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William M. Saxton - One of the best experts on this subject based on the ideXlab platform.
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identification of an axonal kinesin 3 motor for fast Anterograde vesicle transport that facilitates retrograde transport of neuropeptides
Molecular Biology of the Cell, 2008Co-Authors: Rosemarie V Barkus, Olga Klyachko, Dai Horiuchi, Barry J Dickson, William M. SaxtonAbstract:A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic Anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the Anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to Anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to Anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an Anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.
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cytoplasmic dynein the dynactin complex and kinesin are interdependent and essential for fast axonal transport
Molecular Biology of the Cell, 1999Co-Authors: Mary Ann Martin, Stanley J P Iyadurai, Andrew Gassman, Joseph G Gindhart, Thomas S Hays, William M. SaxtonAbstract:In axons, organelles move away from (Anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for Anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and Anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150 Glued (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and Anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150 Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.
Kwo Chang Ueng - One of the best experts on this subject based on the ideXlab platform.
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multiple Anterograde atrioventricular node pathways in patients with atrioventricular node reentrant tachycardia
Journal of the American College of Cardiology, 1996Co-Authors: Ching Tai Tai, Shih Huang Lee, Shihann Chen, Chern En Chiang, Chuen Wang Chiou, Kwo Chang UengAbstract:Abstract Objectives. This study sought to investigate electrophysiologic characteristics and possible anatomic sites of multiple Anterograde slow atrioventricular (AV) node pathways and to compare these findings with those in dual Anterograde AV node pathways. Background. Although multiple Anterograde AV node pathways have been demonstrated by the presence of multiple discontinuities in the AV node conduction curve, the role of these pathways in the initiation and maintenance of AV node reentrant tachycardia (AVNRT) is still unclear, and possible anatomic sites of these pathways have not been reported. Methods. This study included 500 consecutive patients with AVNRT who underwent electrophysiologic study and radiofrequency ablation. Twenty-six patients (5.2%) with triple or more Anterograde AV node pathways were designated as Group I (16 female, 10 male, mean age 48 ± 14 years), and the other 474 patients (including 451 with and 23 without dual Anterograde AV node pathways) were designated as Group II (257 female, 217 male; mean age 52 ± 16 years). Results. Of the 21 patients with triple Anterograde AV node pathways, AVNRT was initiated through the first slow pathway only in 3, through the second slow pathway only in 8 and through the two slow pathways in 9. Of the five patients with quadruple Anterograde AV node pathways, AVNRT was initiated through all three Anterograde slow pathways in three and through the two slower pathways (the second and third slow pathways) in two. After radiofrequency catheter ablation, no patient had inducible AVNRT. Eleven patients (42.3%) in Group I had multiple Anterograde slow pathways eliminated simulataneously at a single ablation site. Eight patients (30.7%) had these slow pathways eliminated at different ablation sites; the slow pathways with a longer conduction time were ablated more posteriorly in the Koch9s triangle than those with a shorter conduction time. The remaining seven patients (27%) had a residual slow pathway after delivery of radiofrequency energy at a single or different ablation sites. The patients in Group I had a longer tachycardia cycle length, poorer retrograde conduction properties and a higher incidence of multiple types of AVNRT than those in Group II. Conclusions. Multiple Anterograde AV node pathways are not rare in patients with AVNRT. However, not all of the Anterograde slow pathways were involved in the initiation and maintenance of tachycardia. Radiofrequency catheter ablation was safe and effective in eliminating critical slow pathways to cure AVNRT.
Harvey M. Friedman - One of the best experts on this subject based on the ideXlab platform.
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Molecular association of herpes simplex virus type 1 glycoprotein E with membrane protein Us9
Archives of Virology, 2016Co-Authors: Sita Awasthi, Harvey M. FriedmanAbstract:Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE), glycoprotein I (gI), and Us9 promote efficient Anterograde axonal transport of virus from the neuron cytoplasm to the axon terminus. HSV-1 and PRV gE and gI form a heterodimer that is required for Anterograde transport, but an association that includes Us9 has not been demonstrated. NS-gE380 is an HSV-1 mutant that has five amino acids inserted after gE residue 380, rendering it defective in Anterograde axonal transport. We demonstrated that gE, gI and Us9 form a trimolecular complex in Vero cells infected with NS-gE380 virus in which gE binds to both Us9 and gI. We detected the complex using immunoprecipitation with anti-gE or anti-gI monoclonal antibodies in the presence of ionic detergents. Under these conditions, Us9 did not associate with gE in cells infected with wild-type HSV-1; however, using a nonionic detergent, TritonX-100, an association between Us9 and gE was detected in immunoprecipitates of both wild-type and NS-gE380-infected cells. The results suggest that the interaction between Us9 and gE is weak and disrupted by ionic detergents in wild-type infected cells. We postulate that the tight interaction between Us9 and gE leads to the Anterograde spread defect in the NS-gE380 virus.
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Anterograde spread of herpes simplex virus type 1 requires glycoprotein e and glycoprotein i but not us9
Journal of Virology, 2009Co-Authors: Helen M Mcgraw, Sita Awasthi, Jason A Wojcechowskyj, Harvey M. FriedmanAbstract:Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus Anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) Anterograde spread using a compartmentalized primary neuron culture system. We found that no Anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 Anterograde spread. However, we did detect Anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, Anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no Anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 Anterograde spread, while Us9 is dispensable.
Lynn W Enquist - One of the best experts on this subject based on the ideXlab platform.
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Visualization of an alphaherpesvirus membrane protein that is essential for Anterograde axonal spread of infection in neurons. MBio 2012, 3. [CrossRef] [PubMed
2016Co-Authors: Matthew P. Taylor, Tal Kramer, Radomir Kratchmarov, M. G. Lyman, Lynn W EnquistAbstract:ABSTRACT Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synapti-cally connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for Anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assem-bled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the Anterograde direction within axons. Additionally, the vast majority of Anterograde-directed cap-sids (labeled with VP26-monomeric red fluorescent protein) and a viral membrane protein (labeled with glycoproteinM fused to mCherry) are cotransported with GFP-Us9 in the Anterograde direction. In contrast, during infection with PRV strains that ex-press nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. These find-ings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for Anterograde spread of infection in neurons. IMPORTANCE Alphaherpesviruses, such as pseudorabies virus (PRV), are parasites of the mammalian nervous system. These vi-ruses spread over long distances in chains of synaptically connected neurons. PRV encodes several proteins that mediate directed virion transport and spread of infection. Us9 is a highly conserved viral membrane protein that is essential for Anterograde neu
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the neuroinvasive profiles of h129 herpes simplex virus type 1 recombinants with putative Anterograde only transneuronal spread properties
Brain Structure & Function, 2015Co-Authors: Greg J Wojaczynski, Lynn W Enquist, Esteban A Engel, Karina E Steren, Patrick J CardAbstract:The use of viruses as transneuronal tracers has become an increasingly powerful technique for defining the synaptic organization of neural networks. Although a number of recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits only one strain, the H129 strain of herpes simplex virus type 1, is reported to selectively spread in the Anterograde direction. However, it is unclear from the literature whether there is an absolute block or an attenuation of retrograde spread of H129. Here, we demonstrate efficient Anterograde spread, and temporally delayed retrograde spread, of H129 and three novel recombinants. In vitro studies revealed no differences in Anterograde and retrograde spread of parental H129 and its recombinants through superior cervical ganglion neurons. In vivo injections of rat striatum revealed a clear bias of Anterograde spread, although evidence of deficient retrograde transport was also present. Evidence of temporally delayed retrograde transneuronal spread of H129 in the retina was observed following injection of the lateral geniculate nucleus. The data also demonstrated that three novel recombinants efficiently express unique fluorescent reporters and have the capacity to infect the same neurons in dual infection paradigms. From these experiments we conclude that H129 and its recombinants not only efficiently infect neurons through Anterograde transneuronal passage, but also are capable of temporally delayed retrograde transneuronal spread. In addition, the capacity to produce dual infection of projection targets following Anterograde transneuronal passage provides an important addition to viral transneuronal tracing technology.
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Glycoproteins gE and gI are required for efficient KIF1A-dependent Anterograde axonal transport of alphaherpesvirus particles in neurons
Journal of virology, 2013Co-Authors: Radomir Kratchmarov, Tal Kramer, Todd M. Greco, Matthew P. Taylor, Ileana M. Cristea, Lynn W EnquistAbstract:Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient Anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates Anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient Anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated Anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A.
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visualization of an alphaherpesvirus membrane protein that is essential for Anterograde axonal spread of infection in neurons
Mbio, 2012Co-Authors: Matthew P. Taylor, Tal Kramer, Radomir Kratchmarov, M. G. Lyman, Lynn W EnquistAbstract:Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synapti- cally connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for Anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assem- bled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional greenfluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the Anterograde direction within axons. Additionally, the vast majority of Anterograde-directed cap- sids (labeled with VP26-monomeric redfluorescent protein) and a viral membrane protein (labeled with glycoprotein M fused to mCherry) are cotransported with GFP-Us9 in the Anterograde direction. In contrast, during infection with PRV strains that ex- press nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. Thesefind- ings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for Anterograde spread of infection in neurons. IMPORTANCE Alphaherpesviruses, such as pseudorabies virus (PRV), are parasites of the mammalian nervous system. These vi- ruses spread over long distances in chains of synaptically connected neurons. PRV encodes several proteins that mediate directed virion transport and spread of infection. Us9 is a highly conserved viral membrane protein that is essential for Anterograde neu- ronal spread of infection. In the absence of Us9, newly replicated viral particles are assembled in the cell body but are not sorted into or transported within axons. Here, we constructed and characterized novel PRV strains that express functional greenfluo- rescent protein (GFP)-Us9 fusion proteins in order to visualize its localization in living neurons during infection. This enabled us to better understand the function of Us9 in facilitating the spread of infection. We show that all viral particles moving in the Anterograde direction are labeled with GFP-Us9, suggesting that the presence of Us9 determines the capacity for directed trans- port within axons.
Connie Cepko - One of the best experts on this subject based on the ideXlab platform.
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vesicular stomatitis virus with the rabies virus glycoprotein directs retrograde transsynaptic transport among neurons in vivo
Frontiers in Neural Circuits, 2013Co-Authors: Kevin T Beier, Arpiar Saunders, Ian A Oldenburg, Bernardo L Sabatini, Connie CepkoAbstract:Defining the connections among neurons is critical to our understanding of the structure and function of the nervous system. Recombinant viruses engineered to transmit across synapses provide a powerful approach for the dissection of neuronal circuitry in vivo. We recently demonstrated that recombinant vesicular stomatitis virus (VSV) can be endowed with Anterograde or retrograde synaptic tracing ability by providing the virus with different glycoproteins. Here we extend the characterization of the transmission and gene expression of VSV with the rabies virus glycoprotein (RABV-G), and provide examples of its activity relative to the Anterograde tracer form of rVSV. rVSV with RABV-G was found to drive strong expression of transgenes and to spread rapidly from neuron to neuron in only a retrograde manner. Depending upon how the RABV-G was delivered, VSV served as a polysynaptic or monosynaptic tracer, or was able to define projections through axonal uptake and retrograde transport. In animals co-infected with rVSV in its Anterograde form, rVSV with RABV-G could be used to begin to characterize the similarities and differences in connections to a given area. rVSV with RABV-G provides a flexible, rapid, and versatile tracing tool that complements the previously described VSV-based Anterograde transsynaptic tracer.
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Anterograde or retrograde transsynaptic labeling of cns neurons with vesicular stomatitis virus vectors
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Kevin T Beier, Arpiar Saunders, Ian A Oldenburg, Bernardo L Sabatini, Kazunari Miyamichi, Nazia Akhtar, Liqun Luo, Sean P J Whelan, Connie CepkoAbstract:To understand how the nervous system processes information, a map of the connections among neurons would be of great benefit. Here we describe the use of vesicular stomatitis virus (VSV) for tracing neuronal connections in vivo. We made VSV vectors that used glycoprotein (G) genes from several other viruses. The G protein from lymphocytic choriomeningitis virus endowed VSV with the ability to spread transsynaptically, specifically in an Anterograde direction, whereas the rabies virus glycoprotein gave a specifically retrograde transsynaptic pattern. The use of an avian G protein fusion allowed specific targeting of cells expressing an avian receptor, which allowed a demonstration of monosynaptic Anterograde tracing from defined cells. Synaptic connectivity of pairs of virally labeled cells was demonstrated by using slice cultures and electrophysiology. In vivo infections of several areas in the mouse brain led to the predicted patterns of spread for Anterograde or retrograde tracers.