scholarly journals Formation of Spindle Poles by Dynein/Dynactin-Dependent Transport of Numa

2000 ◽  
Vol 149 (4) ◽  
pp. 851-862 ◽  
Author(s):  
Andreas Merdes ◽  
Rebecca Heald ◽  
Kumiko Samejima ◽  
William C. Earnshaw ◽  
Don W. Cleveland
Keyword(s):  
Fluorescent Protein ◽  
Gel Filtration ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Green Fluorescent ◽  
Dependent Transport ◽  
Dynactin Complex

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-spe-cific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.

scholarly journals The 14-kDa Dynein Light Chain-Family Protein Dlc1 Is Required for Regular Oscillatory Nuclear Movement and Efficient Recombination during Meiotic Prophase in Fission Yeast

2002 ◽  
Vol 13 (3) ◽  
pp. 930-946 ◽  
Author(s):  
Futaba Miki ◽  
Koei Okazaki ◽  
Mizuki Shimanuki ◽  
Ayumu Yamamoto ◽  
Yasushi Hiraoka ◽  
...  
Keyword(s):  
Light Chain ◽  
Meiotic Prophase ◽  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Dynein Light Chain ◽  
Nuclear Movement ◽  
Green Fluorescent

A Schizosaccharomyces pombe spindle pole body (SPB) protein interacts in a two-hybrid system with Dlc1, which belongs to the 14-kDa Tctex-1 dynein light chain family. Green fluorescent protein-tagged Dlc1 accumulated at the SPB throughout the life cycle. During meiotic prophase, Dlc1 was present along astral microtubules and microtubule-anchoring sites on the cell cortex, reminiscent of the cytoplasmic dynein heavy chain Dhc1. In a dlc1-null mutant, Dhc1-dependent nuclear movement in meiotic prophase became irregular in its duration and direction. Dhc1 protein was displaced from the cortex anchors and the formation of microtubule bundle(s) that guide nuclear movement was impaired in the mutant. Meiotic recombination in the dlc1 mutant was reduced to levels similar to that in the dhc1 mutant. Dlc1 and Dhc1 also have roles in karyogamy and rDNA relocation during the sexual phase. Strains mutated in both the dlc1 and dhc1loci displayed more severe defects in recombination, karyogamy, and sporulation than in either single mutant alone, suggesting that Dlc1 is involved in nuclear events that are independent of Dhc1. S. pombe contains a homolog of the 8-kDa dynein light chain, Dlc2. This class of dynein light chain, however, is not essential in either the vegetative or sexual phases.

scholarly journals The Yeast Dynactin Complex Is Involved in Partitioning the Mitotic Spindle between Mother and Daughter Cells during Anaphase B

1998 ◽  
Vol 9 (7) ◽  
pp. 1741-1756 ◽  
Author(s):  
Jason A. Kahana ◽  
Gabriel Schlenstedt ◽  
Darren M. Evanchuk ◽  
John R. Geiser ◽  
M. Andrew Hoyt ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Cytoplasmic Dynein ◽  
Stable Complex ◽  
Complex Protein ◽  
Mother And Daughter ◽  
Green Fluorescent ◽  
Dynactin Complex

Although vertebrate cytoplasmic dynein can move to the minus ends of microtubules in vitro, its ability to translocate purified vesicles on microtubules depends on the presence of an accessory complex known as dynactin. We have cloned and characterized a novel gene,NIP100, which encodes the yeast homologue of the vertebrate dynactin complex protein p150 glued . Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Δ strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, we find that a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase.

scholarly journals Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts

2005 ◽  
Vol 389 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Tomoya Kotani ◽  
Masakane Yamashita
Keyword(s):  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Middle Part ◽  
Microtubule Organization ◽  
Activity Inhibition ◽  
Green Fluorescent

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-γ-tubulin antibody. The results indicate possible roles of γ-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.

scholarly journals Cell Cycle-dependent Dynamics and Regulation of Mitotic Kinesins in Drosophila S2 Cells

2005 ◽  
Vol 16 (8) ◽  
pp. 3896-3907 ◽  
Author(s):  
Gohta Goshima ◽  
Ronald D. Vale
Keyword(s):  
Cell Cycle ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Active Form ◽  
Nuclear Export Signal ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
And Function

Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule-destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation, respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule-destabilizing kinesins (class 8 and 13 [Klp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with our RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle.

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 Microtubule-Mediated and Microtubule-Independent Transport of Adenovirus Type 5 in HEK293 Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 6899-6908 ◽  
Author(s):  
Carmen Yea ◽  
Joanna Dembowy ◽  
Laura Pacione ◽  
Martha Brown
Keyword(s):  
Viral Genome ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Adenovirus Type ◽  
Spindle Pole ◽  
Hek293 Cells ◽  
Productive Infection ◽  
Microtubule Organizing Center ◽  
293 Cells ◽  
Green Fluorescent

ABSTRACT Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules.

scholarly journals A New Model for Nuclear Envelope Breakdown

2001 ◽  
Vol 12 (2) ◽  
pp. 503-510 ◽  
Author(s):  
Mark Terasaki ◽  
Paul Campagnola ◽  
Melissa M. Rolls ◽  
Pascal A. Stein ◽  
Jan Ellenberg ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Second Phase ◽  
New Model ◽  
Nuclear Envelope Breakdown ◽  
Two Phases ◽  
Green Fluorescent

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

scholarly journals Cytoplasmic Dynein's Mitotic Spindle Pole Localization Requires a Functional Anaphase-promoting Complex, γ-Tubulin, and NUDF/LIS1 in Aspergillus nidulans

2005 ◽  
Vol 16 (8) ◽  
pp. 3591-3605 ◽  
Author(s):  
Shihe Li ◽  
C. Elizabeth Oakley ◽  
Guifang Chen ◽  
Xiaoyan Han ◽  
Berl R. Oakley ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Chromosome Loss ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Spindle Poles ◽  
Dynein Motor ◽  
Chromosome Separation ◽  
Spindle Elongation

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a γ-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that γ-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.

scholarly journals CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization in Aspergillus nidulans

2006 ◽  
Vol 17 (4) ◽  
pp. 2021-2034 ◽  
Author(s):  
Vladimir P. Efimov ◽  
Jun Zhang ◽  
Xin Xiang
Keyword(s):  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Live Cells ◽  
Physiological Conditions ◽  
Physical Interactions ◽  
Cytoplasmic Microtubules ◽  
Growth Promoting ◽  
Green Fluorescent ◽  
Hyphal Tip

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32°C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150Glued dynactin, and NUDF were all seen as plus-end comets at 32°C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32°C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42°C).

scholarly journals Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

2002 ◽  
Vol 158 (5) ◽  
pp. 841-847 ◽  
Author(s):  
Marko J. Kallio ◽  
Victoria A. Beardmore ◽  
Jasminder Weinstein ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Anaphase Promoting Complex ◽  
Late Prophase ◽  
Checkpoint Signaling ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Late Telophase ◽  
Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

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