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

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.

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

2020 ◽  
Vol 133 (22) ◽  
pp. jcs249938
Author(s):  
Gina A. Monzon ◽  
Lara Scharrel ◽  
Ashwin DSouza ◽  
Verena Henrichs ◽  
Ludger Santen ◽  
...  
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 parameters 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 only have a small effect.

Rab3D directs organelle transport by recruiting cytoplasmic dynein motor protein Tctex-1 to secretory membranes of osteoclast

Bone ◽  
2009 ◽  
Vol 44 ◽  
pp. S160 ◽  
Author(s):  
N.J. Pavlos ◽  
J. Xu ◽  
H. Feng ◽  
P. Ng ◽  
T. Cheng ◽  
...  
Keyword(s):  
Motor Protein ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Dynein Motor

scholarly journals Dynactin Is Required for Microtubule Anchoring at Centrosomes

1999 ◽  
Vol 147 (2) ◽  
pp. 321-334 ◽  
Author(s):  
N.J. Quintyne ◽  
S.R. Gill ◽  
D.M. Eckley ◽  
C.L. Crego ◽  
D.A. Compton ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Microtubule Organization ◽  
Cultured Fibroblasts ◽  
Cell Extracts ◽  
Dynein Motor ◽  
Mitotic Spindle Assembly ◽  
Dynactin Complex

The multiprotein complex, dynactin, is an integral part of the cytoplasmic dynein motor and is required for dynein-based motility in vitro and in vivo. In living cells, perturbation of the dynein–dynactin interaction profoundly blocks mitotic spindle assembly, and inhibition or depletion of dynein or dynactin from meiotic or mitotic cell extracts prevents microtubules from focusing into spindles. In interphase cells, perturbation of the dynein–dynactin complex is correlated with an inhibition of ER-to-Golgi movement and reorganization of the Golgi apparatus and the endosome–lysosome system, but the effects on microtubule organization have not previously been defined. To explore this question, we overexpressed a variety of dynactin subunits in cultured fibroblasts. Subunits implicated in dynein binding have effects on both microtubule organization and centrosome integrity. Microtubules are reorganized into unfocused arrays. The pericentriolar components, γ tubulin and dynactin, are lost from centrosomes, but pericentrin localization persists. Microtubule nucleation from centrosomes proceeds relatively normally, but microtubules become disorganized soon thereafter. Overexpression of some, but not all, dynactin subunits also affects endomembrane localization. These data indicate that dynein and dynactin play important roles in microtubule organization at centrosomes in fibroblastic cells and provide new insights into dynactin–cargo interactions.

scholarly journals Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

2018 ◽  
Vol 315 (5) ◽  
pp. E924-E948 ◽  
Author(s):  
Qing Wen ◽  
Elizabeth I. Tang ◽  
Wing-yee Lui ◽  
Will M. Lee ◽  
Chris K. C. Wong ◽  
...  
Keyword(s):  
Germ Cell ◽  
Heavy Chain ◽  
Sertoli Cells ◽  
Cytoplasmic Dynein ◽  
Seminiferous Epithelium ◽  
Organelle Transport ◽  
Cellular Events

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

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 Lissencephaly-1 is a context-dependent regulator of the human dynein complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Janina Baumbach ◽  
Andal Murthy ◽  
Mark A McClintock ◽  
Carly I Dix ◽  
Ruta Zalyte ◽  
...  
Keyword(s):  
Complex Formation ◽  
Binding Protein ◽  
Cytoplasmic Dynein ◽  
Cargo Transport ◽  
Dynein Motor ◽  
Context Dependent ◽  
Shed Light ◽  
Dynamic Microtubule

The cytoplasmic dynein-1 (dynein) motor plays a central role in microtubule organisation and cargo transport. These functions are spatially regulated by association of dynein and its accessory complex dynactin with dynamic microtubule plus ends. Here, we elucidate in vitro the roles of dynactin, end-binding protein-1 (EB1) and Lissencephaly-1 (LIS1) in the interaction of end tracking and minus end-directed human dynein complexes with these sites. LIS1 promotes dynactin-dependent tracking of dynein on both growing and shrinking plus ends. LIS1 also increases the frequency and velocity of processive dynein movements that are activated by complex formation with dynactin and a cargo adaptor. This stimulatory effect of LIS1 contrasts sharply with its documented ability to inhibit the activity of isolated dyneins. Collectively, our findings shed light on how mammalian dynein complexes associate with dynamic microtubules and help clarify how LIS1 promotes the plus-end localisation and cargo transport functions of dynein in vivo.

Cytoplasmic dynein-associated structures move bidirectionallyin vivo

2002 ◽  
Vol 115 (7) ◽  
pp. 1453-1460 ◽  
Author(s):  
Shuo Ma ◽  
Rex L. Chisholm
Keyword(s):  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Anterograde Transport ◽  
Directional Movement ◽  
Motor Activities ◽  
Direction Of Movement ◽  
Linear Movement ◽  
Bidirectional Movement ◽  
Green Fluorescent

Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. The microtubulebased motors, cytoplasmic dynein and kinesin, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement; however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternative binding of different motility factors to the cargo. Alternatively,different motors could associate with the same cargo and have their motor activity regulated. Although several studies have focused on the behavior of specific types of cargoes, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-associated structures display fast linear movement along microtubules in both minus-end and plus-end directions, with velocities similar to that of dynein and kinesin-like motors. In addition, dynein puncta often rapidly reverse their direction. Dynein stably associates with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor-cargo association but rather by switching activity of motors associated with the cargo. These observations suggest that both plus- and minus-end-directed motors associate with a given cargo and that coordinated regulation of motor activities controls vesicle directionality.

scholarly journals Cell cycle control of microtubule-based membrane transport and tubule formation in vitro.

1991 ◽  
Vol 113 (2) ◽  
pp. 347-359 ◽  
Author(s):  
V J Allan ◽  
R D Vale
Keyword(s):  
Cell Cycle ◽  
Membrane Transport ◽  
Cell Cycle Control ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Tubule Formation ◽  
Membrane Structure And Function ◽  
Membrane Networks ◽  
Membrane Components

When higher eukaryotic cells enter mitosis, membrane organization changes dramatically and traffic between membrane compartments is inhibited. Since membrane transport along microtubules is involved in secretion, endocytosis, and the positioning of organelles during interphase, we have explored whether the mitotic reorganization of membrane could involve a change in microtubule-based membrane transport. This question was examined by reconstituting organelle transport along microtubules in Xenopus egg extracts, which can be converted between interphase and metaphase states in vitro in the absence of protein synthesis. Interphase extracts support the microtubule-dependent formation of abundant polygonal networks of membrane tubules and the transport of small vesicles. In metaphase extracts, however, the plus end- and minus end-directed movements of vesicles along microtubules as well as the formation of tubular membrane networks are all reduced substantially. By fractionating the extracts into soluble and membrane components, we have shown that the cell cycle state of the supernatant determines the extent of microtubule-based membrane movement. Interphase but not metaphase Xenopus soluble factors also stimulate movement of membranes from a rat liver Golgi fraction. In contrast to above findings with organelle transport, the minus end-directed movements of microtubules on glass surfaces and of latex beads along microtubules are similar in interphase and metaphase extracts, suggesting that cytoplasmic dynein, the predominant soluble motor in frog extracts, retains its force-generating activity throughout the cell cycle. A change in the association of motors with membranes may therefore explain the differing levels of organelle transport activity in interphase and mitotic extracts. We propose that the regulation of organelle transport may contribute significantly to the changes in membrane structure and function observed during mitosis in living cells.

scholarly journals Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

1993 ◽  
Vol 123 (4) ◽  
pp. 849-858 ◽  
Author(s):  
E A Vaisberg ◽  
M P Koonce ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Detectable Effect ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Dynein Motor ◽  
Motor Enzymes ◽  
Chromosome Attachment

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

scholarly journals Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.

1995 ◽  
Vol 131 (2) ◽  
pp. 411-425 ◽  
Author(s):  
M McGrail ◽  
J Gepner ◽  
A Silvanovich ◽  
S Ludmann ◽  
M Serr ◽  
...  
Keyword(s):  
Temporal Distribution ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Gene Products ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Dynein Motor ◽  
Associated Proteins

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

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