Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

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.

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Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

eLife ◽

10.7554/elife.00943 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 50

Author(s):

Marvin E Tanenbaum ◽

Ronald D Vale ◽

Richard J McKenney

Keyword(s):

Single Molecule ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Motor Proteins ◽

Cytoplasmic Dynein ◽

Eukaryotic Cells ◽

Reverse Direction ◽

Tail Domain ◽

Mitotic Spindles

Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins.

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A cytoplasmic dynein motor in Drosophila: identification and localization during embryogenesis

Journal of Cell Science ◽

10.1242/jcs.107.6.1557 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1557-1569 ◽

Cited By ~ 8

Author(s):

T.S. Hays ◽

M.E. Porter ◽

M. McGrail ◽

P. Grissom ◽

P. Gosch ◽

...

Keyword(s):

Mitotic Spindle ◽

Sedimentation Coefficient ◽

Cytoplasmic Dynein ◽

Binding Activity ◽

Dynein Motor ◽

Embryonic Nervous System ◽

Spindle Microtubules ◽

Nucleotide Specificity ◽

Drosophila Embryos

We have characterized a cytoplasmic dynein motor isoform that is present in extracts of Drosophila embryos. A prominent high molecular weight (HMW) polypeptide (> 400 kDa) is enriched in microtubules prepared from nucleotide-depleted embryonic extracts. Based on its ATP-sensitive microtubule binding activity, 20 S sedimentation coefficient, sensitivity to UV-vanadate and nucleotide specificity, the HMW polypeptide resembles cytoplasmic dyneins prepared from other organisms. The Drosophila cytoplasmic dynein acts as a minus-end motor that promotes microtubule translocation in vitro. A polyclonal antibody raised against the dynein heavy chain polypeptide was used to localize the dynein antigen in whole-mount preparations of embryos by immunofluorescence microscopy. These studies show that the dynein motor is associated with microtubules throughout embryogenesis, including mitotic spindle microtubules and microtubules of the embryonic nervous system.

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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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Dynactin Is Required for Microtubule Anchoring at Centrosomes

The Journal of Cell Biology ◽

10.1083/jcb.147.2.321 ◽

1999 ◽

Vol 147 (2) ◽

pp. 321-334 ◽

Cited By ~ 247

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.

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Interplay of Microtubule Dynamics and Sliding during Bipolar Spindle Formation in Mammalian Cells

Current Biology ◽

10.1016/j.cub.2009.10.056 ◽

2009 ◽

Vol 19 (24) ◽

pp. 2108-2113 ◽

Cited By ~ 27

Author(s):

Swapna Kollu ◽

Samuel F. Bakhoum ◽

Duane A. Compton

Keyword(s):

Mammalian Cells ◽

Microtubule Dynamics ◽

Spindle Formation ◽

Bipolar Spindle

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ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO

The Journal of Cell Biology ◽

10.1083/jcb.50.2.416 ◽

1971 ◽

Vol 50 (2) ◽

pp. 416-431 ◽

Cited By ~ 114

Author(s):

B. R. Brinkley ◽

Joiner Cartwright

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Chinese Hamster ◽

Spindle Axis ◽

Hamster Strain ◽

Early Anaphase ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Elongation Process

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK1) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.

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Kinetochore-microtubule stability governs the metaphase requirement for Eg5

Molecular Biology of the Cell ◽

10.1091/mbc.e14-03-0785 ◽

2014 ◽

Vol 25 (13) ◽

pp. 2051-2060 ◽

Cited By ~ 26

Author(s):

A. Sophia Gayek ◽

Ryoma Ohi

Keyword(s):

Physical Properties ◽

Human Cell ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Human Cell Lines ◽

Bipolar Spindle ◽

Daughter Cells ◽

Microtubule Stability ◽

The Stability ◽

Bipolar Spindles

The mitotic spindle is a bipolar, microtubule (MT)-based cellular machine that segregates the duplicated genome into two daughter cells. The kinesin-5 Eg5 establishes the bipolar geometry of the mitotic spindle, but previous work in mammalian cells suggested that this motor is unimportant for the maintenance of spindle bipolarity. Although it is known that Kif15, a second mitotic kinesin, enforces spindle bipolarity in the absence of Eg5, how Kif15 functions in this capacity and/or whether other biochemical or physical properties of the spindle promote its bipolarity have been poorly studied. Here we report that not all human cell lines can efficiently maintain bipolarity without Eg5, despite their expressing Kif15. We show that the stability of chromosome-attached kinetochore-MTs (K-MTs) is important for bipolar spindle maintenance without Eg5. Cells that efficiently maintain bipolar spindles without Eg5 have more stable K-MTs than those that collapse without Eg5. Consistent with this observation, artificial destabilization of K-MTs promotes spindle collapse without Eg5, whereas stabilizing K-MTs improves bipolar spindle maintenance without Eg5. Our findings suggest that either rapid K-MT turnover pulls poles inward or slow K-MT turnover allows for greater resistance to inward-directed forces.

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Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3576 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3576-3586 ◽

Cited By ~ 77

Author(s):

C H Yang ◽

J Tomkiel ◽

H Saitoh ◽

D H Johnson ◽

W C Earnshaw

Keyword(s):

Dna Binding ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Mammalian Cells ◽

In Vitro Assays ◽

Centromeric Heterochromatin ◽

Structural Protein ◽

Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

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Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

Molecular Biology of the Cell ◽

10.1091/mbc.e08-12-1160 ◽

2009 ◽

Vol 20 (12) ◽

pp. 2885-2899 ◽

Cited By ~ 72

Author(s):

Krysten J. Palmer ◽

Helen Hughes ◽

David J. Stephens

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Cytoplasmic Dynein ◽

Dominant Negative ◽

Automated Image Analysis ◽

Recycling Endosome ◽

Microtubule Network ◽

Dynein Motor ◽

Specific Subset ◽

Biochemical Analyses

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.

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Modeling reveals cortical dynein-dependent oscillations in bipolar spindle length

10.1101/2020.07.10.197285 ◽

2020 ◽

Author(s):

Dayna Mercadante ◽

Amity Manning ◽

Sarah Olson

Keyword(s):

Experimental Data ◽

Mitotic Spindle ◽

Force Generation ◽

Mitotic Progression ◽

Computational Framework ◽

Spindle Formation ◽

Bipolar Spindle ◽

Spindle Dynamics ◽

Force Components ◽

Over Time

AbstractProper formation and maintenance of the mitotic spindle is required for faithful cell division. While much work has been done to understand the roles of the key force components of the mitotic spindle, identifying the consequences of force perturbations in the spindle remains a challenge. We develop a computational framework accounting for the minimal force requirements of mitotic progression. To reflect early spindle formation, we account for microtubule dynamics and interactions with major force-generating motors, excluding chromosome interactions that dominate later in mitosis. We directly integrate our experimental data to define and validate the model, and then use simulations to analyze individual force components over time and their relationship to spindle dynamics, making it distinct from previously published models. Rather than achieving and maintaining a constant bipolar spindle length, oscillations in pole to pole distance occur that coincide with microtubule binding and force generation by cortical dynein. In the context of high kinesin-14 (HSET) activity, we identify the requirement of high cortical dynein activity for bipolar spindle formation.

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