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

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.

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Dynactin is required for bidirectional organelle transport

The Journal of Cell Biology ◽

10.1083/jcb.200210066 ◽

2003 ◽

Vol 160 (3) ◽

pp. 297-301 ◽

Cited By ~ 229

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.131.6.1507 ◽

1995 ◽

Vol 131 (6) ◽

pp. 1507-1516 ◽

Cited By ~ 310

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3717 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3717-3728 ◽

Cited By ~ 248

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.133.3.585 ◽

1996 ◽

Vol 133 (3) ◽

pp. 585-593 ◽

Cited By ~ 120

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.

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Apoptotic Cleavage of Cytoplasmic Dynein Intermediate Chain and P150GluedStops Dynein-Dependent Membrane Motility

The Journal of Cell Biology ◽

10.1083/jcb.153.7.1415 ◽

2001 ◽

Vol 153 (7) ◽

pp. 1415-1426 ◽

Cited By ~ 38

Author(s):

Jon D. Lane ◽

Maïlys A.S. Vergnolle ◽

Philip G. Woodman ◽

Victoria J. Allan

Keyword(s):

Mammalian Cells ◽

Cytoplasmic Dynein ◽

Animal Cells ◽

Dynein Intermediate Chain ◽

Egg Extracts ◽

Microtubule Motor ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Dynactin Complex ◽

Intermediate Chains

Cytoplasmic dynein is the major minus end–directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150Glued. We have found that both CD-IC and p150Glued are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH2-terminal p150Glued binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150Glued in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein–driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein–dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

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Microtubule Dynamics and Organelle Transport in Reticulomyxa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158510 ◽

1990 ◽

Vol 48 (3) ◽

pp. 194-195

Author(s):

Yih-Tai Chen ◽

Ursula Euteneuer ◽

Ken B. Johnson ◽

Michael P. Koonce ◽

Manfred Schliwa

Keyword(s):

Plasma Membrane ◽

Light Microscopy ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Microtubule Dynamics ◽

Model Systems ◽

Organelle Transport ◽

Biochemical Characteristics ◽

Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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Microtubule motors and other maps

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015753x ◽

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.

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Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0103 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3107-3116 ◽

Cited By ~ 29

Author(s):

Anindya Ghosh-Roy ◽

Bela S. Desai ◽

Krishanu Ray

Keyword(s):

Light Chain ◽

Cytoplasmic Dynein ◽

Actin Dynamics ◽

Dynein Light Chain ◽

Actin Assembly ◽

Cellular Functions ◽

Directed Movement ◽

Cellular Processes ◽

Dynactin Complex

Toward the end of spermiogenesis, spermatid nuclei are compacted and the clonally related spermatids individualize to become mature and active sperm. Studies in Drosophila showed that caudal end-directed movement of a microfilament-rich structure, called investment cone, expels the cytoplasmic contents of individual spermatids. F-actin dynamics plays an important role in this process. Here we report that the dynein light chain 1 (DLC1) of Drosophila is involved in two separate cellular processes during sperm individualization. It is enriched around spermatid nuclei during postelongation stages and plays an important role in the dynein-dynactin–dependent rostral retention of the nuclei during this period. In addition, DDLC1 colocalizes with dynamin along investment cones and regulates F-actin assembly at this organelle by retaining dynamin along the cones. Interestingly, we found that this process does not require the other subunits of cytoplasmic dynein-dynactin complex. Altogether, these observations suggest that DLC1 could independently regulate multiple cellular functions and established a novel role of this protein in F-actin assembly in Drosophila.

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Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations

10.1101/2020.06.08.140806 ◽

2020 ◽

Author(s):

Gina A. Monzon ◽

Lara Scharrel ◽

Ashwin DSouza ◽

Ludger Santen ◽

Stefan Diez

Keyword(s):

Cytoplasmic Dynein ◽

Gliding Motility ◽

Force Balance ◽

Stochastic Simulations ◽

Transport Systems ◽

Organelle Transport ◽

Atp Concentration ◽

Dynein Motor ◽

Bidirectional Movement

ABSTRACTThe maintenance of intracellular processes like organelle transport and cell division depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatory mechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameter using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors have only little effect.

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