Browsing by Subject "Cytoskeletal Proteins"
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Item Analysis of the Interaction and Functional Determinants of Arc and Its Regulation of Dynamin(2011-02-01) Byers, Christopher Eugene; Albanesi, Joseph P.Recent evidence indicates that the activity-regulated cytoskeleton-associated protein, Arc, facilitates endocytosis of glutamate receptors and regulates cytoskeletal organization in neuronal dendrites. Both of these functions may be mediated by its reported interaction with dynamin 2, a large (~100 kDa) GTPase that participates in receptor-mediated endocytosis, actin polymerization, and microtubule stabilization. The effects of Arc on dynamin activities have not been characterized. Therefore, I have purified bacterially-expressed Arc and have shown that it enhances dynamin 2, and the neuronal and testes expressed dynamin 3, self-assembly in vitro and stimulates their GTPase activity. However, Arc interacts with dynamin 1, the neuronal isoform required for rapid pre-synaptic vesicle recycling, but does not stimulate its activity or assembly. The Arc-dynamin interaction is strongly dependent on ionic strength and the polymerization state of dynamin, which Arc enhances. Consequently, biophysical studies were used to confirm Arc is capable of forming higher order oligomers. Contrary to published findings, binding studies show Arc interacts with the proline-rich domain of dynamin and not the pleckstrin homology domain. All together, these results provide a mechanism to explain the previously reported role of Arc in glutamate receptor internalization.Item Development of Neocortical Circuits: a Cell Autonomous Examination of mGluR5 and MEF2C(2015-04-08) Loerwald, Kristofer William; Johnson, Jane E.; Meeks, Julian P.; Powell, Craig M.; Huber, Kimberly M.; Gibson, Jay R.Development of neocortical circuits requires both genetic programs and sensory experience-dependent modification of synaptic function. The rules that dictate how synapses develop and respond to changing patterns of input influence both the emergence of receptive fields and the capacity for learning. In turn, the factors that determine the rules for synaptic plasticity are defined by the proteins functioning at the synapse. This project investigates two proteins situated to have wide-reaching impacts on synaptic function. One of the challenges in detailing the roles a protein plays in regulating synapses is discerning not only its acute role on synaptic function, but also its long-term impact on circuit development. Therefore, studying how a protein is engaged by physiological patterns of input in vivo over an extended period of time will provide a broader picture of how it influences synaptic function and circuit development. mGluR5 has previously been implicated in several forms of plasticity that act to directly weaken synaptic function. In this document, I provide evidence that the net-effect of mGluR5 on synaptic function throughout the first few weeks of postnatal development is to promote synaptic input pathway strength, as demonstrated in 2 prominent and well-characterized input pathways to L2/3 pyramidal cells of barrel cortex. Furthermore, I demonstrate a possible role for mGluR5 in a homeostatic mechanism, offsetting the enhanced evoked synaptic input by suppressing both spontaneous transmission and intrinsic excitability. The transcription factor MEF2 also has established roles in regulating synaptic function. However, much less is known about the synaptic mechanisms through which MEF2 mediates its effects. Here, I implicate MEF2C as the critical MEF2 family member involved in regulating synaptic function in L2/3 pyramidal cells in barrel cortex, and provide potential synaptic and molecular mechanisms by which MEF2C regulates pathway input.Item Novel Roles for the Activity-Regulated Genes Arc and Npas4 in Stress- and Cocaine-Induced Plasticity(2016-04-18) Kumar, Jaswinder Singh; Huber, Kimberly M.; Cowan, Christopher W.; Olson, Eric N.; Zinn, Andrew R.Mood, anxiety, and substance abuse disorders are chronic medical illnesses that contribute significantly to morbidity and mortality worldwide. Currently, these conditions are treated symptomatically using pharmacological and psychotherapeutic approaches; however, the efficacy of these modalities is limited by the dearth of understanding of neurobiological mechanisms underlying mental illness. The high rate of mortality associated with mood, anxiety, and substance abuse disorders is compounded by their shared comorbidity, warranting an investigation into potential shared pathophysiological mechanisms. Studies from human patients and rodent models suggest that these mechanisms may be attributed to disrupted structural and functional plasticity in brain regions involved in mood, reward, and motivation, including the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC). However, the molecules and signaling pathways within these structures that regulate these behaviors, and how they are dysregulated in pathological psychiatric conditions, have yet to be fully identified and characterized. Here, we focus on two key proteins that participate in activity-dependent synaptic plasticity, the neuronal Per Arnt Sim Domain protein 4 (NPAS4) and the activity-regulated cytoskeleton-associated protein (Arc). We utilize a series of ethologically relevant behavioral paradigms to identify Arc and NPAS4 as two important mediators of stress, anxiety, and addiction-related behaviors. Npas4 and Arc, two activity-regulated genes, are robustly induced by stressful, anxiogenic stimuli. Loss of either gene confers an antidepressant and anxiolytic response in mice, and these behavioral phenotypes are mediated by local function of these two proteins in limbic forebrain regions. In a related study, we ask whether loss of Arc influences behavioral responses to cocaine administration. We find that Arc knockout (KO) animals exhibit increased sensitivity to the locomotor activating and rewarding effects of cocaine, and these two phenotypes are associated with a selective increase in synaptic strength in the NAc. Taken together, our results highlight a heretofore-unidentified role for Arc and NPAS4 in stress- and anxiety-like behaviors, as well as Arc in cocaine-related behavioral adaptations. We propose that these two molecules play a vital role in regulating synaptic and behavioral plasticity evoked by exposure to stress and drugs of abuse, likely via experience-dependent synaptic remodeling.Item Regulatory Mechanisms of Semaphorin/Plexin/Mical-Mediated F-actin Disassembly and Cellular Remodeling(2017-04-14) Rich, Shannon Kay Good; Johnson, Jane E.; Terman, Jonathan R.; Krämer, Helmut; Alto, NealDynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors - cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology.Item A Sensitive Assay for Monitoring Wild-Type and Mutant Myocilin Secretion(2016-01-19) Zadoo, Serena; Nguyen, Annie; Zode, Gulab; Hullerman, John D.Primary open angle glaucoma (POAG)-associated mutations in myocilin (MYOC) cause protein 'non-secretion', rendering secreted MYOC difficult to quantitatively detect using time-consuming conventional techniques. This study focused on developing an assay which could be used to quickly and easily detect mutant and wild-type (WT) MYOC secretion. We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T cells. The secretion, intracellular soluble and insoluble portions of WT and Y437H MYOC eGLuc2 constructs were evaluated by western blotting and compared to FLAG-tagged constructs. Secreted and soluble MYOC eGLuc2 was measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from HEK-293T and NTM-5 cells to test the general applicability of the assay. MYOC eGLuc2 behaved similarly to FLAG MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 min after a media change. eGLuc2 fused variants followed predicted trends; non-pathogenic variants (D208E, G244V) were secreted at WT-like levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects ranging from 0.20 - 4.0% of WT MYOC levels in HEK-293T cells. These variants were slightly more tolerated in NTM-5 cells, resulting in secretion levels ranging from 1.0 - 12% of WT MYOC levels. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperatures. Interestingly, a combination of growth temperature reduction and cycloheximide treatment of transfected cells indicate that the pool of protein that is rescued at lower temperatures arises from intracellular stores, and is not from newly synthesized MYOC under permissive temperature. Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. The use of the eGLuc2-tagged version of MYOC can be utilized to develop a deeper understanding of MYOC folding and secretion. This newly developed MYOC reporter system has the potential to be used in small molecule and/or genetic high-throughput screens to identify modulators of MYOC secretion.Item A Study on an FMRP-Mediated Translational Switch in the MGluR-Triggered Translation of Arc and Synaptic Plasticity(2012-07-16) Niere, Farr; Huber, Kimberly M.The group 1 metabotropic glutamate receptor (mGluR)-stimulated protein synthesis and long-term synaptic depression (mGluR-LTD) are altered in a mouse model of Fragile X Syndrome, Fmr1 knockout (KO) mouse. Fmr1 encodes the Fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that functions, in part, as a translational suppressor. It is unknown if and how FMRP acutely regulates LTD and/or the rapid synthesis of new proteins required for LTD, such as the activity-regulated cytoskeletal-associated protein (Arc). The protein phosphatase PP2A dephosphorylates FMRP, which contributes to the translational activation of some target mRNAs. Here, I report that PP2A and the dephosphorylation of FMRP at S500 are required for an mGluR-induced, rapid increase in dendritic Arc protein and LTD in rat and mouse hippocampal neurons. In the Fmr1 KO neurons, basal, dendritic Arc protein levels and mGluR-LTD are enhanced, and the mGluR-triggered Arc synthesis is absent. A lentiviral-mediated expression of the wildtype FMRP in Fmr1 KO neurons suppresses basal, dendritic Arc levels and mGluR-LTD, and restores the rapid mGluR-triggered Arc synthesis. A phosphomimic of FMRP (S500D) suppresses steady state dendritic Arc levels but does not rescue the mGluR-induced Arc synthesis. A dephosphomimic of FMRP (S500A) neither suppresses the basal, dendritic Arc levels nor supports the mGluR-induced Arc synthesis. Accordingly, expressing the S500D-FMRP in Fmr1 KO neurons suppresses mGluR-LTD, whereas the S500A-FMRP has no effect. These data support a model whereby a phosphorylated FMRP at S500 functions to suppress the steady state and the mGluR-induced translation of Arc and mGluR-LTD. However, upon mGluR activation of PP2A, FMRP is rapidly dephosphorylated which contributes to the rapid, new synthesis of Arc and mGluR-LTD.