Browsing by Author "Tamkun, Michael, committee member"

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Open Access
Age-dependent decline in Kv4 channels, underlying molecular mechanisms, and potential consequences for coordinated motor function
(Colorado State University. Libraries, 2019) Vallejos, Maximiliano Jose, author; Tsunoda, Susan, advisor; Amberg, Gregory C., committee member; Bouma, Gerrit, committee member; Mykles, Donald, committee member; Tamkun, Michael, committee member
The voltage-gated potassium channel, Kv4, is widely expressed in the central nervous system and it is responsible for a highly conserved rapidly inactivating A-type K+ current. Kv4 channels play a role in the regulation of membrane excitability, contributing to learning/memory and coordinated motor function. Indeed, recent genetic and electrophysiological studies in Drosophila have linked Kv4 A-type currents to repetitive rhythmic behaviors. Because a deterioration in locomotor performance is a hallmark of aging in all organisms, we were interested in examining the effects of age on Kv4/Shal channel protein. In this dissertation, I use Drosophila as a model organism to characterize an age-dependent decline in Kv4/Shal protein levels that contributes to the decline in coordinated motor performance in aging flies. Our findings suggest that accumulation of hydrogen peroxide (H2O2) is amongst the molecular mechanisms that contribute to the age-dependent decline of Kv4/Shal. We show that an acute in vivo H2O2 exposure to young flies leads to a decline of Kv4/Shal protein levels, and that expression of Catalase in older flies results in an increase in levels of Kv4/Shal and improved locomotor performance. We also found that the scaffolding protein SIDL plays a role in maintaining Kv4/Shal protein levels and that SIDL mRNA declines with age, suggesting that an age-dependent loss of SIDL may also lead to Kv4/Shal loss. In behavioral studies, we found that a knockdown of SIDL resulted in a lethal phenotype, leading to a large decline in Drosophila eclosion rates, an event that requires coordinated peristaltic motions. Expression of SIDL or Kv4/Shal in this SIDL knockdown genetic background resulted in a partial rescue; these results are consistent with a model in which SIDL and Kv4/Shal play a role in coordinated peristaltic motions and are required for successful eclosion. The results presented in this dissertation provide new insight into the possible molecular mechanisms that underlie an age-dependent decline in Kv4/Shal protein. We identify two contributing factors: 1) ROS accumulation, and 2) the interacting protein SIDL. Our data also suggests that this age-dependent decline in Kv4/Shal levels is likely to be conserved across species, at least in some brain regions. Because Kv4/Shal channels have been implicated in the regulation of long-term potentiation and in repetitive rhythmic behaviors, the loss of Kv4/Shal may contribute to the age-related decline in learning/memory and motor function.
Embargo
An investigation of synaptic vesicle docking and priming and a proposed method for quantitatively measuring both in Drosophila using electron tomography
(Colorado State University. Libraries, 2023) Twiggs, Jasmin A., author; Reist, Noreen, advisor; Hoerndli, Frederic, committee member; Hoke, Kim, committee member; Tamkun, Michael, committee member
The nervous system, as the body's command center, plays a crucial role in cellular communication within the brain and between the brain and other body systems. Neurons, the individual cellular units, transmit electrical information and communicate with other cells through neurotransmitter release in response to electrical stimuli. Chapter 1 introduces the foundational concepts of neuronal structure and function and delves into the mechanisms underlying neurotransmitter release. Special attention is given to the neuromuscular junction (NMJ), a well-studied chemical synapse crucial for muscle movement. The synaptic vesicle cycle is introduced, with particular emphasis on docking and priming. The significance of active zones, specialized sites for efficient signal transmission, and their associated structural components are underscored. Synaptotagmin, a pivotal protein in calcium-triggered vesicle fusion, is discussed with emphasis on its C2B polylysine motif. Throughout the chapter, the utility of Drosophila as a model system for studying synaptic processes, particularly at the NMJ, is emphasized. In sum, Chapter 1 provides the foundational knowledge essential for comprehending the intricate cellular and molecular facets of synaptic communication within the nervous system, serving as a precursor to subsequent chapters' investigations. Chapter 2 examines synaptotagmin's C2B polylysine motif and its role in synaptic vesicle docking at the Drosophila NMJ. It explores the polylysine motif's potential involvement in endocytosis, demonstrates an unaffected interaction with AP-2, and uses electron microscopy to find no significant changes in vesicle distribution. The findings suggest that the reduced neurotransmitter release in the polylysine mutant is likely due to an impairment in vesicle priming. Chapter 3 introduces a method for studying synaptic vesicle docking and priming in Drosophila, using electron tomography. I address the limitations of conventional electron microscopy and underscore the need for higher-resolution techniques to assess molecular structures that mediate physiological processes. Chapter 3 also emphasizes the significance of the contact area between docked vesicles and the presynaptic membrane as a correlate of vesicle priming. The protocol, expected results, and key considerations are discussed. The methods presented in Chapter 3 offer a promising approach for understanding synaptic processes. In Chapter 4, I discuss key considerations for when standard electron microscopy can be used for assessing vesicle docking. Then, I discuss how the electron tomography method presented in Chapter 3 could not only confirm the results found in Chapter 2, that the synaptotagmin C2B polylysine motif is not implicated in vesicle docking but could also be used to directly test the mutant's role in priming. Specific aims for future studies on the synaptotagmin polylysine mutation in Drosophila are presented, potential results and interpretations are discussed. Finally, I showcase interesting, unpublished findings from electron tomograms I have taken at the Drosophila NMJ and discuss their potential significance.
Open Access
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.
Open Access
Characterizing the diffusional behavior and trafficking pathways of Kv2.1 using single particle tracking in live cells
(Colorado State University. Libraries, 2013) Weigel, Aubrey, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Bartels, Randy, committee member
Studying the diffusion pattern of membrane components yields valuable information regarding membrane structure, organization, and dynamics. Single particle tracking serves as an excellent tool to probe these events. We are investigating of the dynamics of the voltage gated potassium channel, Kv2.1. Kv2.1 uniquely localizes to stable, micro-domains on the cell surface where it plays a non-conducting role. The work reported here examines the diffusion pattern of Kv2.1 and determines alternate functional roles of surface clusters by investigating recycling pathways using single particle tracking in live cells. The movement of Kv2.1 on the cell surface is found to be best modeled by the combination of a stationary and non-stationary process, namely a continuous time random walk in a fractal geometry. Kv2.1 surface structures are shown to be specialized platforms involved in trafficking of Kv channels to and from the cell surface in hippocampal neurons and transfected HEK cells. Both Kv2.1 and Kv1.4, a non-clustering membrane protein, are inserted and retrieved from the plasma membrane at the perimeter of Kv2.1 clusters. From the distribution of cluster sizes, using a Fokker-Planck formalism, we find there is no evidence of a feedback mechanism controlling Kv2.1 domain size on the cell surface. Interestingly, the sizes of Kv2.1 clusters are rather governed by fluctuations in the endocytic and exocytic machinery. Lastly, we pinpoint the mechanism responsible for inducing Kv2.1 non-ergodic dynamics: the capture of Kv2.1 into growing clathrin-coated pits via transient binding to pit proteins.
Open Access
Cholinergic synaptic homeostasis is regulated by Drosophila α7 nicotinic acetylcholine receptors and Kv4 potassium channels
(Colorado State University. Libraries, 2021) Eadaim, Abdunaser Omar, author; Tsunoda, Susan, advisor; Tamkun, Michael, committee member; Amberg, Gergory, committee member; Bouma, Gerrit, committee member; Clay, Colin, committee member; DeLuca, Jennifer, committee member
Homeostatic synaptic plasticity (HSP) is an important mechanism that stabilizes neural activity during changes that occur during development and learning and memory formation, and some pathological conditions. HSP in cholinergic neurons has been implicated in pathological conditions, such as Alzheimer's disease and nicotine addiction. In a previous study in primary Drosophila neuron culture, cholinergic activity was blocked using pharmacological tools and this induced a homeostatic response that was mediated by an increase in the Drosophila α7 (Dα7) nAChR, which was subsequently tuned by an increase in the voltage-dependent potassium channel, Kv4/Shal. In this study, we inhibit cholinergic activity in live flies using temperature-sensitive mutant alleles of the choline acetyltransferase gene (Chats2 mutants). We show that this in vivo activity inhibition induces HSP similarly mediated by Dα7 nAChRs followed by an up-regulation of Kv4/Shal. We show that the up-regulation of Dα7 nAChRs alone is sufficient to induce an increase in Kv4/Shal protein, as well as mRNA. Finally, we test the involvement of transcription factors, dCREB2 and nuclear factor of activated T cells (NFAT) in the up-regulation of Kv4/Shal. In particular, we find that NFAT is required for the inactivity-induced up-regulation of Kv4/Shal channels. Our studies reveal a novel receptor-ion channel system transcriptionally coupled to prevent over-excitation.
Open Access
Compartmentalization of membrane proteins by the actin cytoskeleton
(Colorado State University. Libraries, 2013) Higgins, Jenny, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Bamburg, James, committee member; Azimi-Sadjadi, Mahmood, committee member
Acting as the point of contact for the outside world, the plasma membrane is crucial for cellular signaling events. Proper organization of membrane components is necessary to accomplish this task. Although a number of experiments have demonstrated the compartmentalization of lipids and proteins on the plasma membrane, direct observation of the mechanisms by which the organization occurs has been challenging, in part due to the imaging restrictions of a diffraction-limited system and the dynamic nature of the membrane compartmentalization. Using photoactivated localization microscopy (PALM), a superresolution technique, we have captured the dynamics of compartments formed by the cortical actin cytoskeleton. Live human embryonic kidney (HEK293) cells were imaged with a temporal resolution of 2 s and a spatial resolution of 40 nm. The actin cytoskeleton forms compartments with a mean area of 2.3±0.3 μm2 that are partially outlined by actin bundles. When the PALM images of actin were combined with single particle tracking of membrane proteins, we directly observed the cytoskeleton acting as a barrier to the diffusion of Kv2.1 and Kv1.4, two voltage-gated potassium channels. In addition, we used a novel compartment detection and tracking algorithm to show that Kv2.1 and Kv1.4 channels avoid actin when changing compartments. This work represents the first direct observations of individual membrane protein interactions with barriers formed by the actin cytoskeleton.
Open Access
Detection and measurements of free ubiquitin in fixed cells and characterization of OTUB1 contribution to ubiquitin homeostasis
(Colorado State University. Libraries, 2020) Prada Gomez, Luisa Fernanda, author; Cohen, Robert, advisor; Di Pietro, Santiago, committee member; Markus, Steven, committee member; Tamkun, Michael, committee member
Post-translational modifications with Ubiquitin (Ub) have been found to participate in a wide range of cell functions, including protein degradation, endocytosis, regulation of gene expression and cell cycle progression. Therefore, regulation of free Ub levels is essential to ensure that enough Ub is available for conjugation, while excess Ub does not compete in the large number of processes that depend on binding to ubiquitinated proteins or polyUb. Not surprisingly, changes in Ub pool dynamics can affect the cell functions, and perturbations of free Ub levels have been reported to cause neurological and developmental disorders. Although there are techniques to measure Ub pools in vitro, visualization and quantification of free Ub inside individual cells has not been possible. One way to regulate the intracellular concentration of free Ub, is by means of Deubiquitinating enzymes (DUBs), however specific details about the regulatory mechanism are, in large part, unknown. Most studies about DUBs have focused on enzymatic activity and regulation in vitro, with only few reports on the regulation of Ub homeostasis in vivo. The role of OTUB1 in Ub homeostasis has been hypothesized because its catalytic activity is affected by the ratio of [Ub~E2] to [E2] in response to free Ub concentration. Interaction between OTUB1 and a subset of E2s can stimulate OTUB1 isopeptidase activity, whereas interactions with Ub~E2s can inhibit the ubiquitin transfer from the thioester Ub~E2 adduct. This dissertation describes the successful development of a technique to detect and quantify changes in free Ub levels in fixed cells using a high affinity binding protein. The method was used to quantify changes in Ub levels after proteasome and E1 inhibition and to establish the free Ub distribution in hippocampal neurons. It was shown also that OTUB1 activity is not directly involved in the regulation of free Ub levels under stress conditions. However, a new mechanism for regulation of UBE2D expression levels dependent on OTUB1 was identified. This mechanism is independent of proteasomal degradation and could possibly involve translational regulation.
Open Access
Differential desensitization of pre- and postsynaptic mu opioid receptors regulating proopiomelanocortin neurons of the arcuate nucleus
(Colorado State University. Libraries, 2017) Pennock, Reagan L., author; Hentges, Shane, advisor; Tamkun, Michael, committee member; Vigh, Jozsef, committee member; Krapf, Diego, committee member
The mu opioid receptor (MOR) is the primary target of powerful opiate analgesics such as morphine and codeine. Repeated use of opiates, as may occur in patients with chronic pain, leads to the development of tolerance to the drugs' analgesic effects and may result in the development of dependence. This reduces the effectiveness of opiate-based treatments over extended periods of time, and can result in withdrawal when such a treatment is terminated. Many years of study have been dedicated to understanding the processes that lead to the development of tolerance, as an understanding of the mechanisms underlying tolerance could lead the development of novel therapeutic strategies that prolong the efficacy of opioid-based pain treatments. One particular area of focus has been on acute desensitization of the MOR. Studies of acute desensitization, defined as the loss of receptor function that occurs in the seconds to minutes following activation with an agonist, largely focus on the attenuation of desensitization of desensitization-susceptible MORs found on the somato-dendritic region of neurons in various parts of the nervous system. In these studies, we will focus on characterizing desensitization-resistant MORs located on the axon terminal region of GABAergic neurons that form synapses with hypothalamic proopiomelanocortin (POMC) neurons. Activation of presynaptic MORs, as well as other Gαi/o-coupled GPCRs located on presynaptic terminals, results in an inhibition of GABA release, which causes a subsequent inhibition of the amplitude or frequency of inhibitory postsynaptic currents (IPSCs). Our findings demonstrate that apparent resistance to desensitization by presynaptic MORs, measured as a sustained inhibition of IPSC amplitude or frequency, cannot be explained by a large receptor reserve, nor can desensitization become detectable after chronic treatment with the opiate morphine. It was also found that resistance to desensitization is a common, but not universal, property of Gαi/o-coupled G-protein coupled receptors located on presynaptic terminals. Comparison of desensitization-resistant MORs with desensitization-susceptible GABAB receptors revealed that both populations of receptors have similar receptor-effector coupling, and that resistance or susceptibility to desensitization is unaffected by experimental conditions that isolate either Ca2+-independent spontaneous release or Ca2+-dependent synchronous release. These findings provide evidence that resistance or susceptibility to desensitization is not dependent on particular receptor-effector coupling, and is likely receptor delimited. The previous findings suggest that resistance to desensitization by the MOR may be conferred by altered physical properties of presynaptic receptors relative to their postsynaptic counterparts. A likely way that these physical differences could manifest would be through differential mobility of pre- and postsynaptic receptors. To provide proof of principle that such measurements can be made, single-particle tracking of MORs containing an N-terminal FLAG tag was performed the AtT20 cell line. MOR diffusion was measured before and after activation with a maximal, desensitizing concentration of the full MOR agonist DAMGO. In the absence of DAMGO, FLAG-MORs could be found in either a mobile or immobile state. After ten minutes in the presence of DAMGO the fraction of immobile FLAG-MORs was increased, but both mobile and immobile receptors were still present. Because ten minutes in a maximal concentration of DAMGO is sufficient to cause MOR desensitization to reach a maximum and for the internalization of most desensitized receptors to occur, the findings demonstrate that steady-state signaling of the MOR may be maintained by both mobile and immobile receptors. These findings provide a basis for future studies comparing the mobility of pre- and postsynaptic MORs in neurons, as well as determining the role of mobile and immobile MORs in signaling pathways recruited by the receptor.
Open Access
Exercise training improves exercise capacity despite persistent muscle mitochondrial dysfunction in the taz shRNA mouse model of human Barth Syndrome
(Colorado State University. Libraries, 2013) Claiborne, Michael Scott, author; Chicco, Adam J., advisor; Hamilton, Karyn, committee member; Miller, Benjamin, committee member; Tamkun, Michael, committee member
Barth Syndrome is a mitochondrial disease associated with exercise intolerance and cardioskeletal myopathy resulting from mutations in the tafazzin (taz) gene. The present study characterized skeletal muscle mitochondrial function and exercise capacity of a taz shRNA mouse model of Barth Syndrome (90% taz-deficient), and examined the effect of exercise training on these parameters. Mitochondrial respiratory function was assessed, in mitochondria freshly isolated from hindlimb muscles, using an Oroboros O2K respirometer with pyruvate + malate as substrates, oligomycin as an ATP synthase inhibitor, and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) to establish maximal activity. A pre-training GXT revealed profound exercise intolerance, which corresponded to reduced respiratory capacity, citrate synthase (CS) and ETC complex 1 protein content of muscle mitochondria in the taz vs. age-matched wild-type (WT) mice. Based on the pre-training GXT, exercise training was conducted at 12-17 m/min, 0% grade for 60 min/d, 5d/wk, for 4 wks. Exercise training elicited a 99% increase in GXT run time in the taz mice (P < 0.01 vs. pre-training), but failed to increase times to those of sedentary WT mice. Training significantly decreased state 3 respiratory capacity of muscle mitochondria from exercised mice (wild type sedentary (WTS): 4992.59 ± 371.35, wild type exercised (WTX): 3779.60 ± 561.43, taz sedentary (TazS): 2978.50 ± 383.53, TazS: 1827.55 ± 525.17 (pmolO2/(s*mg), P = 0.02, Sed. vs. Ex.), and significantly decreased mitochondrial CS activity in taz mice (WTS: 4.48 ± 0.51, WTX: 3.87 ± 0.69, TazS: 3.21 ± 0.54, taz exercised (TazX): 1.63 ± 0.69 (relative absorbance/gram of protein) (RU/g), P = 0.01). Training also tended to reduce mitochondrial lactate dehydrogenase (LDH) and monocarboxylate transporter 1 (MCT1) activities, MnSOD content, and 4-hydroxnonenal-protein adducts (index of oxidative stress), but tended to increase mitochondrial UCP3 in exercised WT and taz mice. Interestingly, training significantly increased muscle levels of CS (WTS: 1.491 ± 0.112, WTX: 1.792 ± 0.143, TazS: 1.325 ± 0.108, TazX: 1.550 ± 0.143 (RU/g), P = 0.05 Sed. v. Ex.), suggesting increased muscle mitochondrial content with training. This study indicates that exercise training improves functional capacity of taz deficient mice and induces selective mitochondrial protein remodeling during mitochondrial biogenesis that perhaps mitigates oxidative stress while adapting to increased metabolic demand.
Open Access
Investigating new protein components of the endocytic machinery in Saccharomyces cerevisiae
(Colorado State University. Libraries, 2016) Farrell, Kristen, author; Di Pietro, Santiago, advisor; Bamburg, James, committee member; Krapf, Diego, committee member; Tamkun, Michael, committee member
Clathrin-mediated endocytosis is an essential eukaryotic process which allows cells to control membrane lipid and protein content, signaling processes, and uptake of nutrients among other functions. About 60 proteins have been identified that compose the endocytic machinery in Saccharyomes cerevisiae, or budding yeast. Clathrin-mediated endocytosis is highly conserved between yeast and mammals in terms of both protein content and timing of protein arrival. First, there is an immobile phase in which clathrin and other coat components concentrate at endocytic sites. Second, another wave of proteins assembles about 20 seconds before localized actin polymerization. Third, a fast mobile stage of endocytosis occurs coinciding with local actin polymerization and culminates with vesicle scission. Fourth, most coat proteins disassemble from the internalized vesicle. Despite the knowledge of so many endocytic proteins, gaps still remain in the complete understanding of the endocytic process. We attempt to fill some of these gaps with a screen of the yeast GFP library for novel endocytic-related proteins using confocal fluorescence microscopy. We identified proteins colocalizing with RFP-tagged Sla1, a clathrin adaptor that serves as a well-known marker of endocytic sites. Ubx3 and Tda2, two unstudied proteins, were selected for further investigation based on high degree of colocalization with Sla1. Ubx3 shows fluorescent patch dynamics similar to an endocytic coat protein. Ubx3 is dependent on clathrin for patch lifetime and binds clathrin via a W-box, the first identification of this clathrin binding motif in a non-mammalian species. Uptake assays performed in a knockout strain of Ubx3 display a reduction in both bulk endocytosis by fluorescent dye Lucifer Yellow and cargo-specific endocytosis by methionine transporter Mup1. The Ubx3 knockout cells also show a significant increase in lifetime of early endocytic protein Ede1, and removing its UBX domain alone results in similar defects to the Ubx3 knockout. The endocytic defect may be due to lack of recruitment of ubiquitin regulator AAA ATPase Cdc48 to the endocytic site. Inactivation of Cdc48 reduces Lucifer yellow uptake to minimal levels and causes aggregates of early endocytic protein Ede1-GFP. This is the first identification of a UBX domain-containing protein in clathrin-mediated endocytosis. Tda2 appears at the tail end of each endocytic site, suggesting a function in late stage endocytosis. A Tda2 knockout strain shows similar reductions in bulk and cargo dependent endocytosis through Lucifer yellow and Mup1 uptake assays. Tda2 appears unaffected by clathrin disruption, but is no longer recruited to the endocytic site when cells are treated with the actin depolymerizing agent LatA, suggesting it is associated with the actin cytoskeleton. A crystal structure of Tda2 reveals it is a homolog of mammalian dynein light chain TcTex-1. Tda2 is associated with a larger protein complex in the cytosol but does not co-purify with dynein and is unaffected by addition of the microtubule depolymerizing drug Nocodazole. Tda2 has similar localization to actin capping proteins Cap1/2 which localize to the plus end of actin filaments near the plasma membrane. Tda2 deletion increases Cap1 patch lifetime but reduces its fluorescent intensity. Aim21 fluorescent intensity at endocytic sites is reduced to half without Tda2. When Aim21 is deleted, Tda2 is no longer recruited to endocytic sites and the large Tda2-containing complex is no longer present in the cytosol. Tda2 is a newly identified component of the actin cytoskeleton in stable complex with Aim21. This is the first identification of a TcTex type dynein light chain in yeast and the first dynein light chain associated with clathrin-mediated endocytosis. Thus, we have identified two novel components of endocytic machinery by screening the yeast GFP library. The successful identification of previously uncharacterized endocytic proteins indicates the unique advantages of the GFP library screening. Many previous screens for endocytic proteins rely on the yeast knockout library or cargo accumulation, which have many disadvantages. The GFP library screening method has potential for use with other cellular processes that have distinct cellular localizations and established fluorescent markers. The GFP library also has potential for use in a screen for cargo proteins dependent on clathrin-mediated endocytosis. Additionally, more proteins of the endocytic machinery may be further characterized from the list of Sla1-colocalizing proteins identified in our screen.
Open Access
IQGAP1 is a novel effector of gonadotropin-releasing hormone receptor signaling
(Colorado State University. Libraries, 2023) Alqahtani, Huda A., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Tamkun, Michael, committee member; DeLuca, Jennifer, committee member
Stimulation of gonadotropin-releasing hormone (GnRH) receptors on the surface of anterior pituitary gonadotrope cells is a key signaling event for the hypothalamic-pituitary-gonadal axis. One important downstream component of GnRH receptor signaling is activation of the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase), which is essential for the production of the gonadotropin luteinizing hormone. Evidence suggests that GnRH receptors reside in low-density plasma membrane domains where they participate in multiprotein signaling complexes. Here we used quantitative proteomics to identify proteins associated with low-density plasma membrane domains and to measure changes in their relative abundance in these domains in response to GnRH. Using αT3-1 gonadotropes, we identified 537 proteins in detergent-free subcellular fractions containing low-density plasma membranes. SILAC (stable isotope labeling by amino acids in cell culture) in combination with mass spectrometry demonstrated that GnRH, within 10 min, altered the association of 87 proteins with this plasma membrane fraction. Ontology analysis revealed that GnRH promoted an enrichment of actin cytoskeletal and adherens junction-related proteins including the molecular scaffold IQGAP1 and the small GTPase Rac1. Subsequent investigation revealed that the association between Rac1 and IQGAP1 increased with GnRH receptor stimulation and that GnRH increased Rac1 activity. Demonstrating functional relevance, inhibiting Rac1 reduced GnRH-dependent ERK activation. Our data reveals an upstream activation of signaling and structural molecules, including Ca2+, CDC42 and Rac1, E-cadherin, N-cadherin, and β-catenin. We also identified interactions between the scaffold protein IQGAP1 and these molecules, indicating that IQGAP1 is a fundamental regulator of GnRH-dependent signaling in gonadotropes. Furthermore, our data shows that IQGAP1 has a transcriptional regulatory role in gonadotropes treated with GnRH. In sum, these data indicate that IQGAP1 complexed with Rac1 modulates ERK activity and as such serves as an essential effector in modulating cell polarity and cell-cell contacts in gonadotropes. Altogether, our proteomics data show that acute stimulation of GnRH receptors (3 nM for 10 min) alters the PAM fraction abundance of proteins, such as IQGAP1, mechanistically linked to gonadotrope activation.
Open Access
Na+ -activated K+ channels protect against overexcitation and seizure-like behavior in Drosophila
(Colorado State University. Libraries, 2021) Byers, Nathan S., author; Tsunoda, Susan, advisor; Garrity, Deborah, committee member; Hentges, Shane, committee member; Hoerndli, Frederic, committee member; Tamkun, Michael, committee member
Na+-activated K+ channels (KNa) encode K+ channels that are activated by internal Na+ and are widely expressed throughout the mammalian central nervous system. Based on the biophysical properties of the channels, it has long been postulated that they act as a reserve mechanism to combat neuronal overexcitation. Specifically, early electrophysiological recordings suggested that only when intracellular Na+ levels rise significantly, for instance in neuropathological conditions, do KNa channels become active. More recent evidence suggests that they may function under normal physiological circumstances by means of binding cytoplasmic factors and via the persistent Na+ current. However, to date it is unclear if KNa channels function to prevent overexcitation in vivo. Therefore, research in my dissertation sets out to test the hypothesis that KNa channels protect against overexcitation in Drosophila models of epilepsy. Drosophila contain one gene encoding a KNa channel, dSlo2. In the third chapter of this dissertation, I examine expression of dSlo2 channels throughout the nervous system. Findings from this chapter show that dSlo2 channels are expressed in cholinergic neurons, the main excitatory neuron of the Drosophila brain. Furthermore, dSlo2 channels were excluded from GABAergic neurons. I additionally found that dSlo2 channels are localized to axonal regions of multiple neuronal subtypes in the nervous system. Thus, these results suggest that as K+ channels widely and preferentially expressed in excitatory neurons in the brain, dSlo2 channels may function to dampen neuronal, and perhaps behavioral, excitability. In Chapter 4, I test the hypothesis that dSlo2 channels protect against behavioral abnormalities caused by cholinergic overexcitation. I first show that the loss of dSlo2 exacerbates behavioral deficits and death associated with prolonged exposure to a cholinergic agonist, Imidacloprid. Furthermore, I found that adult flies lacking dSlo2 exhibit mechanically induced seizure-like behavior following feeding of Imidacloprid, which does not occur in wild-type flies. Combined, these results suggest that dSlo2 channels do indeed protect against cholinergic overexcitation. It has previously been shown that mammalian KNa channels are activated by a persistent Na+ current (INaP) in neurons, suggesting that these channels may ameliorate behavioral consequences of an increased INaP in vivo. In Chapter 5, I test the hypothesis that dSlo2 channels protect against Drosophila seizure-like behavior induced by an increased INaP. I find that the loss of dSlo2 significantly exacerbates seizure-like behavior in multiple Drosophila epileptic models, including a model for human generalized epilepsy with febrile seizures plus (GEFS+). Additionally, the absence of dSlo2 worsens seizure-like behavior when flies are exposed to Veratridine, a pharmacological agent known to increase INaP. Interestingly, the loss of dSlo2 also revealed a spontaneous seizure phenotype in INaP-affected seizure models that was otherwise absent. Altogether, these results are consistent with the model that KNa channels are activated by INaP, and protect against seizure-like behavior actuated by increased INaP. Overall, the work in my dissertation expands our understanding of the role of KNa channels. These findings suggest that KNa channels may play a protective role for many neuropathological diseases associated with an increased INaP, such as epilepsy, amyotrophic lateral sclerosis, neuropathic pain, and ischemia.
Open Access
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.
Open Access
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.
Open Access
Regulation of local L-type calcium channel signaling in anterior pituitary gonadotropes
(Colorado State University. Libraries, 2017) Dang, An Khanh, author; Amberg, Greg, advisor; Clay, Colin, advisor; Tamkun, Michael, committee member; Navratil, Amy, committee member; Duval, Dawn, committee member
The binding of gonadotropin-releasing hormone (GnRH) to its receptor initiates signaling cascades in gonadotropes which result in enhanced luteinizing hormone (LH) and follicle stimulating hormone (FSH) biosynthesis and secretion. Most dramatic is the sharp rise in LH secretion ("LH surge") that precedes and is necessary for follicular maturation and ovulation. Ca2+ influx activates mitogen-activated protein kinases (MAPKs) which lead to increased transcription of LH and FSH genes. Interestingly, previous research suggests that two MAPK signaling pathways, ERK and JNK, are activated by either Ca2+ influx through L-type Ca2+ channels or by global Ca2+ signals originating from intracellular stores, respectively. These discrete Ca2+ sources for divergent signaling cascades provides a mechanism in which gonadotropes can decode different pathways for appropriate gonadotropin release during various stages of the ovulatory cycle. However, direct evidence supporting an underlying subplasmalemmal local Ca2+ signaling through L-type Ca2+ channels distinct from intracellular Ca2+ was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence (TIRF) microscopy to visualize discrete sites of Ca2+ influx (Ca2+ sparklets) in gonadotrope-derived αT3-1 cells in real time. These localized GnRH-induced Ca2+ influxes are mediated by L-type Ca2+ channels and important for downstream ERK activation. In addition, precise structural and molecular elements to create a microenvironment suitable for localized subplasmalemmal L-type Ca2+ channel signaling was necessary for gonadotrope function, in which GnRH-dependent stimulation of L-type Ca2+ channel influx was found to require PKC and a dynamic actin cytoskeleton. More recently, we have further elucidated molecular mechanisms modulating localized L-type Ca2+ channel influx. Reactive oxygen species (ROS) are cognate signaling molecules that mediate cell function, but their role in regulating Ca2+ in gonadotropes is unknown. We have explored GnRH regulation of both NADPH oxidase complexes and mitochondrial sources of ROS and assessed ROS modulation of L-type Ca2+ channel activity in gonadotropes. We identified GnRH-induced spatially localized ROS "puncta" in αT3-1 cells, and ROS increased local Ca2+ channel activity in both αT3-1 cells and primary mouse gonadotropes. In addition, GnRH increased mitochondrial oxidation activity at the subplasmalemmal surface and mitochondrial ROS increased localized L-type Ca2+ channel influx. Also, active L-type Ca2+ channels were associated with subplasmalemmal mitochondria. Taken together, this dissertation explored the first direct evidence for localized L-type Ca2+ channel signaling in αT3-1 cells and elucidated signaling mechanisms in gonadotropes. Specifically, cellular organization via an intact cytoskeletal platform and ROS regulated L-type Ca2+ channel sparklet activity that are important for the downstream ERK activation and gonadotropin gene expression that regulates reproduction.
Open Access
S-nitrosylation mediates synaptic plasticity in the retina
(Colorado State University. Libraries, 2015) Tooker, Ryan E., author; Vigh, Jozsef, advisor; Tamkun, Michael, committee member; Hentges, Shane, committee member; Hoke, Kim, committee member
Over the course of an entire day, our visual system must accommodate intensities of light that can change by a factor of 10¹⁰. In order to do so, the retina adapts to large, daily changes in natural light intensity by shifting its dynamic range of coding. For example, as morning light intensity increases, the retina implements multiple strategies that result in decreases in overall sensitivity in order to avoid saturation. However, adaptation to bright environments poses the inherent risk of losing visual information carried by dim/weak signals in complex natural scenes. Here we studied whether the light-evoked increase in retinal nitric oxide (NO) production is followed by NO-mediated, direct post-translational modification of proteins called S-nitrosylation and if it contributes to the modulation of the dynamic range of vision. In the central nervous system, including the retina, S-nitrosylation has not been considered to be significant under physiological conditions, and instead, has been primarily associated with neurodegenerative diseases. In this study, we provide immunohistochemical and proteomic evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner. Functionally, we report a novel form of activity-dependent synaptic plasticity via S-nitrosylation: a “weighted potentiation” that selectively increases the output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. Importantly, the NO action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Our data strongly suggest that in the retina, light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant post-translational modification affecting a wide range of proteins under physiological conditions. S-nitrosylation may function to extend the dynamic range of vision by counteracting the decreases in retinal sensitivity during light adaptation ultimately preventing the loss of visual information carried by dim scotopic signals. Finally, our results may set the framework for exploring the role of S-nitrosylation in certain neurodegenerative retinal diseases that are associated with toxic levels of NO.
Open Access
Single molecule fluorescence measurements of complex systems
(Colorado State University. Libraries, 2017) Sadegh, Sanaz, author; Krapf, Diego, advisor; Tamkun, Michael, committee member; Chong, Edwin, committee member; Prasad, Ashok, committee member
Single molecule methods are powerful tools for investigating the properties of complex systems that are generally concealed by ensemble measurements. Here we use single molecule fluorescent measurements to study two different complex systems: 1/ƒ noise in quantum dots and diffusion of the membrane proteins in live cells. The power spectrum of quantum dot (QD) fluorescence exhibits 1/ƒ noise, related to the intermittency of these nanosystems. As in other systems exhibiting 1/ƒ noise, this power spectrum is not integrable at low frequencies, which appears to imply infinite total power. We report measurements of individual QDs that address this long-standing paradox. We find that the level of 1/ƒβ noise for QDs decays with the observation time. We show that the traditional description of the power spectrum with a single exponent is incomplete and three additional critical exponents characterize the dependence on experimental time. A broad range of membrane proteins display anomalous diffusion on the cell surface. Different methods provide evidence for obstructed subdiffusion and diffusion on a fractal space, but the underlying structure inducing anomalous diffusion has never been visualized due to experimental challenges. We addressed this problem by imaging the cortical actin at high resolution while simultaneously tracking individual membrane proteins in live mammalian cells. Our data show that actin introduces barriers leading to compartmentalization of the plasma membrane and that membrane proteins are transiently confined within actin fences. Furthermore, superresolution imaging shows that the cortical actin is organized into a self-similar fractal.
Open Access
Super-resolution imaging and modeling of murine sperm during capacitation process
(Colorado State University. Libraries, 2019) Xu, Xinran, author; Krapf, Diego, advisor; Munsky, Brian, committee member; Pezeshki, Ali, committee member; Tamkun, Michael, committee member
The effort to achieve better spatial resolution beyond the diffraction limit has been dedicated for many years. In the past decade, super-resolution microscopy methods have successfully advanced into extremely powerful tools to reveal hidden three-dimensional structures and properties in various biological complex systems. Here we use single-molecule localization based three-dimensional super-resolution microscopy to study the mouse sperm capacitation process, a critical step in gaining the fertilization ability. On top of that, we construct a stochastic model to represent this signaling pathway in order to be able to predict the cellular event within the capacitation. The major subjects we are interested in can be categorized into two parts: actin-based cytoskeleton and capacitation-associated signaling proteins. In the midpiece, we discovered that F-actin forms a highly specialized double helical structure, which has been the very first observation among species and has disappeared in the principal piece. Similarly, the distinctive compartments regarding actin-binding proteins have also been visualized in the mouse sperm tail. Additionally, the structure as well as localization of capacitation central mediator, protein kinase A have been investigated to address the significance of spatial positioning during the capacitation event. As the capacitation end point reporter, tyrosine phosphorylation localization has been studied to help identify its real upstream kinase among other candidates. As in many regulating processes, second messenger Ca2+ plays a vital role in the capacitation process, which needs to be conveyed by the sperm specific calcium channel CatSper. We show the structural relation of a small GTPase Cdc42 to CatSper, implying its key role in transporting Ca2+. Considering that major critical signaling molecules are well characterized in the complex capacitation network, we choose a different method–stochastic modeling, other than experimental studies, surpassing the need for probing the behavior of a large number of individual cells over time, to describe capacitation process and furthermore to predict the behavior of sperm. With the known pathways of those signaling molecules in hand, we are able to build a stochastic model by utilizing chemical master equations. A couple sets of experimental measurements are used to assist in quantifying the model.
Open Access
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.
Open Access
The autism-associated loss of δ-catenin function disrupts social behavior
(Colorado State University. Libraries, 2023) Mendez-Vazquez, Hadassah, author; Kim, Seonil, advisor; Tamkun, Michael, committee member; Myers, Brent, committee member; Chanda, Soham, committee member
Social impairment is a key symptom of several neuropsychiatric disorders, including autism spectrum disorder (ASD), anxiety, depression, and schizophrenia. Despite the increasing prevalence of these disorders the physiological, cellular, and molecular factors underlying social dysfunction are still poorly understood. In humans, mutations in the δ-catenin gene have been linked to severe forms of ASD. δ-catenin is a post-synaptic scaffolding protein that is expressed in excitatory synapses and functions as an anchor for N-cadherin and the AMPA receptor (AMPAR) subunit GluA2 at the postsynaptic density. A glycine 34 to serine (G34S) mutation in the δ-catenin gene was identified in ASD patients and induces a loss of δ-catenin function, which may mediate ASD pathogenesis. The mechanism by which this G34S mutation causes loss of δ-catenin function to induce ASD remains unclear. Initial findings revealed that the G34S mutation increases glycogen synthase kinase 3β (GSK3β)-dependent δ-catenin degradation to reduce δ-catenin levels. Moreover, we found that mice possessing the G34S δ-catenin mutation have significantly reduced synaptic cortical δ-catenin and GluA2 levels. The G34S mutation was also found to differentially alter glutamatergic activity in cortical excitatory and inhibitory cells. Furthermore, G34S δ-catenin mutant mice exhibit markedly impaired social behavior, which is a characteristic feature of ASD. Most significantly, we found that inhibition of GSK3β is sufficient to reverse the G34S-induced loss of δ-catenin function in cells and mice. Altogether, our study reveals that the loss of δ-catenin function arising from the ASD-associated G34S mutation induces social dysfunction via disruptions in glutamatergic activity, and that GSK3β inhibition can reverse abnormal δ-catenin G34S-induced glutamatergic activity and social deficits.