scholarly journals Dynactin is required for bidirectional organelle transport

2003 ◽  
Vol 160 (3) ◽  
pp. 297-301 ◽  
Author(s):  
Sean W. Deacon ◽  
Anna S. Serpinskaya ◽  
Patricia S. Vaughan ◽  
Monica Lopez Fanarraga ◽  
Isabelle Vernos ◽  
...  
Keyword(s):  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Opposite Polarity ◽  
Model System ◽  
Organelle Transport ◽  
Transport Activity ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Dynactin Complex ◽  
Binding Subunit

Kinesin II is a heterotrimeric plus end–directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II–mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II–associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530–793 of XKAP and aa 600–811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

scholarly journals Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.

1995 ◽  
Vol 131 (6) ◽  
pp. 1507-1516 ◽  
Author(s):  
K T Vaughan ◽  
R B Vallee
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Direct Interaction ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Cell Extracts ◽  
Complex Protein ◽  
Microtubule Motor ◽  
Dynactin Complex ◽  
Intermediate Chains

Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end-directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC-binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.

scholarly journals Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends

1999 ◽  
Vol 112 (10) ◽  
pp. 1437-1447 ◽  
Author(s):  
K.T. Vaughan ◽  
S.H. Tynan ◽  
N.E. Faulkner ◽  
C.J. Echeverri ◽  
R.B. Vallee
Keyword(s):  
Chromosome Segregation ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Interacting Proteins ◽  
Functional Interaction ◽  
Organelle Movement ◽  
Linear Arrays ◽  
Microtubule Motor ◽  
Lower Temperature ◽  
Dynactin Complex

Cytoplasmic dynein is a minus end-directed microtubule motor responsible for centripetal organelle movement and several aspects of chromosome segregation. Our search for cytoplasmic dynein-interacting proteins has implicated the dynactin complex as the cytoplasmic dynein ‘receptor’ on organelles and kinetochores. Immunofluorescence microscopy using a total of six antibodies generated against the p150Glued, Arp1 and dynamitin subunits of dynactin revealed a novel fraction of dynactin-positive structures aligned in linear arrays along the distal segments of interphase microtubules. Dynactin staining revealed that these structures colocalized extensively with CLIP-170. Cytoplasmic dynein staining was undetectable, but extensive colocalization with dynactin became evident upon transfer to a lower temperature. Overexpression of the dynamitin subunit of dynactin removed Arp1 from microtubules but did not affect microtubule-associated p150Glued or CLIP-170 staining. Brief acetate treatment, which has been shown to affect lysosomal and endosomal traffic, also dispersed the Golgi apparatus and eliminated the microtubule-associated staining pattern. The effect on dynactin was rapidly reversible and, following acetate washout, punctate dynactin was detected at microtubule ends within 3 minutes. Together, these findings identify a region along the distal segments of microtubules where dynactin and CLIP-170 colocalize. Because CLIP-170 has been reported to mark growing microtubule ends, our results indicate a similar relationship for dynactin. The functional interaction between dynactin and cytoplasmic dynein further suggests that this these regions represent accumulations of cytoplasmic dynein cargo-loading sites involved in the early stages of minus end-directed organelle transport.

Microtubule motors and other maps

1990 ◽  
Vol 48 (3) ◽  
pp. 1-1
Author(s):  
Richard B. Vallee
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Structural Components ◽  
Microtubule Associated Proteins ◽  
Microtubule Motors ◽  
Associated Proteins ◽  
The Subject ◽  
Intracellular Motility

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

scholarly journals Microtubule-based Endoplasmic Reticulum Motility in Xenopus laevis: Activation of Membrane-associated Kinesin during Development

1999 ◽  
Vol 10 (6) ◽  
pp. 1909-1922 ◽  
Author(s):  
Jon D. Lane ◽  
Victoria J. Allan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Differential Interference Contrast Microscopy ◽  
Animal Cells ◽  
Microtubule Motor ◽  
Directed Motility ◽  
Conventional Kinesin

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts ofXenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopusegg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development inXenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute ∼19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower (∼0.4 μm/s) than minus end-directed motility (∼1.3 μm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

scholarly journals Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

1999 ◽  
Vol 10 (11) ◽  
pp. 3717-3728 ◽  
Author(s):  
MaryAnn Martin ◽  
Stanley J. Iyadurai ◽  
Andrew Gassman ◽  
Joseph G. Gindhart ◽  
Thomas S. Hays ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Heavy Chain ◽  
Cytoplasmic Dynein ◽  
Genetic Interactions ◽  
Organelle Transport ◽  
Fast Axonal Transport ◽  
Anterograde Transport ◽  
Dynein Heavy Chain ◽  
Conventional Kinesin ◽  
Dynactin Complex

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 p150Glued(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 p150Glued 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 orcDhc64C mutations were stronger than those betweenGlued 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.

scholarly journals Cell cycle regulation of dynein association with membranes modulates microtubule-based organelle transport.

1996 ◽  
Vol 133 (3) ◽  
pp. 585-593 ◽  
Author(s):  
J Niclas ◽  
V J Allan ◽  
R D Vale
Keyword(s):  
Cell Cycle ◽  
Membrane Surface ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
A Cell ◽  
Cycle Dependent ◽  
Intermediate Chain

Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.

scholarly journals Dynein, Dynactin, and Kinesin II's Interaction with Microtubules Is Regulated during Bidirectional Organelle Transport

2000 ◽  
Vol 151 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Eric L. Reese ◽  
Leah T. Haimo
Keyword(s):  
Pigment Granule ◽  
Cytoplasmic Dynein ◽  
Binding Activity ◽  
The State ◽  
Organelle Transport ◽  
Microtubule Binding ◽  
Pigment Granules ◽  
Microtubule Motors

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

scholarly journals Microtubule-organizing center formation at telomeres induces meiotic telomere clustering

2013 ◽  
Vol 200 (4) ◽  
pp. 385-395 ◽  
Author(s):  
Masashi Yoshida ◽  
Satoshi Katsuyama ◽  
Kazuki Tateho ◽  
Hiroto Nakamura ◽  
Junpei Miyoshi ◽  
...  
Keyword(s):  
Chromosome Pairing ◽  
Light Chain ◽  
Homologous Chromosome ◽  
Cytoplasmic Dynein ◽  
Dynein Light Chain ◽  
Microtubule Organizing Center ◽  
Homologous Chromosome Pairing ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Organizing Center

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering requires the interaction of telomeres with the nuclear membrane proteins SUN (Sad1/UNC-84) and KASH (Klarsicht/ANC-1/Syne homology). The mechanism by which telomeres gather remains elusive. In this paper, we show that telomere clustering in fission yeast depends on microtubules and the microtubule motors, cytoplasmic dynein, and kinesins. Furthermore, the γ-tubulin complex (γ-TuC) is recruited to SUN- and KASH-localized telomeres to form a novel microtubule-organizing center that we termed the “telocentrosome.” Telocentrosome formation depends on the γ-TuC regulator Mto1 and on the KASH protein Kms1, and depletion of either Mto1 or Kms1 caused severe telomere clustering defects. In addition, the dynein light chain (DLC) contributes to telocentrosome formation, and simultaneous depletion of DLC and dynein also caused severe clustering defects. Thus, the telocentrosome is essential for telomere clustering. We propose that telomere-localized SUN and KASH induce telocentrosome formation and that subsequent microtubule motor-dependent aggregation of telocentrosomes via the telocentrosome-nucleated microtubules causes telomere clustering.

scholarly journals The Interaction of Neurofilaments with the Microtubule Motor Cytoplasmic Dynein

2004 ◽  
Vol 15 (11) ◽  
pp. 5092-5100 ◽  
Author(s):  
Oliver I. Wagner ◽  
Jennifer Ascaño ◽  
Mariko Tokito ◽  
Jean-Francois Leterrier ◽  
Paul A. Janmey ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Specific Interaction ◽  
Cytoplasmic Dynein ◽  
Live Cells ◽  
Slow Axonal Transport ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Physical Interactions

Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein. We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components. AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex. Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC. This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein. Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron.

membrane traffic: Microtubule motor cycles and kinectin

1996 ◽  
Vol 54 ◽  
pp. 732-733
Author(s):  
Michael P. Sheetz
Keyword(s):  
Membrane Traffic ◽  
Growth Conditions ◽  
Cytoplasmic Dynein ◽  
Microtubule Motors ◽  
Transport Cycle ◽  
Microtubule Motor ◽  
Associated Proteins ◽  
The Many ◽  
Dependent Transport

Although membrane traffic can occur in the absence of microtubules, the rapid metabolism of many eucaryotic cells requires that vesicular organelles are actively transported. Microtubule-dependent transport is particularly robust in actively growing cells and the volume of membrane transported increases dramatically in going from static to growth conditions. Both inward and outward movement are regulated simultaneously implying coordinate activation of both motors or a transport cycle. Relatively large complexes of the microtubule motors and associated proteins are responsible for powering the movements (reviewed in Vallee and Sheetz, 1996). Of the many motors that have been described, kinesin and cytoplasmic dynein are the most abundant and have been the focus of these studies. Included in the microtubule motor complexes are motor receptors and activating factors. A membranous motor receptor, kinectin, was identified on the basis of its interaction with kinesin (Toyoshima et al., 1992). An antibody to kinectin blocks both kinesin binding to organelles and kinesin-dependent motility of those organelles (Kumar et al., 1995).

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