The Experts below are selected from a list of 24771 Experts worldwide ranked by ideXlab platform
Kristen J Verhey - One of the best experts on this subject based on the ideXlab platform.
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MAP7 regulates axon morphogenesis by recruiting Kinesin-1 to microtubules and modulating organelle transport.
eLife, 2018Co-Authors: Stephen R. Tymanskyj, Benjamin H Yang, Kristen J VerheyAbstract:Neuronal cell morphogenesis depends on proper regulation of microtubule-based transport, but the underlying mechanisms are not well understood. Here, we report our study of MAP7, a unique microtubule-associated protein that interacts with both microtubules and the motor protein Kinesin-1. Structure-function analysis in rat embryonic sensory neurons shows that the Kinesin-1 interacting domain in MAP7 is required for axon and branch growth but not for branch formation. Also, two unique microtubule binding sites are found in MAP7 that have distinct dissociation kinetics and are both required for branch formation. Furthermore, MAP7 recruits Kinesin-1 dynamically to microtubules, leading to alterations in organelle transport behaviors, particularly pause/speed switching. As MAP7 is localized to branch sites, our results suggest a novel mechanism mediated by the dual interactions of MAP7 with microtubules and Kinesin-1 in the precise control of microtubule-based transport during axon morphogenesis.
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autoinhibition of Kinesin 1 is essential to the dendrite specific localization of golgi outposts
Journal of Cell Biology, 2018Co-Authors: Michael T Kellihe, Daichi Kamiyama, O Huang, Kristen J Verhey, Yang Yue, Jill WildongeAbstract:Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor Kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how Kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. Our in vivo structure-function analysis of endogenous Drosophila melanogaster Kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt Kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates Kinesin-1 localization. Uninhibited Kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of Kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. Our data indicate that Kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.
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autoinhibition of Kinesin 1 is essential to the dendrite specific localization of golgi outposts
bioRxiv, 2018Co-Authors: Michael T Kellihe, Daichi Kamiyama, O Huang, Kristen J Verhey, Yang Yue, Jill WildongeAbstract:Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor Kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how Kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. Our in vivo structure-function analysis of endogenous Drosophila Kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt Kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates Kinesin-1 localization. Uninhibited Kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of Kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. Our data indicate that Kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.
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Effects of α-Tubulin K40 Acetylation and Detyrosination on Kinesin-1 Motility in a Purified System
Biophysical Journal, 2014Co-Authors: Neha Kaul, Virupakshi Soppina, Kristen J VerheyAbstract:Long-range transport in cells is achieved primarily through motor-based transport along a network of microtubule tracks. Targeted transport by Kinesin motors can be correlated with posttranslational modifications (PTMs) of the tubulin subunits in specific microtubules. To directly examine the influence of specific PTMs on Kinesin-1 motility, we generated tubulin subunits that were either enriched in or lacking acetylation of α-tubulin lysine 40 (K40) or detyrosination of the α-tubulin C-terminal tail. We show that K40 acetylation does not result in significant changes in Kinesin-1’s landing rate or motility parameters (velocity and run length) across experimental conditions. In contrast, detyrosination causes a moderate increase in Kinesin-1’s landing rate. The fact that the effects of detyrosination are dampened by prior K40 acetylation indicates that the combination of PTMs may be an important aspect of the functional output of microtubule heterogeneity. Importantly, our results indicate that the moderate influences that single PTMs have on Kinesin-1 in vitro do not explain the strong correlation between specific PTMs and Kinesin-1 transport in cells. Thus, additional mechanisms for regulating Kinesin-1 transport in cells must be explored in future work.
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Recycling of Kinesin-1 motors by diffusion after transport.
PLoS ONE, 2013Co-Authors: T. Lynne Blasius, Nathan A. Reed, Boris M. Slepchenko, Kristen J VerheyAbstract:Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of Kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of Kinesin-1 motors after completion of transport in neuronal cells. We find that Kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of Kinesin-1 motors fits a “loose bucket brigade” where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual Kinesin-1 motors are utilized for multiple rounds of transport.
Stefan Diez - One of the best experts on this subject based on the ideXlab platform.
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Mitochondria-adaptor TRAK1 promotes Kinesin-1 driven transport in crowded environments.
Nature Communications, 2020Co-Authors: Verena Henrichs, Stefan Diez, Lenka Grycova, Cyril Barinka, Zuzana Nahacka, Jiri Neuzil, Jakub Rohlena, Marcus Braun, Zdenek LanskyAbstract:Intracellular trafficking of organelles, driven by Kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, Kinesin-1 performs micron-long runs. Under crowding conditions, however, Kinesin-1 motility is drastically impeded. It is thus unclear how Kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates Kinesin-1 and increases robustness of Kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates Kinesin-1 navigation around obstacles, ii) increases the probability of Kinesin-1 passing through cohesive islands of tau and iii) increases the run length of Kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the Kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating Kinesin-1 motility in various cellular environments. Intracellular trafficking of organelles is driven by Kinesin-1 stepping along microtubules, but crowding conditions impede Kinesin-1 motility. Here authors demonstrate that TRAK1, an adaptor protein essential for mitochondrial trafficking, activates Kinesin-1 and increases robustness of Kinesin-1 stepping on crowded microtubule surfaces.
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Mitochondria-adaptor TRAK1 promotes Kinesin-1 driven transport in crowded environments.
Nature communications, 2020Co-Authors: Verena Henrichs, Stefan Diez, Lenka Grycova, Cyril Barinka, Zuzana Nahacka, Jiri Neuzil, Jakub Rohlena, Marcus Braun, Zdenek LanskyAbstract:Intracellular trafficking of organelles, driven by Kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, Kinesin-1 performs micron-long runs. Under crowding conditions, however, Kinesin-1 motility is drastically impeded. It is thus unclear how Kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates Kinesin-1 and increases robustness of Kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates Kinesin-1 navigation around obstacles, ii) increases the probability of Kinesin-1 passing through cohesive islands of tau and iii) increases the run length of Kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the Kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating Kinesin-1 motility in various cellular environments.
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Mitochondria-adaptor TRAK1 promotes Kinesin-1 driven transport in crowded environments
2020Co-Authors: Verena Henrichs, Stefan Diez, Lenka Grycova, Cyril Barinka, Zuzana Nahacka, Jiri Neuzil, Jakub Rohlena, Marcus Braun, Zdenek LanskyAbstract:Intracellular trafficking of organelles, driven by Kinesin-1 stepping along microtubules, underpins essential processes including neuronal activity. In absence of other proteins on the microtubule surface, Kinesin-1 performs micron-long runs. Under protein crowding conditions, however, Kinesin-1 motility is drastically impeded. It is thus unclear how Kinesin-1 acts as an efficient transporter in crowded intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates Kinesin-1 and increases its robustness of stepping in protein crowding conditions. Interaction with TRAK1 i) facilitated Kinesin-1 navigation around obstacles, ii) increased the probability of Kinesin-1 passing through cohesive envelopes of tau and iii) increased the run length of Kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the Kinesin-1-TRAK1 complex. We propose adaptor-mediated tethering as a mechanism regulating Kinesin-1 motility in various cellular environments.
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Transport by Kinesin-1 Motors Diffusing on a Lipid Bilayer
Biophysical Journal, 2016Co-Authors: Rahul Grover, Janine Fischer, Petra Schwille, Stefan DiezAbstract:In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors such as Kinesin-1 have recently been well characterized in vitro as single molecules or as ensembles, rigidly attached to surfaces of non-biological substrates. However, the collective transport by membrane-anchored motors, i.e. motors attached to a fluid lipid membrane, is poorly understood. Here, we investigate the influence of Kinesin-1 motors’ anchorage to a diffusive lipid bilayer on its collective transport characteristics. Towards this end we reconstituted ‘membrane-anchored’ gliding motility assays using truncated Kinesin-1 motors with a streptavidin-binding-peptide (SBP) tag that can attach to streptavidin-loaded biotinylated supported lipid bilayers (SLBs). The diffusing Kinesin-1 motors were found to propel microtubules upon ATP addition. Notably, we found the transport velocity of the microtubules to be dependent on the number of motors as well as on their diffusivity in the lipid bilayer. Microtubule velocity increased with increasing motor density reaching up to the single-motor stepping velocity, but decreased with increasing motor diffusivity. We reason, that the transport efficiency of motors linked to a diffusive SLB is reduced because the membrane-anchored motors slip backwards in the lipid membrane while stepping on a microtubule. This effect, which we directly observed using single-molecule fluorescence microscopy, results in a decreased transport velocity. Our results illustrate the importance of the motor-cargo coupling which potentially provides cells with an additional means of regulating the efficiency of cargo transport. Moreover, our ‘membrane-anchored’ gliding motility assays can be used to study the effects of lipid diffusivity (e.g. the presence of lipid micro-domains and rafts), lipid composition, and adaptor proteins on the collective dynamics of different motors.
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Small Crowders Slow Down Kinesin-1 Stepping by Hindering Motor Domain Diffusion.
Physical Review Letters, 2015Co-Authors: Krzysztof Sozanski, Felix Ruhnow, Stefan Diez, Agnieszka Wiśniewska, Marcin Tabaka, Robert HołystAbstract:The dimeric motor protein Kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of Kinesin-1: small crowders stop the motor at a viscosity of 5 mPa·s-corresponding to a hydrodynamic load in the sub-fN (~10^{-4} pN) range-whereas large crowders have no impact even at viscosities above 100 mPa·s. This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor determining Kinesin's functionality. Our results emphasize the role of diffusion in the Kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.
Christopher L Berger - One of the best experts on this subject based on the ideXlab platform.
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Phosphoregulation of Tau modulates inhibition of Kinesin-1 motility.
Molecular Biology of the Cell, 2017Co-Authors: Jamie Stern, Gregory J Hoeprich, Gerardo Morfini, Dominique V. Lessard, Christopher L BergerAbstract:Regulation of axonal transport includes control of the microtubule-associated protein Tau. Site-specific pseudophosphorylation of Tau modulates its ability to inhibit Kinesin-1 motility by both shi...
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Phosphoregulation of Tau modulates inhibition of Kinesin-1 motility.
Molecular biology of the cell, 2017Co-Authors: Jamie Stern, Gregory J Hoeprich, Gerardo Morfini, Dominique V. Lessard, Christopher L BergerAbstract:Microtubule-based axonal transport is tightly regulated by numerous pathways, ensuring appropriate delivery of specific organelle cargoes to selected subcellular domains. Highlighting the importance of this process, pathological evidence has linked alterations in these pathways to the pathogenesis of several neurodegenerative diseases. An important regulator of this system, the microtubule-associated protein Tau, has been shown to participate in signaling cascades, modulate microtubule dynamics, and preferentially inhibit Kinesin-1 motility. However, the cellular means of regulating Tau's inhibition of Kinesin-1 motility remains unknown. Tau is subject to various posttranslational modifications, including phosphorylation, but whether phosphorylation regulates Tau on the microtubule surface has not been addressed. It has been shown that tyrosine 18 phosphorylated Tau regulates inhibition of axonal transport in the disease state. Tyrosine 18 is both a disease- and nondisease-state modification and is therefore an attractive starting point for understanding control of Tau's inhibition of Kinesin-1 motility. We show that pseudophosphorylation of tyrosine 18 reduces 3RS-Tau's inhibition of Kinesin-1 motility. In addition, we show that introduction of negative charge at tyrosine 18 shifts Tau's previously described static-dynamic state binding equilibrium toward the dynamic state. We also present the first evidence of Tau's static-dynamic state equilibrium under physiological conditions.
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Kinesin 2 s neck linker is critical to navigating obstacles on the microtubule surface more efficiently than Kinesin 1
Biophysical Journal, 2014Co-Authors: Christopher L Berger, Gregory J Hoeprich, Andrew R Thompson, William O. HancockAbstract:A number of cargo during intracellular transport are known to be bound to both Kinesin-1 and Kinesin-2, but the advantage of having two similarly plus-end directed motors on a single cargo is not clear. Kinesin-1 is known to be sensitive to alterations in the microtubule track, including those arising from post-translational modifications, changes in nucleotide state, and the presence of microtubule associated proteins (MAPs) such as Tau. Less is known about effects of microtubule lattice modifications on Kinesin-2 motility. Kinesin-2, which contains three additional amino acids in its neck-linker compared with Kinesin-1, has reduced stepping coordination between motor domains, which decreases its processivity on paclitaxel-stabilized microtubules. We hypothesize these differences in Kinesin-2's structure and function allows it to more easily navigate obstacles on the microtubule surface, such as Tau, compared to Kinesin-1. To directly test this hypothesis, we used single molecule imaging with TIRF microscopy to measure motility from different Kinesin-1 and Kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two isoforms of Tau known to differentially affect Kinesin-1 motility. Our results demonstrate that Kinesin-2, unlike Kinesin-1, is insensitive in the presence of either Tau isoform on paclitaxel-stabilized microtubules. Swapping the neck-linkers between Kinesin-1 and Kinesin-2 resulted in a switch in the sensitivity to Tau between the two motors: the Kinesin-1 construct containing a Kinesin-2 neck-linker became insensitive to Tau, while the Kinesin-2 construct containing a Kinesin-1 neck-linker became sensitized to the presence of Tau. Thus, while Kinesin-2 is less processive than Kinesin-1, it is better optimized through its longer neck-linker to navigate obstacles on the microtubule surface, such as Tau, allowing the two motors to work together for the efficient delivery of cargo in the complex intracellular environment.
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Kinesin 2 navigates microtubule obstacles more efficiently than Kinesin 1
Biophysical Journal, 2013Co-Authors: Gregory J Hoeprich, William O. Hancock, Tony Jiang, Christopher L BergerAbstract:During axonal transport, an ensemble of molecular motors, including Kinesin-1 and Kinesin-2, navigate a complex microtubule landscape to deliver cargo to their target destinations within the cell. It has previously been shown in vitro that the neuronal microtubule associated proteins, 3RS-tau and 4RL-tau, reduce Kinesin-1 processivity on taxol-stabilized GDP microtubules, but not on microtubules stabilized with GMPCPP (a slowly hydrolyzable GTP analog ). Furthermore, Kinesin-1 processivity is also reduced on GMPCPP microtubules relative to taxol-stabilized microtubules, suggesting the microtubule lattice modulates interactions with both Kinesin-1 and tau (McVicker et al., (2011) J Biol Chem 286:42873). However, the effects of tau and the microtubule lattice structure on Kinesin-2 processivity are still unknown. Kinesin-2 is known to have a longer neck-linker than Kinesin-1, resulting in reduced coordination between motor domains and decreased processivity on taxol-stabilized microtubules (Shastry et al., (2010) Curr Biol 20:939). We hypothesize that these differences in Kinesin-2 function make it less sensitive to alterations in the microtubule lattice than Kinesin-1, and allow it to more easily navigate obstacles, such as tau, on the microtubule surface. To directly test this hypothesis, we used single molecule imaging with TIRF microscopy to measure Kinesin-2 motility as it stepped along microtubules in different nucleotide states (GDP or GMPCPP) in the absence or presence of 3RS-tau and 4RL-tau. Our results demonstrate that, in contrast to Kinesin-1, Kinesin-2 processivity is unchanged on taxol-stabilized vs. GMPCPP microtubules and is insensitive to the presence of either 3RS-tau or 4RL-tau. Thus, while Kinesin-2 is less processive than Kinesin-1, it may be better optimized to navigate around obstacles on different microtubule lattice structures, allowing the two motors to work together for the efficient delivery of cargo in the complex environment of the neuronal axon.
Michael Way - One of the best experts on this subject based on the ideXlab platform.
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Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3.
Structure, 2018Co-Authors: Joseph J.b. Cockburn, Sophie J. Hesketh, Peter Mulhair, Maren Thomsen, Mary J. O'connell, Michael WayAbstract:Summary Kinesin-1 transports numerous cellular cargoes along microtubules. The Kinesin-1 light chain (KLC) mediates cargo binding and regulates Kinesin-1 motility. To investigate the molecular basis for Kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a Kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating Kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate Kinesin-1 through related molecular mechanisms.
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A Kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome
The EMBO Journal, 2011Co-Authors: Mark P. Dodding, Richard Mitter, Ashley C. Humphries, Michael WayAbstract:Transport of cargoes by Kinesin‐1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit Kinesin‐1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define Kinesin‐1 binding. We now show that a bipartite tryptophan‐based Kinesin‐1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for Kinesin‐1‐dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan‐based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the Kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases.
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Coupling viruses to dynein and Kinesin-1
The EMBO Journal, 2011Co-Authors: Mark P. Dodding, Michael WayAbstract:It is now clear that transport on microtubules by dynein and Kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and Kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and Kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and Kinesin-1 motors are coordinated and regulated during bi-directional transport.
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Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection.
Cell Host & Microbe, 2011Co-Authors: Sten Strunze, Michael Way, Martin F. Engelke, I-hsuan Wang, Daniel Puntener, Karin Boucke, Sibylle Schleich, Philipp Schoenenberger, Christoph J. Burckhardt, Urs F. GreberAbstract:Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor Kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and Kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the Kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the Kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic dextran polymers. Thus, Kinesin-1 uncoats viral DNA and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.
Mark P. Dodding - One of the best experts on this subject based on the ideXlab platform.
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A small-molecule activator of Kinesin-1 drives remodeling of the microtubule network
Proceedings of the National Academy of Sciences, 2017Co-Authors: Thomas S. Randall, Anneri Sanger, Roberto A. Steiner, Yan Y Yip, Daynea J. Wallock-richards, Karin Pfisterer, Weronika Ficek, Andrew J. Beavil, Maddy Parsons, Mark P. DoddingAbstract:The microtubule motor Kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, Kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between Kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits Kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates Kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics.
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The Dynamic Localization of Cytoplasmic Dynein in Neurons Is Driven by Kinesin-1
Neuron, 2016Co-Authors: Alison E. Twelvetrees, Stefano Pernigo, Anneri Sanger, Pedro Guedes-dias, Mark P. Dodding, Roberto A. Steiner, Giampietro Schiavo, Erika L F HolzbaurAbstract:Cytoplasmic dynein, the major motor driving retrograde axonal transport, must be actively localized to axon terminals. This localization is critical as dynein powers essential retrograde trafficking events required for neuronal survival, such as neurotrophic signaling. Here, we demonstrate that the outward transport of dynein from soma to axon terminal is driven by direct interactions with the anterograde motor Kinesin-1. In developing neurons, we find that dynein dynamically cycles between neurites, following Kinesin-1 and accumulating in the nascent axon coincident with axon specification. In established axons, dynein is constantly transported down the axon at slow axonal transport speeds; inhibition of the Kinesin-1-dynein interaction effectively blocks this process. In vitro and live-imaging assays to investigate the underlying mechanism lead us to propose a new model for the slow axonal transport of cytosolic cargos, based on short-lived direct interactions of cargo with a highly processive anterograde motor.
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Structural basis for Kinesin-1:cargo recognition
Science, 2013Co-Authors: Stefano Pernigo, Anneri Lamprecht, Roberto A. Steiner, Mark P. DoddingAbstract:Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how Kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here, we present the crystal structure of the tetratricopeptide repeat domain of Kinesin light chain 2 in complex with a cargo peptide harboring a "tryptophan-acidic" motif derived from SKIP (SifA-Kinesin interacting protein), a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical, and cellular assays allow us to propose a framework for intracellular transport based on the binding by Kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for Kinesin-1:cargo recognition.
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A Kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome
The EMBO Journal, 2011Co-Authors: Mark P. Dodding, Richard Mitter, Ashley C. Humphries, Michael WayAbstract:Transport of cargoes by Kinesin‐1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit Kinesin‐1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define Kinesin‐1 binding. We now show that a bipartite tryptophan‐based Kinesin‐1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for Kinesin‐1‐dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan‐based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the Kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases.
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Coupling viruses to dynein and Kinesin-1
The EMBO Journal, 2011Co-Authors: Mark P. Dodding, Michael WayAbstract:It is now clear that transport on microtubules by dynein and Kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and Kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and Kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and Kinesin-1 motors are coordinated and regulated during bi-directional transport.
