The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Richard B Vallee - One of the best experts on this subject based on the ideXlab platform.
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Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment
2016Co-Authors: Julian Scherer, Richard B ValleeAbstract:Virus capsids provide genome protection from environmental challenges but are also poised to execute a program of composi-tional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus serotype 5 (AdV5) capsid protein, hexon, directly recruits the motor protein Cytoplasmic Dynein following virion entry. Dynein recruitment is cru-cial for capsid transport to the nucleus and requires the transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH but undergoes more subtle conformational changes. These changes are indicated by increased sensitivities to SDS-mediated dissociation and dispase proteolysis. Both effects are reversed at neutral pH, as is Dynein binding by low-pH-treated hexon. Dis-pase cleavage, which we findmaps to a specific site within hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry but completely inhibits its transport to the nucleus. In addition, an AdV5mutant containing HVR1 of AdV48 is unable to bind Dynein and is strongly inhibited in the postentry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus. IMPORTANCE The adenovirus serotype 5 (AdV5) capsid protein hexon recruits the molecular motor protein Cytoplasmic Dynein in a pH-de
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control of Cytoplasmic Dynein force production and processivity by its c terminal domain
Nature Communications, 2015Co-Authors: Matthew P Nicholas, Richard B Vallee, Peter Hook, Sibylle Brenner, Caitlin L Wynne, Arne GennerichAbstract:Cytoplasmic Dynein from the yeast S. cerevisiae behaves distinctly from mammalian Dyneins, despite structural conservation. Here, Nicholas et al. identify a C-terminal domain in mammalian Dynein that restricts force generation and travel distance, which, when removed, allows mammalian Dynein to behave like its yeast counterpart.
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Multiple modes of Cytoplasmic Dynein regulation
Nature Cell Biology, 2012Co-Authors: Richard B Vallee, Richard J. Mckenney, Kassandra M. Ori-mckenneyAbstract:In performing its multiple cellular functions, the Cytoplasmic Dynein motor is subject to complex regulation involving allosteric mechanisms within the Dynein complex, as well as numerous extramolecular interactions controlling subcellular targeting and motor activity. Recent work has distinguished high- and low-load regulatory modes for Cytoplasmic Dynein, which, combined with a diversity of targeting mechanisms, accounts for a very broad range of functions.
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A Cytoplasmic Dynein tail mutation impairs motor processivity
Nature cell biology, 2010Co-Authors: Kassandra M. Ori-mckenney, Steven P. Gross, Richard B ValleeAbstract:Mutations in the tail of the Cytoplasmic Dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at odd angles (Loa) has impaired retrograde axonal transport, but the molecular deficiencies in the mutant Dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified Dynein from wild-type mice and the Legs at odd angles mutant mice. Using biochemical, single-molecule, and live-cell-imaging techniques, we find a marked inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the Dynein from mutant mice. These results suggest a potential role for the Dynein tail in motor function, and provide direct evidence for a link between single-motor processivity and disease.
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adenovirus transport via direct interaction of Cytoplasmic Dynein with the viral capsid hexon subunit
Cell Host & Microbe, 2009Co-Authors: Helen K Bremner, Julian Scherer, Steven P. Gross, Michael Vershinin, Richard B ValleeAbstract:Early in infection, adenovirus travels to the nucleus as a naked capsid using the microtubule motor Cytoplasmic Dynein. How the Dynein complex is recruited to viral cargo remains unclear. We find that Cytoplasmic Dynein and its associated proteins dynactin and NudE/NudEL, but not LIS1 or ZW10, colocalized with incoming, postendosomal adenovirus particles. However, in contrast to physiological cargos, Dynein binding to adenovirus was independent of these Dynein-associated proteins. Dynein itself directly interacted through its intermediate and light intermediate chains with the adenovirus capsid subunit hexon in a pH-dependent manner. Expression of hexon or injection of anti-hexon antibody inhibited virus transport but not physiological Dynein function. These results identify hexon as a direct receptor for Cytoplasmic Dynein and demonstrate that hexon recruits Dynein for transport to the nucleus by a mechanism distinct from that for physiological Dynein cargo.
Erika L F Holzbaur - One of the best experts on this subject based on the ideXlab platform.
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mutant superoxide dismutase disrupts Cytoplasmic Dynein in motor neurons
Neuroreport, 2005Co-Authors: Lee A Ligon, Bernadette H Lamonte, Karen Wallace, Nicholas Weber, Robert G Kalb, Erika L F HolzbaurAbstract:Cytoplasmic Dynein and dynactin drive retrograde axonal transport in neurons, and mutations in Dynein/dynactin cause motor neuron degeneration. To test whether defects in Dynein/dynactin function are involved in the neurodegenerative disease amyotrophic lateral sclerosis, we examined neurotracer transport from muscle to motor neuron in a transgenic mouse model of amyotrophic lateral sclerosis. Significant inhibition was observed, which was temporally correlated with declines in muscle strength. No decrease in Dynein/ dynactin expression was observed, but immunohistochemistry s uggests that Dynein associates with aggregates of mutant Cu/Zn superoxide dismutase I. Expression of mutant Cu/Zn superoxide dismutase I in primary motor neurons altered the cellular localization of Dynein, suggesting an inhibition of Dynein/dynactin function. Thus, inhibition of Dynein/dynactin function may have a role in motor neuron degeneration in amyotrophic lateral sclerosis.
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a direct interaction between Cytoplasmic Dynein and kinesin i may coordinate motor activity
Journal of Biological Chemistry, 2004Co-Authors: Lee A Ligon, Mariko Tokito, Jeffrey M Finklestein, Francesca E Grossman, Erika L F HolzbaurAbstract:Cytoplasmic Dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between Cytoplasmic Dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of Dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize Dynein to cellular sites of function. There is also a vesicle population with both Dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.
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Cytoplasmic Dynein and dynactin in cell division and intracellular transport
Current Opinion in Cell Biology, 1999Co-Authors: Sher Bahadur Karki, Erika L F HolzbaurAbstract:Since the initial discovery of Cytoplasmic Dynein, it has become apparent that this microtubule-based motor is involved in several cellular functions including cell division and intracellular transport. Another multisubunit complex, dynactin, may be required for most, if not all, Cytoplasmic Dynein-driven activities and may provide clues to Dynein's functional diversity. Recent genetic and biochemical findings have illuminated the cellular roles of Dynein and dynactin and provided insight into the functional mechanism of this complex motor.
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the interaction between Cytoplasmic Dynein and dynactin is required for fast axonal transport
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Clare M Watermanstorer, Sher Karki, Sergei A Kuznetsov, Joel S Tabb, Dieter G Weiss, George M Langford, Erika L F HolzbaurAbstract:Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic Dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and Cytoplasmic Dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150Glued subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150Glued disrupted the binding of Cytoplasmic Dynein to dynactin and also inhibited the association of Cytoplasmic Dynein with organelles. In contrast, the anti-p150Glued antibodies had no effect on the binding of dynactin to microtubules nor on Cytoplasmic Dynein-driven microtubule gliding. These results indicate that the interaction between Cytoplasmic Dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and Cytoplasmic Dynein during vesicle transport.
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functional analysis of dynactin and Cytoplasmic Dynein in slow axonal transport
The Journal of Neuroscience, 1996Co-Authors: James F. Dillman, Lewis P Dabney, Sher Bahadur Karki, Bryce Mark Paschal, Erika L F Holzbaur, Kevin K PfisterAbstract:The neuron moves protein and membrane from the cell body to the synapse and back via fast and slow axonal transport. Little is known about the mechanism of microtubule movement in slow axonal transport, although Cytoplasmic Dynein, the motor for retrograde fast axonal transport of membranous organelles, has been proposed to also slide microtubules down the axon. We previously showed that most of the Cytoplasmic Dynein moving in the anterograde direction in the axon is associated with the microfilaments and other proteins of the slow component b (SCb) transport complex. The dynactin complex binds Dynein, and it has been suggested that dynactin also associates with microfilaments. We therefore examined the role of Dynein and dynactin in slow axonal transport. We find that most of the dynactin is also transported in SCb, including dynactin, which contains the neuron-specific splice variant p135 Glued , which binds Dynein but not microtubules. Furthermore, SCb Dynein binds dynactin in vitro . SCb Dynein, like Dynein from brain, binds microtubules in an ATP-sensitive manner, whereas brain dynactin binds microtubules in a salt-dependent manner. Dynactin from SCb does not bind microtubules, indicating that the binding of dynactin to microtubules is regulated and suggesting that the role of SCb dynactin is to bind Dynein, not microtubules. These data support a model in which dynactin links the Cytoplasmic Dynein to the SCb transport complex. Dynein then may interact transiently with microtubules to slide them down the axon at the slower rate of SCa.
Thomas S Hays - One of the best experts on this subject based on the ideXlab platform.
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the drosophila tctex 1 light chain is dispensable for essential Cytoplasmic Dynein functions but is required during spermatid differentiation
Molecular Biology of the Cell, 2004Co-Authors: Madeline Serr, Eric A Newman, Thomas S HaysAbstract:Variations in subunit composition and modification have been proposed to regulate the multiple functions of Cytoplasmic Dynein. Here, we examine the role of the Drosophila ortholog of tctex-1, the 14-kDa Dynein light chain. We show that the 14-kDa light chain is a bona fide component of Drosophila Cytoplasmic Dynein and use P element excision to generate flies that completely lack this Dynein subunit. Remarkably, the null mutant is viable and the only observed defect is complete male sterility. During spermatid differentiation, the 14-kDa light chain is required for the localization of a nuclear “cap” of Cytoplasmic Dynein and for proper attachment between the sperm nucleus and flagellar basal body. Our results provide evidence that the function of the 14-kDa light chain in Drosophila is distinct from other Dynein subunits and is not required for any essential functions in early development or in the adult organism.
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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.
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Cytoplasmic Dynein is required for the nuclear attachment and migration of centrosomes during mitosis in drosophila
Journal of Cell Biology, 1999Co-Authors: John T Robinson, Maura Mcgrail, Edward Wojcik, Mark A Sanders, Thomas S HaysAbstract:Cytoplasmic Dynein is a multisubunit minus-end–directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that Dynein function is essential in metazoan organisms. However, whether the essential function of Dynein reflects a mitotic requirement, and what specific mitotic tasks require Dynein remains controversial. Drosophila is an excellent genetic system in which to analyze Dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the Dynein heavy chain gene, Dhc64C, to reveal the contributions of the Dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type Cytoplasmic Dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immu-nofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that Dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for Dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.
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the microtubule motor Cytoplasmic Dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in drosophila
Development, 1997Co-Authors: Maura Mcgrail, Thomas S HaysAbstract:During animal development cellular differentiation is often preceded by an asymmetric cell division whose polarity is determined by the orientation of the mitotic spindle. In the fruit fly, Drosophila melanogaster, the oocyte differentiates in a 16-cell syncytium that arises from a cystoblast which undergoes 4 synchronous divisions with incomplete cytokinesis. During these divisions, spindle orientation is highly ordered and is thought to impart a polarity to the cyst that is necessary for the subsequent differentiation of the oocyte. Using mutations in the Drosophila Cytoplasmic Dynein heavy chain gene, Dhc64C, we show that Cytoplasmic Dynein is required at two stages of oogenesis. Early in oogenesis, Dynein mutations disrupt spindle orientation in dividing cysts and block oocyte determination. The localization of Dynein in mitotic cysts suggests spindle orientation is mediated by the microtubule motor Cytoplasmic Dynein. Later in oogenesis, Dynein function is necessary for proper differentiation, but does not appear to participate in morphogen localization within the oocyte. These results provide evidence for a novel developmental role for the Cytoplasmic Dynein motor in cellular determination and differentiation.
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phylogeny and expression of axonemal and Cytoplasmic Dynein genes in sea urchins
Molecular Biology of the Cell, 1994Co-Authors: Barbara H Gibbons, Thomas S Hays, David J Asai, Wen Jing Y Tang, I R GibbonsAbstract:Transcripts approximately 14.5 kilobases in length from 14 different genes that encode for Dynein heavy chains have been identified in poly(A)+ RNA from sea urchin embryos. Analysis of the changes in level of these Dynein transcripts in response to deciliation, together with their sequence relatedness, suggests that 11 or more of these genes encode Dynein isoforms that participate in regeneration of external cilia on the embryo, whereas the single gene whose deduced sequence closely resembles that of Cytoplasmic Dynein in other organisms appears not to be involved in this regeneration. The four consensus motifs for phosphate binding found previously in the beta heavy chain of sea urchin Dynein are present in all five additional isoforms for which extended sequences have been obtained, suggesting that these sites play a significant role in Dynein function. Sequence analysis of a approximately 400 amino acid region encompassing the putative hydrolytic ATP-binding site shows that the Dynein genes fall into at least six distinct classes. Most of these classes in sea urchin have a high degree of sequence identity with one of the Dynein heavy chain genes identified in Drosophila, indicating that the radiation of the Dynein gene family into the present classes occurred at an early stage in the evolution of eukaryotes. Evolutionary changes in Cytoplasmic Dynein have been more constrained than those in the axonemal Dyneins.
Kevin K Pfister - One of the best experts on this subject based on the ideXlab platform.
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The Mammalian Cytoplasmic Dynein Complexes
2013Co-Authors: Kevin K Pfister, Stephen M King, Paresh R Shah, Holger Hummerich, Andreas Russ, James Cotton, Azlina Ahmad Annuar, Elizabeth M. C. FisherAbstract:(A) Cytoplasmic Dynein. (Left panel) Polypeptides of immunoaffinity-purified rat brain Cytoplasmic Dynein. Polypeptide mass (in kDa) is indicated on the right side of the gel, and the consensus family names are indicated on the left. (Right panel) Structural model for the association of the Cytoplasmic Dynein complex subunits. The core of the Cytoplasmic Dynein complex is made of two DYNC1H1 heavy chains which homodimerize via regions in their N-termini. The motor domains are at the C-termini of the heavy chains, the large globular heads of ~350 kDa that are composed of a ring of seven densities surrounding a central cavity; six of the densities are AAA domains (numbered 1–6). AAA domain 1 is the site of ATP hydrolysis. The microtubule-binding domain is a projection found on the opposite side of the ring between AAA domains 4 and 5. C is the C-terminus of the heavy chain that would form the 7th density. Two DYNC1I intermediate chains (IC74) and DYNC1LI light intermediate chains bind at overlapping regions of the N-terminus of the heavy chain, overlapping with the heavy chain dimerization domains. Dimers of the three light chain families; DYNLT, the Tctex1 light chains; DYNLRB, the Roadblock light chains; and DYNLL, the LC8 light chains, bind to the intermediate chain dimers.(B) Cytoplasmic Dynein 2 complex, structural model for subunit association. This Dynein complex has a unique role in IFT and is sometimes known as IFT Dynein. Structural predictions indicate that the heavy chain, DYNC2H1, is similar to the Cytoplasmic and axonemal Dyneins. The only known subunit of this complex is a 33- to 47-kDa polypeptide, DYNC2LI1, which is related to the Cytoplasmic Dynein light intermediate chains. No intermediate chain or light chains have yet been identified [16].
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a neuron specific Cytoplasmic Dynein isoform preferentially transports trkb signaling endosomes
Journal of Cell Biology, 2008Co-Authors: Kenneth R Myers, Tiffany M Carr, Michael K Humsi, Bareza A Rasoul, Rosalind A Segal, Kevin K PfisterAbstract:Cytoplasmic Dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of Dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional Dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–Dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by Cytoplasmic Dynein containing the neuron-specific IC-1B isoform and not by Dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain Dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define Dynein populations that are selectively recruited to transport distinct cargoes.
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Cytoplasmic Dynein mediates adenovirus binding to microtubules
Journal of Virology, 2004Co-Authors: Samir Kelkar, Kevin K Pfister, Ronald G Crystal, Philip L LeopoldAbstract:During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, Cytoplasmic Dynein, has been implicated in the retrograde movement of Ad. We hypothesized that Cytoplasmic Dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% ± 3.5% to 80.7% ± 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained Cytoplasmic Dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of Cytoplasmic Dynein-dependent microtubule interactions. Immunodepletion of Cytoplasmic Dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both Dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though Cytoplasmic Dynein-mediated binding.
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functional analysis of dynactin and Cytoplasmic Dynein in slow axonal transport
The Journal of Neuroscience, 1996Co-Authors: James F. Dillman, Lewis P Dabney, Sher Bahadur Karki, Bryce Mark Paschal, Erika L F Holzbaur, Kevin K PfisterAbstract:The neuron moves protein and membrane from the cell body to the synapse and back via fast and slow axonal transport. Little is known about the mechanism of microtubule movement in slow axonal transport, although Cytoplasmic Dynein, the motor for retrograde fast axonal transport of membranous organelles, has been proposed to also slide microtubules down the axon. We previously showed that most of the Cytoplasmic Dynein moving in the anterograde direction in the axon is associated with the microfilaments and other proteins of the slow component b (SCb) transport complex. The dynactin complex binds Dynein, and it has been suggested that dynactin also associates with microfilaments. We therefore examined the role of Dynein and dynactin in slow axonal transport. We find that most of the dynactin is also transported in SCb, including dynactin, which contains the neuron-specific splice variant p135 Glued , which binds Dynein but not microtubules. Furthermore, SCb Dynein binds dynactin in vitro . SCb Dynein, like Dynein from brain, binds microtubules in an ATP-sensitive manner, whereas brain dynactin binds microtubules in a salt-dependent manner. Dynactin from SCb does not bind microtubules, indicating that the binding of dynactin to microtubules is regulated and suggesting that the role of SCb dynactin is to bind Dynein, not microtubules. These data support a model in which dynactin links the Cytoplasmic Dynein to the SCb transport complex. Dynein then may interact transiently with microtubules to slide them down the axon at the slower rate of SCa.
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Cytoplasmic Dynein is associated with slow axonal transport
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: James F. Dillman, Lewis P Dabney, Kevin K PfisterAbstract:Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether Dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which Cytoplasmic Dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving Dynein was associated with slow transport, whereas only approximately 15% of the Dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of Dynein through contiguous regions of the optic nerve and tract showed that Dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that Cytoplasmic Dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how Dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon.
Xin Xiang - One of the best experts on this subject based on the ideXlab platform.
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accumulation of Cytoplasmic Dynein and dynactin at microtubule plus ends in aspergillus nidulans is kinesin dependent
Molecular Biology of the Cell, 2003Co-Authors: Jun Zhang, Reinhard Fischer, Xin XiangAbstract:The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both Cytoplasmic Dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and Cytoplasmic Dynein require each other for microtubule plus-end accumulation, and the presence of Cytoplasmic Dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of Dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed Dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of Cytoplasmic Dynein and dynactin, but not of NUDF.
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the aspergillus Cytoplasmic Dynein heavy chain and nudf localize to microtubule ends and affect microtubule dynamics
Current Biology, 2001Co-Authors: Gongshe Han, Ronald N Morris, Jun Zhang, Bo Liu, Wenqi Zuo, Xin XiangAbstract:Cytoplasmic Dynein is a multisubunit, minus end-directed microtubule motor that uses dynactin as an accessory complex to perform various in vivo functions including vesicle transport, spindle assembly, and nuclear distribution [1]. We previously showed that in the filamentous fungus Aspergillus nidulans, a GFP-tagged Cytoplasmic Dynein heavy chain (NUDA) forms comet-like structures that exhibited microtubule-dependent movement toward and back from the hyphal tip [2]. Here we demonstrate that another protein in the NUDA pathway, NUDF, which is homologous to the human LIS1 protein involved in brain development [3, 4], also exhibits such dynamic behavior. Both NUDA and NUDF are located at the ends of microtubules, and this observation suggests that the observed dynamic behavior is due to their association with the dynamic microtubule ends. To address whether NUDA and NUDF play a role in regulating microtubule dynamics in vivo, we constructed a GFP-labeled alpha-tubulin strain and used it to compare microtubule dynamics in vivo in wild-type A. nidulans versus temperature-sensitive loss-of-function mutants of nudA and nudF. The mutants showed a lower frequency of microtubule catastrophe, a lower rate of shrinkage during catastrophe, and a lower frequency of rescue. The microtubules in the mutant cells also paused longer at the hyphal tip than wild-type microtubules. These results indicate that Cytoplasmic Dynein and the LIS1 homolog NUDF affect microtubule dynamics in vivo.
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dynamics of Cytoplasmic Dynein in living cells and the effect of a mutation in the dynactin complex actin related protein arp1
Current Biology, 2000Co-Authors: Xin Xiang, Donald A Winkelmann, Ronald N MorrisAbstract:Abstract Cytoplasmic Dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly [1]. To study the dynamic behavior of Cytoplasmic Dynein in the filamentous fungus Aspergillus nidulans , we replaced the gene for the Cytoplasmic Dynein heavy chain, nudA, with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP–nudA . The GFP–NUDA fusion protein is fully functional in vivo : strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy showed GFP–NUDA to be in comet-like structures that moved in the hyphae toward the growing tip. Retrograde movement of some GFP–NUDA comets after they arrived at the tip was also observed. These dynamics of GFP–NUDA were not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP–NUDA tip-ward movement is similar to the rate of Cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP–NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP–NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex.
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Cytoplasmic Dynein is involved in nuclear migration in aspergillus nidulans
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Xin Xiang, S M Beckwith, N R MorrisAbstract:Nuclear migration plays an important role in the growth and development of many organisms including the multinuclear fungus Aspergillus nidulans. We have identified four genes, nudA, nudC, nudF, and nudG, in which temperature-sensitive mutations affect nuclear distribution. In this report, we describe the cloning of the nudA gene by complementation of the mutant phenotype by using a chromosome VIII-specific cosmid library. A genomic fragment of nudA hybridized to an mRNA of approximately 14 kb. Sequencing analysis of nudA revealed four ATP-binding sites that are characteristic of the Cytoplasmic Dynein heavy chain. The amino acid sequence of the nudA gene product shows 52% overall identity with the rat brain Cytoplasmic Dynein heavy chain. Our study provides in vivo evidence that Dynein, a microtubule motor molecule, plays a role in the nuclear migration process.
