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Browsing by Author "Amberg, Gregory, advisor"

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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 (Naáµ¥) 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 Naáµ¥ 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 Naáµ¥ channels is linked to many diseases including epilepsy, cardiac arrhythmias, and pain disorders. Despite the importance of Naáµ¥ channels, knowledge of their trafficking and cell-surface dynamics is severely limited. Research in this area has been hampered by the lack of modified Naáµ¥ constructs suitable for investigations into neuronal Naáµ¥ cell biology. This dissertation demonstrates the successful creation of modified Naáµ¥1.6 cDNAs that retain wild-type function and trafficking following expression in cultured rat hippocampal neurons. The Naáµ¥1.6 isoform is emphasized because it 1) is the most abundant Naáµ¥ 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 Naáµ¥1.6 were investigated. In contrast to the current dogma that Naáµ¥ channels are localized to the AIS of neurons through diffusion trapping and selective endocytosis, the experiments presented here demonstrate that Naáµ¥1.6 is directly delivered to the AIS via a vesicular delivery mechanism. The modified Naáµ¥1.6 constructs were also used to investigate the distribution and cell-surface dynamics of Naáµ¥1.6. Somatic Naáµ¥1.6 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 Naáµ¥ localization mechanism. Channels within the nanoclusters appear to be stably bound on the order of minutes to hours, while non-clustered Naáµ¥1.6 channels are mobile. Novel single-particle tracking photoactivation localization microscopy (spt-PALM) analysis of Naáµ¥1.6-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 Naáµ¥ channel isoform. The approaches developed during the course of this dissertation research will further our understanding of Naáµ¥1.6 cell biology under both normal and pathological conditions.
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    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.
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    L-type calcium channel-dependent signaling impacts GnRH receptor function and intercellular communication in cultured gonadotropes
    (Colorado State University. Libraries, 2020) Drennan, Meggan L., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Garrity, Deborah, committee member; Kelp, Nicole, committee member
    The hypothalamic-pituitary-gonadal (HPG) axis is a negative feedback biological system critical in fertility, reproduction and development. Gonadotropin-releasing hormone (GnRH) is first released by the hypothalamus and binds to GnRH receptors (GnRH-R) on gonadotrope cells of the anterior pituitary gland where the receptors must mediate a variety of pulsatile signals. The gonadotropin hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are subsequently released by the pituitary and act upon the ovaries and testes, further producing gonadal steroids to be circulated throughout the body. GnRH pulse frequency and amplitude determine successful gonadotropin release, which is ultimately regulated by the GnRH-R. The GnRH-R is a heterotrimeric G-protein coupled 7-transmembrane domain receptor with Gα, β, and γ subunits. Ligand binding initiates an intracellular cascade that leads to a global increase of cytosolic calcium concentration by way of calcium influx through voltage-gated calcium (Cav) channels, and intracellular calcium release from endoplasmic reticulum (ER) stores. Gonadotropes depend on intracellular calcium concentration to carry out their specific physiological function, such as transcription of gonadotropin subunits, hormone biosynthesis and release. Calcium flux is a normal and important aspect of cellular function, including cell-cell communication. Calcium oscillations have been well documented in multiple cell types, with different patterns being induced with distinct treatments. Observations in this line of research include the following: different oscillatory patterns lead to different physiological outcomes, the rate at which internal calcium is secreted from the ER can greatly impact these patterns, and IP3 receptor clustering on the ER results in localized changes in calcium concentration rather than a marked global difference, implicating a spatial stochasticity. These oscillations have shown evidence of paracellular coupling at gap junctions, as well as synchrony following extracellular diffusion. Chapter two of this thesis details experiments investigating calcium oscillations using a membrane-targeted calcium indicator. Immortalized αT3-1 cells were transfected with a membrane-targeted GCaMP and TIRF microscopy was used to capture fluorescent calcium activity. Cells were treated with GnRH as well as various pharmaceutical treatments that would exploit L-type Cav channel function and manipulate normal intracellular calcium release. An array of observations was recorded. Qualitatively, there was an overall increase in calcium activity in the majority of cells after GnRH treatment. Drug-induced inhibition of calcium influx and intracellular calcium release diminished calcium activity entirely. Further, synchronized activity was captured among several cell groups, showing both pre-established synchrony and GnRH-induced synchronized peaking. Further research should be conducted to better understand the full mechanism underlying these behavioral responses, but these experiments provide a foundation for this work. Chapter three highlights experiments using a GFP-tagged GnRH-R in αT3-1 gonadotropes in order to investigate GnRH binding-induced receptor mobility and clustering. Treatment groups were identical to the previous chapter. SRRF and binary analysis were used to characterize receptor activity. Descriptively, clustering of receptors was seen, especially when calcium activity was limited, but more appropriate methods of quantitative analysis should be explored in order to go beyond these observations in processed images. This thesis concludes overall that GnRH-induced calcium oscillation patterning and receptor clustering are far more complex and difficult to study than initially thought. Much more research is needed to determine any conclusive findings, however, these experiments may serve as a stepping stone toward obtaining the answers sought.
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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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    Reactive oxygen species regulate activity-dependent transport and delivery of AMPA receptors to synapses
    (Colorado State University. Libraries, 2022) Doser, Rachel, author; Hoerndli, Frederic, advisor; Amberg, Gregory, advisor; Di Pietro, Santiago, committee member; Vigh, Jozsef, committee member
    In neurons, changes in the subcellular localization of the AMPA subtype of ionotropic glutamate receptors (AMPARs) is necessary for learning and memory. AMPARs are primarily translated in the cell body and transported through dendrites to their destinations by molecular motors. This means that their localization, and therefore synaptic function and plasticity, requires proper intracellular transport to the synapse. Our lab and others have shown that AMPAR transport is regulated by activity-dependent calcium signaling. Neuronal activity and intracellular transport are energy demanding requiring high rates of ATP production from which reactive oxygen species (ROS), a class of chemically reactive molecules, are a normal byproduct. For my dissertation work, I build upon this knowledge by demonstrating a physiological role for ROS in the regulation of AMPAR transport in the transparent genetic model C. elegans. In Chapter 2, we show that slight increases in ROS decreased transport and synaptic delivery of AMPARs by attenuating calcium influx. Diminished ROS levels also decreased AMPAR transport but via a calcium-independent mechanism. This prompted us to assess how ROS signaling is initiated in vivo, which was the aim of experiments in Chapter 3. The results from these experiments demonstrated that calcium buffering and ROS production at mitochondria are positively regulated by activity. So, the rest of Chapter 3 was devoted to experiments addressing how AMPAR transport dynamics are impacted by localized ROS signaling originating at mitochondria. In the neurite, we found that localized increases in ROS from mitochondria regulate the stopping of AMPAR transport events by potentially altering local calcium influx. Further investigation on how local calcium influx, mitochondrial ROS production and AMPAR transport are interrelated in vivo necessitates optimization of optogenetic tools for high spatial and temporal control of cytoplasmic calcium levels. To this end, Chapter 4 overviews the optimization and characterization of a few optogenetic approaches for simultaneously manipulating and measuring neuronal activity in vivo. This work begins to detail how ROS signaling regulates synaptic function and plasticity which describes a novel mechanism in which metabolic rate indirectly regulates synaptic activity. Understanding this mechanism would provide insight into why altered glutamatergic synaptic transmission accompanies elevated calcium/ROS and mitochondrial dysfunction in neuronal aging and degeneration.