Browsing by Subject "Nerve Tissue Proteins"
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Item Identification of a Novel ERK 1/2-Interacting A-Kinase Anchoring Protein(2009-06-17) Jivan, Arif; Cobb, MelanieInitially identified in Chlamydomonas, radial spoke protein 3 (RSP3) is one of at least twenty identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be identified and fully characterized. Mammalian RSP3 was found to interact with ERK2 through a yeast two-hybrid screen designed to identify interactors that have a higher affinity for the phosphorylated, active form of ERK2. Confirming this finding, the human homolog long form, RSP3H, co-immunoprecipitates with ERK1/2 in HEK293 cells. Human RSP3, and its larger alternative start site gene product, radial spoke protein 3 homolog (RSP3H), are phosphorylated by ERK1/2 on threonine 286 in vitro and in cells. RSP3/RSP3H are also phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Additionally, we showed that human RSP3H functions as an A-kinase anchoring protein (AKAP), and its ability to bind to the regulatory subunits of PKA, RII and RII, is regulated by ERK1/2 activity and phosphorylation. Interestingly, expression analysis of mRNA suggests RSP3/RSP3H are also present in cells that are thought to contain a single primary cilium but not motile cilia. Immunofluorescence staining of primary cilia-containing cells indicates that RSP3/RSP3H localize to nuclear punctae, specifically promyelocytic leukemia (PML) bodies, suggesting a non-cilia related role for RSP3/RSP3H in these cells. Functionally, RSP3/RSP3H may localize ERK1/2 to a distinct site of action within the cell and serve as a point of convergence of cAMP-dependent and PKA-mediated influence upon ERK1/2 downstream signaling or vice versa. These data are the first to establish a connection between ERK1/2 and what was once ostensibly thought to only be a ciliary component as well as to identify a novel ERK1/2-interacting AKAP.Item Regulation and Lineage Analysis of Neurog1 in the Developing Spinal Cord(2007-05-23) Quinones-Figueroa, Herson Isaac; Johnson, Jane E.The bHLH transcription factor Neurog1 is involved in neuronal differentiation and cell-type specification in distinct regions of the developing nervous system. I developed mouse models that efficiently drive expression of GFP or Cre recombinase in all Neurog1 (Ngn1, NeuroD3) domains. Deleting highly conserved sequences from a BAC containing 113kb 5' and 71kb 3' genomic sequence surrounding the Neurog1 coding region allowed the identification of enhancer elements required to drive Neurog1 expression. I show that a 3.8 kb fragment located 4.2 kb 5' of Neurog1 is required for efficient reporter expression in all Neurog1 domains. This sequence contains previously identified enhancer elements for midbrain, hindbrain and dorsal neural tube, and has two sequences conserved from human to fish. A 16kb fragment containing 8.9 kb 5' and 5.2 kb 3' of the Neurog1 coding sequence was not sufficient to drive expression in all domains. Reporter expression was observed in the dorsal neural tube, the midbrain, hindbrain and trigeminal ganglia, but was missing in the olfactory epithelium, dorsal root ganglia, dorsal telencephalon, and ventral neural tube. A 2.3 kb enhancer element located 8 kb 5' of the Neurog1 coding region was identified that is necessary to direct expression in the ventral neural tube. In addition, these mouse models allowed both short-term and long-term lineage analyses. I show that derivatives of Neurog1-expressing progenitor cells in the neural tube largely comprise the interneuron populations dI2, dI6, V0, V1, and V2, and to a lesser extent motorneurons. This is seen in the co-expression of GFP driven by Neurog1 regulatory sequences with the neuronal identity markers Brn3a, Islet1/2, Lhx1/5, Lhx3, Pax2, and Chx10. Genetic fate mapping in vivo using Cre recombinase reveals that although Neurog1-expressing cells primarily give rise to neurons, minor populations of oligodendrocytes and astrocytes are also identified in the lineage by adult stages in the spinal cord. Adding temporal control to the fate mapping strategy demonstrates that the neurons are generated from Neurog1-expressing cells prior to E13, and glial cells after E13, placing Neurog1 in lineage restricted precursor cells during embryogenesis.Item Role of Neuroligin in Synapse Formation and Autism(2006-08-11) Chubykin, Alexander Anatoly; Sudhof, Thomas C.Neuroligins mediate synaptogenesis through formation of a trans-synaptic complex with presynaptic neurexins. Interaction of neuroligin 1 with neurexins is regulated by alternative splicing of both neuroligin 1 (at splice site B) and of neurexins (at splice site #4). Full-length neuroligin 1 that binds only beta -neurexin more potently promotes synapse formation in hippocampal neurons, whereas neuroligin 1 lacking splice site B, which binds both alpha - and beta -neurexins, is more efficient at synapse expansion. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta and blocked synapse formation. Neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synaptogenic activity. In hippocampal neurons, overexpressed neuroligin 1 enhances excitatory but not inhibitory synaptic responses, and increases the ratio of NMDA to AMPA receptor-mediated synaptic currents. In contrast, genetic deletion of neuroligin 1 in mice decreases NMDA receptor-mediated synaptic currents and the NMDA/AMPA receptor ratio. Contrary to neuroligin 1, neuroligin 2 potentiates inhibitory but not excitatory synaptic responses. The synaptic actions of neuroligin 1 are suppressed by chronic blockade of NMDA receptors or of CaM-kinase II. Neuroligin 1 with an autistic-spectrum syndrome mutation decreases excitatory synaptic responses, consistent with a role for endogenous neuroligin 1 in synapse development. Taken together, our data suggest that neuroligin-neurexin interaction regulated by their alternative splicing promotes formation of specific synapses; synaptogenic function of neuroligin is regulated by NMDA receptor and Cam-kinase II activation, suggesting a critical role for neuroligins in synaptic plasticity and modulation of neural circuits.Item Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release(2007-05-22) Dai, Han; Rizo-Rey, JoseNeurotransmitter release is mediated by synaptic vesicle exocytosis at presynaptic nerve terminals. This process is extremely fast and strictly regulated by the intracellular Ca2+ concentration. To achieve this exquisite regulation, many proteins are involved. The central fusion machinery includes the SNARE proteins synaptobrevin, syntaxin and SNAP-25, as well as Munc18. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered release. Interestingly, most of these regulatory proteins contain C2 domains, versatile protein modules with Ca2+-dependent and Ca2+- independent activities. However, the mechanism of regulation by these C2-domain proteins remains unclear. My research has focused on understanding the structural and functional properties of two types of C2-domain proteins, synaptotagmins and RIMs. With NMR spectroscopy and biochemical assays, we demonstrated that synaptotagmin 4 is a Ca2+ sensor in Drosophila but not in rat, in contrast to the prediction based on sequence alignments. X-ray crystallography revealed that changes in the orientations of critical Ca2+-ligands render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. We also analyzed the structural and biochemical properties of the RIM2 C2A domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM2 C2A domain and Ca2+, phospholipid, synaptotagmin 1, and SNAP-25. However, the crystal structure of RIM C2A domain exhibits a strikingly dipolar distribution of the surface electrostatic potential. Several lines of evidence from the crystal structure suggested a potential target binding site around the bottom 310-helix. With fluorescence microscopy and microfluidic channel technology, we demonstrated that synaptotagmin 1 binds simultaneously to phospholipids and the SNARE complex reconstituted in membranes in the presence of Ca2+, forming a quaternary SSCAP complex, and that the membrane penetration of synaptotagmin 1 into phospholipids is independent of the reconstituted SNARE complex. We also showed that synaptotagmin 1 displaces complexin from the reconstituted SNARE complex in the presence of Ca2+, and that the synaptotagmin 1 C2B domain is primarily responsible for SNARE binding. Moreover, NMR spectroscopy and site-directed mutagenesis studies yielded structural information of the potential binding interface, allowing us to use computational modeling and docking to generate a preliminary model of the SSCAP complex.Item Structural and Functional Studies of Munc18 and SNARE Proteins(2011-08-26T17:35:42Z) Xu, Yi; Rizo-Rey, JoseRelease of neurotransmitters is a tightly regulated process and a key event in interneuron communication. Release involves a series of steps, including vesicles docking to the active zone of the plasma membrane, priming to a readily releasable state, and Ca2+-triggered membrane fusion. These steps are tightly controlled by an intricate protein machinery. Essential components of this machinery are proteins from the Sec1/Munc18 (SM) and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) families. SNAREs function by forming a four-helix bundle called SNARE complex; assembly of the SNARE complex brings two membranes together and is key for membrane fusion. The function of SM proteins is less clear. The neuronal SM proteins Munc18-1 was identified and linked to synaptic vesicle fusion due to its tight binding to syntaxin-1. The strict requirement of Munc18-1 for release is illustrated by the observation that, in mice, deletion of Munc18-1 abolished neurotransmitter release completely. Munc18-1 binds to the closed conformation of syntaxin-1 as well as to assembled SNARE complexes containing open syntaxin-1. Analysis of point mutations on Munc18-1 showed that binding of Munc18-1 to the Habc domain of open syntaxin-1 is critical for synaptic vesicle priming but not for the release step. The fact that Munc18-1 and complexin-1 could bind simultaneously to the SNARE complex suggested Munc18-1 remained bound to a macrocomplex that is poised for Ca2+ triggering of fusion. Analysis using diverse biophysical approaches revealed that Munc18-1 indeed binds to the C-terminus of the synaptobrevin SNARE motif and to the SNARE four-helix bundle. Both interactions have similar affinities and the N-terminal region of syntaxin-1 competes with the SNARE four-helix bundle and synaptobrevin for Munc18-1 binding, suggesting that the interaction between Munc18-1 and the SNARE four-helix bundle involves the same cavity of Munc18-1 that binds to syntaxin-1. To directly test Munc18-1’s role in fusion, I reconstituted v- and t-SNAREs into separate liposomes. Fusion between these proteoliposomes in the presence of Munc18-1 was monitored with a lipid mixing assay. Intriguingly, I found that enhanced lipid mixing caused by rat Munc18-1 alone was observed. Biochemistry studies showed that denaturation of rat Munc18-1 or squid Munc18-1 causes membrane lipid mixing in the absence of SNAREs proteins.Item Structural and Functional Studies of the Munc13 MUN Domain and the RIM C2B Domain(2007-12-17) Guan, Rong; Rizo-Rey, JoseNeurotransmitter release is essential for normal brain function and is achieved through exocytosis of synaptic vesicles. Many proteins are involved the regulation of neurotransmission. The central fusion machinery includes the SNARE proteins and Munc18- 1. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered neurotransmitter release, such as the key priming factors RIMs and Munc13s. Munc13s are essential for vesicle priming. RIMs form a protein scaffold in the presynaptic nerve terminal. My studies have focused on the structures and functions of the Munc13 MUN domain and the RIM C2B domain. I have studied the structure and function of the Munc13 MUN domain. On one hand, I have tried to determine the three dimensional structure of the Munc13 MUN domain by Xray crystallography. I have successfully obtained crystals of the Munc13-1 MUN domain, Munc13-3 MUN domain and a fragment containing the Munc13-1 C1, C2B and MUN domains. These crystals will be further optimized to enable structure determination. On the other hand, I have tried to identify the binding partners of the MUN domain using various methods. Cross-linking experiments revealed an interaction between the Munc13-1 MUN domain and endogenous Munc18-1. In addition, cofloatation assays revealed an interaction between MUN and reconstituted SNARE complex. Detailed analysis using cofloatation assays suggested both MUN and complexin can compete with Munc18-1 for SNARE complex binding in a membrane environment. Our studies also suggested that the membrane environment can modulate the strength of protein-protein interactions remarkably, which emphasize the importance to include membranes in the studies of protein-protein interactions involved in neurotransmission. I have also analyzed the structural and biochemical properties of the RIM1 C2B domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM1 C2B domain and Ca2+, phospholipids, or its putative binding partners, synaptotagmin 1 and liprins. X-ray crystallography revealed the existence of a RIM1 C2B domain homodimer, which was confirmed by analytical ultracentrifugation and NMR spectroscopy. Our results suggested a model that RIM1 C2B dimerization might facilitate the Munc13 C2A homodimer to Munc13 C2A/RIM zinc finger heterodimer switch during synaptic vesicle priming.