolt, premolt, and post-molt animals and changes in expression of Gl-MIH and mTOR signaling pathway components were investigated. There was a significant effect of molt stage on Gl-MIH and mTOR signaling pathway gene expression in the ESG of G. lateralis. Continuous elevation of MIH transcript abundance during pre and post molt indicates that MIH titers in the hemolymph are not regulated by changes in transcript abundance. Molting also significantly increased expression of Gl-Akt, Gl-mTOR, Gl-Rheb, and Gl-S6K in one or more molt stages. Akt inhibits the tuberous sclerosis complex allowing for the activation of Rheb. Rheb is a GTPase that binds and activates the mechanistic target of rapamycin (mTOR). mTOR activates S6 kinase (S6K), increasing protein synthesis. ESG of naturally molting green crabs, Carcinus maenas, were also collected from intermolt, early premolt, and post molt animals. Molting had little effect on gene expression in C. maenas, confirming previous findings that molt progression is regulated post transcriptionally. This dissertation identifies a novel nitric oxide (NO) binding protein in the SG of C. maenas. The hypothesis is that NO negatively regulates MIH secretion from the SG thereby controlling molt progression. This unidentified endogenous binding protein allows NO to be present in the SG for a prolonged period and can therefore continually regulate neuropeptide release. Localization of the enzyme that produces NO (nitric oxide synthase; NOS) and MIH in the SG of C. maenas, G. lateralis, and Metacarcinus magister is consistent with the hypothesis that NO is a regulator of neuropeptide release in the crustacean SG. The second goal of this dissertation was to explore why some crustacean populations or individuals within a population are refractory to molt induction. The Bodega Bay population of C. maenas is refractory to molt induction techniques and a similar phenomenon is observed in G. lateralis. Some G. lateralis induced to molt via MLA did not enter premolt 90 days post induction and were classified as "blocked." These animals underwent a second molt induction technique, eyestalk ablation (ESA), and YO, brain (Br), and thoracic ganglia (TG) were collected at 1, 3, and 7 days post ESA. Gene expression of MIH and mTOR signaling pathway genes was examined in all three tissues (see Figure 3 for MIH signaling pathway components and there interactions). Results from this experiment suggested that a similar mechanism of molt resistance exists between C. maenas and G. lateralis. ESA did not increase hemolymph ecdysteroid titers of blocked animals, whereas ESA significantly increased ecdysteroid titers in control and intermolt animals. Gl-MIH expression in the ESG and expression of many Gl-MIH signaling components in the YO were upregulated in blocked animals, suggesting that the blocked animals were in a "hyper-repressed" state, and therefore resistant to molt induction by ESA and MLA. In both species, MIH is expressed in the Br and TG. The hypothesis is that MIH secretion from these other central nervous system (CNS) tissues contributes to a resistance to molt induction techniques. Expression of MIH signaling pathway genes is unchanged in the Br and TG in response to ESA. These data suggest that MIH does not activate a signaling pathways in CNS tissues but like the ESG, MIH is synthesized and secreted from these tissues. This experiment also supports the growing body of literature that mTOR inhibition activates downstream transcription factors which are important in maintaining energy homeostasis in times of environmental stress.
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    Angiotensin-II signaling in the pars reticulata GABA-ergic neurons in the substantia nigra and its implications in nigral neurotransmission
    (Colorado State University. Libraries, 2021) Singh, Maibam Ratan, author; Amberg, Gregory C., advisor; Vigh, Jozsef, committee member; Tsunoda, Susan, committee member; Tamkun, Michael, committee member; Garrity, Deborah, committee member
    Renin-Angiotensin-system is one of the most widely studied hormonal systems in the peripheral system and is primarily associated with the essential function of regulating blood pressure, fluid and electrolyte balance in the body. Most of the drugs used to treat hypertension currently are targeted towards one or more components of the RAS system. However, increasing studies have presented evidence of local RAS in tissues completely independent of the humoral system. In the CNS, in addition to highly vascularized areas in the brain lacking the blood-brain-barrier (BBB) such as the circumventricular organs, all RAS components have also been found in the brain regions inside the BBB and are suspected to be involved in neuronal differentiation, neurotransmission, and learning and memory. Increasing studies have reported the interaction of brain RAS with pathophysiological mechanisms of many neurological and psychiatric illnesses. However, this extrarenal effect of RAS is only beginning to gain some scientific attention, and the underlying mechanisms are far from elucidated. All the RAS components are strongly expressed in the midbrain, especially the substantia nigra. Accumulating evidence in recent years has implicated Angiotensin-II (Ang-II), the primary effector peptide of RAS, in the selective degeneration of dopaminergic neurons in the substantia nigra compacta (SNc) in animal models of Parkinson's disease. Ang-II is believed to induce G-protein signaling through Ang-II type 1 receptor (AT1-R) and increase cellular oxidative stress, intracellular calcium load and activate apoptotic pathways in SNc dopaminergic neurons. Interestingly, studies have also shown Ang-II mediated striatal dopamine release in rats. These studies suggest that Ang-II signaling can induce both intracellular effects and influence dopaminergic neuronal output in the midbrain. However, if Ang-II signaling exists in other neuronal cell types in the substantia nigra is not known. Substantia nigra is comprised of two primary cell types: dopaminergic and GABAergic neurons. The majority of dopaminergic neurons are located in the SNc, and the SNr is comprised of GABAergic projection neurons with few interspersed dopaminergic neurons. Besides being one of the major output neurons of basal ganglia, SNr GABAergic projection neurons also provide significant inhibitory input to the neighboring SNc dopaminergic neurons, not through a direct axonal projection like its other target areas but via its extensive network of axon collaterals. Inhibitory input from the SNr GABAergic neurons contributes to the essential balance between afferent excitatory and inhibitory inputs to SNc dopaminergic neurons that tightly regulates their cellular activity and output. Indeed, SNr GABAergic neurons are necessary for the voluntary control of movement and are implicated in basal ganglia dysfunctions associated with movement disorders such as Parkinson's disease. RAS components are also expressed in the SNr GABAergic neurons, but it is not known if Ang-II signaling exists in these cells and what effects it may have on intranigral neurotransmission and dopaminergic cell activity. Here we used a combination of electrophysiology, imaging, and optogenetics to characterize and investigate the role of Ang-II in local neurotransmission in the substantia nigra. We found a heterogeneous effect of Ang-II in the nigral dopaminergic and GABAergic neurons. Ang-II suppressed both electrically and light-evoked activity of SNr GABAergic neurons through a combination of mechanisms: enhancement of postsynaptic GABAa receptors and increasing the action potential duration. On the contrary, Ang-II had no noticeable direct effect on the activity of SNc dopaminergic neurons and its GABAa receptors. This provides the first evidence of novel Ang-II signaling in SNr GABAergic neurons and its heterogeneous effect in the two nigral cell types. Interestingly, in contrast to observed suppression of SNr GABAergic neuronal activity by Ang-II, under phasic photoactivation of SNr GABAergic neurons, Ang-II enhanced the feedforward inhibitory input to SNc dopaminergic neurons. This shows a non-linear effect of Ang-II on population output of nigral GABAergic neurons and may indicate the involvement of an intricate intranigral network formed by the axon collaterals of SNr GABAergic neurons that can further modulate its effect on postsynaptic targets.
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    Autism-associated δ-catenin G34S mutation promotes GSK3β-mediated premature δ-catenin degradation inducing neuronal dysfunction
    (Colorado State University. Libraries, 2019) Nip, Kaila, author; Kim, Seonil, advisor; Bamburg, James, committee member; Tsunoda, Susan, committee member
    δ-catenin is a crucial component of a synaptic scaffolding complex, which regulates synaptic structure and function in neurons. Loss of δ-catenin function is strongly associated with severe autism spectrum disorder (ASD) in female-enriched multiple families. In particular, a G34S (Glycine 34 to Serine) mutation in the δ-catenin gene has been identified in ASD patients and suggested to exhibit loss-of-function. The G34S mutation is located in the amino terminal region of δ-catenin, where there are no known protein interaction domains and post-translational modifications. Notably, the Group-based Prediction System predicts that the G34S mutation is an additional target for GSK3β-mediated phosphorylation, which may result in protein degradation. Therefore, we hypothesize the G34S mutation accelerates δ-catenin degradation, resulting in loss of δ-catenin function in ASD. Indeed, we found significantly lower G34S δ-catenin levels compared to wild-type (WT) δ-catenin when expressed in cells lacking endogenous δ-catenin, which is rescued by genetic inhibition of GSK3β. By using Ca2+ imaging in cultured mouse hippocampal neurons, we further revealed overexpression of WT δ-catenin is able to significantly increase neuronal Ca2+ activity. Conversely, Ca2+ activity remains unaffected in G34S δ-catenin overexpression, which is reversed by pharmacological inhibition of GSK3β using lithium. This suggests the G34S mutation of δ-catenin provides an additional GSK3β-mediated phosphorylation site, which could promote δ-catenin premature degradation, resulting in loss-of-function effects on neuronal Ca2+ activity in ASD. In addition, inhibition of GSK3β activity is able to reverse G34S-induced loss of δ-catenin function. Thus, inhibition of GSK3β may be a potential therapeutic treatment for δ-catenin-associated ASD patients.
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    Changing dogma regarding the conformation of electron transferring menaquinone (MK)
    (Colorado State University. Libraries, 2017) Magallanes, Estela Serrano, author; Crans, Debbie C., advisor; Menoni, Carmen S., committee member; Tsunoda, Susan, committee member
    Menaquinone-9 (MK-9) is the natural substrate containing a naphthoquinone and an isoprenyl side-chain with nine isoprene units that carry out the electron transfer for the Mycobacterium tuberculosis. We present studies aiming to understand the chemical and biochemical properties of hydrophobic MK molecules. Specifically, we are investigating the MK derivative with two isoprene units, MK-2, because it provides us with the base structure containing the naphthoquinone unit and the isoprene side-chain. Its synthesis is relatively simple because the precursors are commercially available, which allows for large scale preparation and detailed characterization of the molecular structure under different conditions. Using 1D and 2D 1H NMR studies we are establishing that MKs have different conformations depending on the specific environmental conditions. Similarly, we show using 1H-1H 2D NOESY NMR studies that the association of MK with the surfactant- water interface of reverse micelles, which is a model membrane system, modify the conformation of the menaquinone derivative. Finally, the redox potentials of MK-2 was measured in the three different solvents (DMSO, CH3CN and pyridine). We hypothesize that the redox potential is correlated to the conformational of the MK. We observed that the redox potentials varied with solvent. The observed folded structures of MK derivatives stand in contrast to the linear conformation shown in life science text books.
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    Characterizing the target of ivermectin, the glutamate-gated chloride channel, and other insecticide targets as candidate antigens for an anti-mosquito vaccine
    (Colorado State University. Libraries, 2015) Meyers, Jacob, author; Partin, Kathryn, advisor; Foy, Brian, advisor; Vigh, Jozsef, committee member; Tsunoda, Susan, committee member
    The latest WHO World Malaria Report estimates that, in 2013, there were 198 million cases worldwide causing 584,000 malaria-related deaths. Current malaria control programs primarily target malaria vectors through the use of long lasting insecticide treated bed nets and indoor residual spraying of pyrethroid-based insecticides. However, pyrethroid resistance is becoming widespread in many An. gambiae populations across Africa (Ranson et al., 2011; Trape et al., 2011). Out of recent efforts to find new vector-targeting interventions with novel modes of action, the endectocide ivermectin (IVM) has arisen as a new candidate to control malaria transmission. IVM, when imbibed by vectors from host-treated blood meals, has proven to efficiently kill or disable An. gambiae s.s. both in the lab and the field (Kobylinski et al., 2010; Sylla et al., 2010). More recently, IVM mass drug administrations in multiple locations across west Africa have been shown to temporarily reduce the proportion of P. falciparum-infected An. gambiae in IVM-treated villages (Kobylinski et al., 2011; Alout et al., 2014). The primary target of IVM is the invertebrate glutamate-gated chloride channel (GluCl) (Cully et al., 1994; Cully et al., 1996; Janssen et al., 2007; McCavera et al., 2009; Janssen et al., 2010; Moreno et al., 2010). The purpose of the first chapter of this thesis was to characterize GluCl from An. gambiae in order to understand the physiological role of GluCl and how IVM may be affecting mosquito physiology. Cloning of the An. gambiae GluCl (AgGluCl) revealed unique splicing sites and products not previously predicted. We expressed AgGluCl clones in Xenopus laevis oocytes to measure its electrophysiological activity in response to glutamate and IVM. We also examined AgGluCl isoform-specific transcript levels across different tissues, ages, blood feeding status and gender and GluCl tissue expression in adult An. gambiae. Given that GluCl can be targeted by drugs found in a blood meal and that GluCl is not expressed in mammals, we wanted to test the efficacy of AgGluCl as a candidate mosquitocidal vaccine antigen. We administered a polyclonal anti-AgGluCl immunoglobulin G (anti-AgGluCl IgG) to An. gambiae mosquitoes through a blood meal or directly into the hemocoel by intrathoracic injections and found it significantly reduced An. gambiae survivorship. By co-administering anti-AgGluCl IgG with a known GluCl agonist, IVM, we discovered anti-AgGluCl IgG reverses the mosquitocidal effects of IVM. Our results describing the mosquitocidal properties of anti-AgGluCl IgG suggest that other neuronal proteins could be used as candidate antigens for a mosquitocidal vaccine. The An. gambiae GABA-gated chloride channel (resistance to dieldrin; AgRDL) is another member of the cys-loop ligand-gated ion channels with a similar structure and physiological function to AgGluCl. The An. gambiae voltage-gated sodium channel (AgVGSC) is the target of dichlorodiphenyltrichloroethane (DDT) and the pyrethroid class of insecticides (Soderlund and Bloomquist, 1989). VGSCs are also the target of multiple classes of spider, scorpion and snail toxins, demonstrating that peptides binding to VGSC extracellular residues can affect channel function (Nicholson, 2007; King et al., 2008; Stevens et al., 2011; Klint et al., 2012). Preliminary results shows that IgG targeting AgRDL or AgVGSC similarly reduce An. gambiae survivorship. Finally we tested anti-AgGluCl IgG against A. aegypti and C. tarsalis to see if this strategy has broad potential across both Anopheline and Culicine mosquitoes. However, blood meals containing anti-AgGluCl IgG had no effect on A. aegypti or C. tarsalis survivorship. We determined that this was due to a barrier in antibody translocation from the blood meal to the hemolymph. Since the IgG target, AgGluCl, is only expressed in the hemocoel, antibody translocation was required for mosquito toxicity.
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    Clustering of non-conducting Kv2.1 channels induces endoplasmic reticulum/plasma membrane junctions and forms cell-surface trafficking hubs
    (Colorado State University. Libraries, 2014) Fox, Philip Douglas, author; Tamkun, Michael M., advisor; Amberg, Gregory C., committee member; Vigh, Jozsef, committee member; Tsunoda, Susan, committee member; Garrity, Deborah M., committee member
    The voltage-gated K+ channel, Kv2.1, is expressed widely in the mammalian CNS, where it carries the majority of the delayed-rectifier current. The Kv2.1 current facilitates high-frequency action potential firing by promoting the repolarization of the membrane potential and subsequent recovery of voltage-gated Na+ channels from inactivation. Furthermore, Kv2.1 displays a unique cell-surface localization to dense, micron-sized clusters which are sensitive to neuronal insults such as glutamate excitotoxicity. The following dissertation presents original research extending our knowledge of the Kv2.1 K+ channel. The majority of Kv2.1 channels are held in a non-conducting state which is incapable of fluxing K+ in response to membrane potential depolarization. These non-conducting channels tend to localize to the micron-sized clusters which distinguish Kv2.1. Non-conducting, clustered Kv2.1 channels remodel the cortical endoplasmic reticulum (cER) into tight connections with the plasma membrane (PM), likely through a direct interaction. Trafficking of membrane proteins, both exo- and endocytosis are localized to the perimeter of the Kv2.1-induced ER/PM contacts by virtue of remodeling the cER underneath the Kv2.1 clusters. Thus the clustering of Kv2.1 functions to bring protein trafficking and intermembrane signaling together at the neuronal soma.
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    Electrophysiological analysis of Kv2 channel regulation by non-canonical and canonical mechanisms
    (Colorado State University. Libraries, 2020) Maverick, Emily E., author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member; Vigh, Jozsef, committee member
    Kv2 channels are the most abundant voltage-gated potassium channels in the mammalian nervous system and entire body. These channels regulate action potential firing and apoptosis via their canonical conducting functions. However, Kv2 channels also play a non-conducting role in the cells in which they are expressed. Specifically, they form junctions between the endoplasmic reticulum and plasma membranes, and these junctions regulate a myriad of cellular process. Several studies have now shown that many Kv2.1 channels expressed on the plasma membranes of mammalian cells do not respond canonically to changes in membrane voltage. Instead of opening to allow potassium efflux, the pores of these non-canonical channels are locked in a non-conducting state. This state has likely evolved to prevent electrical paralysis that would otherwise be conferred upon cells expressing high levels of completely functional Kv2 channels. The mechanism bringing about the non-conducting state of Kv2.1 channels is unknown. The work described in the first part of this dissertation was carried out with the ultimate goal of revealing the mechanism of the Kv2.1 channel non-conducting state. I describe an improved, all-electrophysiological method to quantify the numbers of nonconducting Kv channels expressed in heterologous systems. I validate this approach by measuring the fraction of non-conducting Kv2.1 channels that arise when expressed in HEK293 cells. I go on to use this approach to show evidence for a non-conducting state in the second Kv2 isoform, Kv2.2, for the first time. I find that like Kv2.1, the Kv2.2 nonconducting state is dependent on the density of channels in the membrane. Surprisingly, I also find that two Shaker-related channels, Kv1.4 and Kv1.5 also show density dependence in the fraction of channels that conduct. These results suggest that the mechanism underlying the non-conducting state is more common than we thought, and I discuss hypotheses that should be tested in the future. In the last part of this dissertation I describe the effects of the assembly of Kv2 channels with a newly discovered family of Kv β subunits, the AMIGOs. The experiments in this portion of the dissertation focus on each AMIGO's ability to modulate canonical, conducting Kv2 channels, as well as Kv2's ability to alter AMIGO trafficking and localization. I find that both Kv2.1 and Kv2.2 promote AMIGO trafficking to the plasma membrane and alter their localization there. I also find that while all three AMIGO isoforms promote Kv2 channel opening, AMIGO2 confers an additional stabilizing effect on the open state by slowing inactivation and deactivation. In all, the work in this dissertation expands on our current understanding of Kv channel function. These findings should guide future experiments to probe both canonical and non-canonical functions of Kv channels.
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    High resolution optical analysis of Nav1.6 localization and trafficking
    (Colorado State University. Libraries, 2015) Akin, Elizabeth Joy, author; Tamkun, Michael, advisor; Amberg, Gregory, advisor; Di Pietro, Santiago, committee member; Krapf, Diego, committee member; Tsunoda, Susan, committee member
    Voltage-gated sodium (Naᵥ) channels are responsible for the depolarizing phase of the action potential in most nerve cell membranes. As such, these proteins are essential for nearly all functions of the nervous system including thought, movement, sensation, and many other basic physiological processes. Neurons precisely control the number, type, and location of these important ion channels. The density of Naᵥ channels within the axon initial segment (AIS) of neurons can be more than 35-fold greater than that in the somatodendritic region and this localization is vital to action potential initiation. Dysfunction or mislocalization of Naᵥ channels is linked to many diseases including epilepsy, cardiac arrhythmias, and pain disorders. Despite the importance of Naᵥ channels, knowledge of their trafficking and cell-surface dynamics is severely limited. Research in this area has been hampered by the lack of modified Naᵥ constructs suitable for investigations into neuronal Naᵥ cell biology. This dissertation demonstrates the successful creation of modified Naᵥ1.6 cDNAs that retain wild-type function and trafficking following expression in cultured rat hippocampal neurons. The Naᵥ1.6 isoform is emphasized because it 1) is the most abundant Naᵥ channel in the mammalian brain, 2) is involved in setting the action potential threshold, 3) controls repetitive firing in Purkinje neurons and retinal ganglion cells, 4) and can contain mutations causing epilepsy, ataxia, or mental retardation. Using single-molecule microscopy techniques, the trafficking and cell-surface dynamics of Naᵥ1.6 were investigated. In contrast to the current dogma that Naᵥ channels are localized to the AIS of neurons through diffusion trapping and selective endocytosis, the experiments presented here demonstrate that Naᵥ1.6 is directly delivered to the AIS via a vesicular delivery mechanism. The modified Naᵥ1.6 constructs were also used to investigate the distribution and cell-surface dynamics of Naᵥ1.6. Somatic Naᵥ1.6 channels were observed to localize to small membrane regions, or nanoclusters, and this localization is ankyrinG independent. These sites, which could represent sites of localized channel regulation, represent a new Naᵥ localization mechanism. Channels within the nanoclusters appear to be stably bound on the order of minutes to hours, while non-clustered Naᵥ1.6 channels are mobile. Novel single-particle tracking photoactivation localization microscopy (spt-PALM) analysis of Naᵥ1.6-Dendra2 demonstrated that the nanoclusters can be modeled as energy wells and the depth of these interactions increase with neuronal age. The research presented in this dissertation represents the first single-molecule approaches to any Naᵥ channel isoform. The approaches developed during the course of this dissertation research will further our understanding of Naᵥ1.6 cell biology under both normal and pathological conditions.
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    Molecular mechanisms regulating Kv2.1-induction of endoplasmic reticulum / plasma membrane contact sites
    (Colorado State University. Libraries, 2019) Johnson, Ben, author; Tamkun, Michael, advisor; Amberg, Gregory, committee member; Di Pietro, Santiago, committee member; Prenni, Jessica, committee member; Tsunoda, Susan, committee member
    Kv2 voltage gated potassium channels localize to 'clusters' on the soma, axon initial segment, and dendritic arbor of hippocampal neurons. For decades the molecular mechanism behind this localization pattern was unknown. In 2015 our lab determined that this behavior was due to the channels interacting with an unknown endoplasmic reticulum resident protein and thereby forming endoplasmic reticulum / plasma membrane (ER/PM) junctions. The channel clusters covering the surface of cells represented those domains. The work in this dissertation examines in increased detail the mechanism, regulation, and possible functions associated with these sites. ER/PM junctions are domains with a variety of roles. They regulate both calcium and lipid homeostasis, they are involved in vesicular trafficking, and they oversee a host of cell signaling pathways. Junctions represent 12% of the neuronal soma surface and are also present in both the axon and the dendritic arbor. These are sites that exhibit a high degree of dynamic flux, both in composition and in structure. Residency of junction proteins is governed by the calcium concentration of the ER, the calcium concentration of the cytosol, the activity of the excitable cell, and the lipid composition of the PM. In turn these residents influence the nature of the junction, determining the function and nanoarchitecture of these domains. In this work we use a proximity-based biotinylation approach to identify VAMP-associated proteins (VAPs) as the Kv2 channel interactor responsible for the formation of ER/PM junctions. We characterize the amino acid motif necessary to generate interaction between the two proteins, finding an unconventional FFAT motif located in the channel C-terminus. We examine the protein composition of these novel junctions by investigating their relationship with other known ER/PM tethers such as Nir2, STIM1 and the junctophilins. We use super resolution imaging techniques to observe ER membrane behavior at these locations and study how that behavior changes during the concentration of additional protein residents. Lastly, we investigate the mechanisms underlying Kv2-VAP junction disassembly during neuronal activity and insult. We find that Kv2.1-VAP unbinding during glutamate stimulation is mediated by serine residues downstream of the Kv2.1 FFAT motif. This dispersal of Kv2-VAP ER/PM junctions during calcium influx is mirrored by junctophilin-induced junction disassembly, suggesting a common mechanism regulating ER/PM junctions throughout the hippocampus. This dissertation examines a novel microdomain formed by Kv2 channels and presents data describing how this domain is created and regulated on a molecular level. It represents the first in-depth study of this topic.
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    Novel insights into protein synthesis rates in the brain following two lifespan-extending treatments
    (Colorado State University. Libraries, 2018) Reid, Justin, author; Hamilton, Karyn, advisor; Miller, Benjamin, advisor; Tsunoda, Susan, committee member
    The number of individuals 65 years or older is rapidly increasing. Aging is the predominant risk factor for chronic disease and disability. Dramatic increases in the number of individuals living with chronic disease or disability will present unique societal challenges. Accordingly, much research has focused on treatments that slow the aging process to prevent many chronic diseases simultaneously. Treatments using two pharmaceuticals, rapamycin and rapamycin plus metformin, have been shown to extend lifespan and improve health in model organisms. Neurodegenerative diseases represent an important subset of debilitating chronic diseases, for which treatment is currently limited. The use of slowed aging treatments in research of neurological function may provide insight into causes of neurodegenerative disease. Protein homeostasis (proteostasis) is crucial for cell and organismal health. Loss of proteostasis is characteristic of aging and chronic disease, and slowed aging treatments improve proteostasis-related outcomes. Protein synthesis is a necessary component of proteostasis. The effect of rapamycin and rapamycin plus metformin on protein synthesis rates in vivo is unexplored. Investigation into the effect of both slowed aging treatments on protein synthesis rates in the brain could inform effects on neuronal health, which may have implications for neurodegeneration. The purpose of the current study is to establish the use of deuterium oxide as a stable isotopic tracer for brain protein synthesis rates in vivo, and to determine the effect of two slowed aging treatments on brain protein synthesis rates. Supportive measurements related to proteostasis were also made. Deuterium oxide labeling allowed for measurement of subcellular brain protein synthesis rates with ample sensitivity to detect sex differences and responses to treatment. The results demonstrated a strong influence of sex in response to both rapamycin and rapamycin plus metformin. Both slowed aging treatments had differing effects on protein synthesis as well as other markers of proteostasis. This study is the first to demonstrate the use of deuterium oxide for protein synthesis rates in the brain, which represents a novel methodology for evaluating proteostasis in neuronal tissue. Further, this is the first study to explore and reveal the effects of rapamycin and rapamycin with metformin on protein synthesis rates in the brain. Future studies using these methods and slowed aging interventions in models of neurodegenerative disease may prove insightful in determining causes, pathologies, and treatments of age-related neurological disorders.
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    Oxidant-dependent regulation of L-type calcium channel activity by angiotensin in vascular smooth muscle
    (Colorado State University. Libraries, 2015) Chaplin, Nathan L., author; Amberg, Gregory, advisor; DeLuca, Jennifer, committee member; Tamkun, Michael, committee member; Tsunoda, Susan, committee member
    Resistance arteries are a major point of physiological regulation of blood flow. Increases in vessel wall stress or sympathetic activity stimulate vascular wall angiotensin signaling, resulting in smooth muscle contraction which directly increases peripheral resistance. Calcium influx through voltage-gated L-type calcium channels underlies vascular smooth muscle contraction. Roughly half of calcium influx in these cells occurs through a small number of persistently active channels, whose activity increases with membrane depolarization. The number of channels gating in this manner is increased by activation of angiotensin receptors on the cell membrane, and basal L-type channel activity is increased during hypertension. Reactive oxygen species are also generated by vascular smooth muscle in response to vessel stretch and by several paracrine signaling pathways including angiotensin signaling. Oxidative stress and augmented calcium handling resulting from chronic angiotensin signaling in the vasculature each contribute to enhanced vessel reactivity, pathological inflammation and vessel remodeling associated with hypertension. This study uses a multidisciplinary approach to investigate the role of hydrogen peroxide in angiotensin signaling in vascular smooth muscle. Using calcium- and redox-sensitive fluorescent indicators, local generation of hydrogen peroxide by NAD(P)H oxidase and mitochondria are shown to synergistically promote PKC-dependent persistent gating of plasma membrane L- type calcium channels in response to angiotensin II. We show that broad inhibition of hydrogen peroxide signaling by catalase and targeted inhibition of mitochondrial reactive oxygen species production attenuates cerebral resistance artery constriction to angiotensin. We further demonstrate the role of endothelium-independent mitochondrial reactive oxygen species in development of enhanced vessel tone and smooth muscle calcium in a murine model of hypertension. Together, these findings contribute to the understanding of intracellular calcium and oxidative signaling in vascular physiology and disease and may provide insight into local signaling dynamics involving these second messengers in various other systems.
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    Reevaluating the functional role of the C₂A domain of synaptotagmin in neurotransmitter release
    (Colorado State University. Libraries, 2020) Bowers, Matthew Robert, author; Reist, Noreen, advisor; Tsunoda, Susan, committee member; Di Pietro, Santiago, committee member; Tamkun, Michael, committee member
    Efficient cell-to-cell communication is critical for nervous system function. Fast, synchronous neurotransmission underlies this communication. Following depolarization of the nerve terminal, Ca2+ enters the presynaptic cell and drives fusion of vesicles with the membrane, releasing neurotransmitter. The synaptic vesicle protein, synaptotagmin, was identified as the Ca2+ sensor for this fast, synchronous neurotransmitter release. It is hypothesized that Ca2+ binding by synaptotagmin acts as an electrostatic switch. At rest, vesicles are in a state of variable priming with repulsion between negatively charged residues in synaptotagmin and the negatively charged presynaptic membrane acting as a brake to prevent fusion of the vesicles. Following binding of positively charged Ca2+ ions, this electrostatic repulsion is switched to attraction, allowing hydrophobic residues in synaptotagmin to insert into the presynaptic membrane. This insertion is thought to lower the energy barrier for fusion, resulting in the synchronous fusion of many vesicles and the chemical propagation of a signal to the postsynaptic cell. Synaptotagmin is composed primarily of two C2 domains that have negatively charged Ca2+ binding pockets, C2A and C2B. The C2B domain is thought to be the primary functional domain, with C2A playing a supporting role. While using point mutations to the C2A domain to investigate the functional roles of specific residues of the protein, I discovered that the C2A domain, may, in fact, be much more important than anticipated. In chapter 2, I created mutations disrupting the membrane penetrating hydrophobic residues of the C2A domain. Mutation of these residues was hypothesized to only partially disrupt evoked release. Surprisingly, mutation of both residues in tandem resulted in the most dramatic phenotype of a C2A domain mutation to date. This dramatic decrease in synaptic transmission is the first instance of a C2A domain mutation resulting in a phenotype worse than the synaptotagmin null mutant. In chapter 3, I generated mutations to various combinations of Ca2+-binding aspartate residues in the Ca2+ binding pocket of C2A. These mutations are hypothesized to prevent Ca2+ binding, while simultaneously neutralizing the charge of the pocket, essentially mimicking constitutive Ca2+ binding. Again surprisingly, evoked release was dramatically decreased in some of the mutants, suggesting C2A Ca2+ binding mutants disrupt Ca2+ dependent synchronous release, a finding that increases our understanding of Ca2+ binding by the domain and contradicts some interpretations of previous reports. My findings provide key mechanistic insights into the function of this critical protein. For one, investigation of the role of the C2A hydrophobic residues revealed that the downstream effector interactions mediated by these hydrophobic residues are critical to drive synchronous vesicle fusion. Also, investigation of the role of the critical Ca2+-binding residues in the C2A domain revealed that these residues each play a distinct role in driving vesicle fusion, while further suggesting Ca2+ binding by C2A is more important than originally posited. Most interestingly though, I believe the sum of my findings disproves the long-held belief that C2A is purely a facilitatory domain, prompting many questions about how these two C2 domains may work together to promote neurotransmitter release in tandem.
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    Super-resolution imaging reveals mechanisms of glutamate transporter localization near neuron-astrocyte contacts
    (Colorado State University. Libraries, 2021) Leek, Ashley N., author; Tamkun, Michael M., advisor; Hentges, Shane T., committee member; Tjalkens, Ronald B., committee member; Tsunoda, Susan, committee member
    Astrocytes contact neurons at several locations, including somatic clusters of Kv2.1 potassium channels and synapses across the brain. A primary function of astrocytes at these locations is to limit the action of extracellular glutamate. Astrocytic glutamate transporters, such as Glt1, ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. Additionally, they act to limit glutamate induced hyperexcitability by preventing the spread of glutamate to extrasynaptic receptors. The role of Glt1 in limiting neuronal hyperactivity relies heavily on the localization and diffusion of the transporter in the membrane, however, little is known about the mechanisms governing these properties. The work presented in this dissertation examines the mechanisms of Glt1 localization near Kv2.1-mediated neuron-astrocyte contact sites. To that end, in Chapter 2, we used super-resolution imaging to analyze the localization of two splice forms of Glt1, Glt1a and Glt1b. In cultures of primary astrocytes, we find that Glt1a, but not Glt1b, is specifically localized over cortical actin filaments. We go on to discover that this localization is dependent on the Glt1a C-terminus, where Glt1a and Glt1b differ, as exogenous expression of the Glt1a C-terminus was able to prevent localization of Glt1a to cortical actin filaments. In the somatosensory cortex, astrocyte Glt1 forms net-like structures around neuronal Kv2.1 clusters, however the cause of this Glt1 localization pattern is unknown. In Chapter 3, using super-resolution imaging of mixed cultures of astrocytes and neurons, we replicate findings of astrocyte Glt1 in a net-like localization around neuronal Kv2.1 clusters. We discover that both astrocyte actin and ER were excluded from the region across from neuronal Kv2.1 clusters. The actin-Glt1a relationship discussed in Chapter 2 is likely responsible for the net-like appearance of Glt1, as astrocytic Glt1 and actin colocalize in nets around Kv2.1 clusters at points of neuron-astrocyte contact. Neuronal control over the astrocyte cytoskeleton appears central to this Glt1a localization, although the mechanism of this control is still unknown. Together, these data describe a novel interaction between the Glt1a C-terminus and cortical actin filaments, which localizes Glt1 near neuronal structures involved in detecting ischemic insult. Although the mechanism of neuronal control over the astrocyte cytoskeleton remains a mystery, presumably cell-cell contact has a major influence. Contacts between neurons and astrocytes at Kv2.1 clusters could be mediated by the Kv2.1 β-subunit, AMIGO, which acts a cell adhesion molecule. Only one member of the AMIGO family of proteins is known to be an auxiliary β-subunit for Kv2 channels and to modulate Kv2.1 electrical activity. However, the AMIGO family has two additional members of ∼50% similarity that have not yet been characterized as Kv2 β-subunits. In Chapter 4, we show that the surface trafficking and localization of all three AMIGOs are controlled by their interaction with both Kv2.1 and Kv2.2 channels. Additionally, assembly of each AMIGO with either Kv2 alters important electrophysiological properties of these channels. The coregulatory effects of Kv2s and AMIGOs likely fine-tune both electrical and cell adhesion properties of the neurons in which they are expressed. Altogether, the work presented in this dissertation further defines the composition of Kv2.1-induced neuron-astrocyte contact sites, representing the first significant addition to this field in more than a decade.
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    Synaptotagmin in asynchronous neurotransmitter release and synaptic disease
    (Colorado State University. Libraries, 2018) Shields, Mallory Catherine, author; Reist, Noreen, advisor; Garrity, Deborah, committee member; Tamkun, Michael, committee member; Tsunoda, Susan, committee member
    The majority of cell-to-cell communication relies on the stimulated release of neurotransmitter. Two forms of Ca2+-dependent stimulated release, synchronous and asynchronous, have been identified. Synchronous release is the initial release that occurs within milliseconds of stimulation. Critical for efficient synaptic communication, synchronous release is the dominant form of release at most synapses. Alternatively, asynchronous release occurs over longer time periods, with implications in synaptic plasticity and development. However, its mechanisms are poorly understood. Both synchronous and asynchronous release rely on Ca2+ sensors to confer their distinct characteristics. Synaptotagmin 1 is widely accepted as the Ca2+ sensor for fast, synchronous release, but its role in asynchronous release is unclear. Previous studies have led to the hypothesis that synaptotagmin 1, particularly Ca2+ binding by its C2A domain, is needed to inhibit aberrant asynchronous fusion events. However, recent studies have raised questions regarding the interpretation of the results that led to this conclusion. In chapter 2, I have directly tested the effect of Ca2+ binding by synaptotagmin 1's C2A domain on asynchronous release utilizing an alternant Ca2+-binding mutant. This novel mutation was designed to block Ca2+ binding without introducing the artifacts of the original Ca2+-binding mutation. By investigating asynchronous events in vivo at the Drosophila neuromuscular junction, I found no significant effect on asynchronous release when C2A Ca2+ binding was blocked. Thus, I conclude that Ca2+ binding by synaptotagmin's C2A domain is not needed for regulation of asynchronous release, in contrast to the previous study that inadvertently introduced an artifact described below. To prevent Ca2+ binding, the original aspartate to asparagine mutations (sytD-N) removed some of the negatively-charged residues that coordinate Ca2+. This simultaneously introduced aberrant fusion events, because it also interrupted the electrostatic repulsion between synaptotagmin's negatively-charged C2A Ca2+-binding pocket and the negatively-charged presynaptic membrane which is required to clamp constitutive SNARE-mediated fusion. Previous Reist lab results demonstrate that the sytD-N mutations in the C2A domain are likely behaving as ostensibly constitutively bound Ca2+. Indeed, I report that the sytD-N mutation displays slower release kinetics. To directly test if this mutation is the cause of the increase in asynchronous events, I generated additional mutations that prevent interactions with the presynaptic membrane coupled to the originally published sytD-N mutations. In chapter 3 of this dissertation, I investigated these novel mutations at the Drosophila neuromuscular junction. I reported no increase in asynchronous release relative to control, providing evidence that the increased asynchronous events in sytD-N mutants are a result of the original mutation acting as an asynchronous sensor. Together, my results contradict the current hypothesis in the field and provide the likely mechanism for the increased asynchronous release observed in the original study. This dissertation also investigated the relatively new role for synaptotagmin mutations in the etiology of neuromuscular disease. With increased availability of high-throughput sequencing, over 20 candidate genes have been implicated in different forms of congential myasthenic syndromes. These inherited disorders are caused by mutations in genes needed for effective neuromuscular signaling. Two families, presenting with similar myasthenic syndromes, carry point mutations in the C2B Ca2+ binding pocket of synaptotagmin, expressed as an autosomal dominant disorder. One of theses families contains a proline to leucine substitution (sytP-L) a residue that had not been previously investigated for synaptotagmin function. In chapter 4, I investigated the functional importance of this mutation and created a disease model for this familial condition by driving the expression of a homolous proline-leucine synaptotagmin substitution in the central nervous system of Drosophila. I demonstrated that the proline residue plays a functional role in efficient transmitter release by testing its function in an otherwise synaptotagmin null genetic background. Additionally, this mutation displayed characteristics similar to the human disorder when expressed in a heterozygous synaptotagmin background, similar to the familial expression. Namely, the sytP-L mutants exhibited a decreased release probability, which resulted in decreased evoked responses that facilitate upon high frequency stimulation, a rightward shift in Ca2+ sensitivity, and behavioral deficits, including decreased motor output and increased fatigability. Thus, these studies establish the causative nature of the sytP-L mutation in this rare form of congenital myasthenic syndrome and highlight the utility of the Drosophila system for disease modeling.