scholarly journals Nudel Modulates Kinetochore Association and Function of Cytoplasmic Dynein in M Phase

2007 ◽  
Vol 18 (7) ◽  
pp. 2656-2666 ◽  
Author(s):  
Yun Liang ◽  
Wei Yu ◽  
Yan Li ◽  
Lihou Yu ◽  
Qiangge Zhang ◽  
...  
Keyword(s):  
Protein Transport ◽  
Cytoplasmic Dynein ◽  
Mitotic Progression ◽  
Kinetochore Protein ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
M Phase ◽  
And Function ◽  
Force Generator

The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly (∼78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.

scholarly journals Cytoplasmic dynein participates in meiotic checkpoint inactivation in mouse oocytes by transporting cytoplasmic mitotic arrest-deficient (Mad) proteins from kinetochores to spindle poles

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Dong Zhang ◽  
Shen Yin ◽  
Man-Xi Jiang ◽  
Wei Ma ◽  
Yi Hou ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Mitotic Arrest ◽  
Checkpoint Proteins ◽  
Meiotic Progression ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Oocyte Meiosis ◽  
And Function

The present study was designed to investigate the localization and function of cytoplasmic dynein (dynein) during mouse oocyte meiosis and its relationship with two major spindle checkpoint proteins, mitotic arrest-deficient (Mad) 1 and Mad2. Oocytes at various stages during the first meiosis were fixed and immunostained for dynein, Mad1, Mad2, kinetochores, microtubules, and chromosomes. Some oocytes were treated with nocodazole before examination. Anti-dynein antibody was injected into the oocytes at germinal vesicle (GV) stage before the examination of its effects on meiotic progression or Mad1 and Mad2 localization. Results showed that dynein was present in the oocytes at various stages from GV to metaphase II and the locations of Mad1 and Mad2 were associated with dynein’s movement. Both Mad1 and Mad2 had two existing states: one existed in the cytoplasm (cytoplasmic Mad1 or cytoplasmic Mad2), which did not bind to kinetochores, while the other bound to kinetochores (kinetochore Mad1 or kinetochore Mad2). The equilibrium between the two states varied during meiosis and/or in response to the changes of the connection between microtubules and kinetochores. Cytoplasmic Mad1 and Mad2 recruited to chromosomes when the connection between microtubules and chromosomes was destroyed. Inhibition of dynein interferes with cytoplasmic Mad1 and Mad2 transportation from chromosomes to spindle poles, thus inhibits checkpoint silence and delays anaphase onset. These results indicate that dynein may play a role in spindle checkpoint inactivation.

scholarly journals LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains

Open Biology ◽  
2018 ◽  
Vol 8 (6) ◽  
pp. 170202 ◽  
Author(s):  
Sirong Ou ◽  
Mei-Hua Tan ◽  
Ting Weng ◽  
HoiYeung Li ◽  
Cheng-Gee Koh
Keyword(s):  
Protein Transport ◽  
Protein Localization ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Centrosomal Protein ◽  
And Function ◽  
Cytoskeleton Dynamics ◽  
Dynein Light Intermediate Chains ◽  
Intermediate Chains ◽  
Mitotic Spindle Pole

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.

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 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 The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

2009 ◽  
Vol 29 (14) ◽  
pp. 3975-3990 ◽  
Author(s):  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Serine Threonine Kinase ◽  
Spindle Formation ◽  
Spindle Poles ◽  
And Function ◽  
Interfering Rna ◽  
Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

scholarly journals SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

2014 ◽  
Vol 204 (7) ◽  
pp. 1099-1109 ◽  
Author(s):  
Yagmur Turgay ◽  
Lysie Champion ◽  
Csaba Balazs ◽  
Michael Held ◽  
Alberto Toso ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Dominant Negative ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Microtubule Depolymerization ◽  
Nuclear Envelope Breakdown ◽  
And Migration ◽  
Delayed Removal

SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression.

scholarly journals The translational landscape of the splicing factor SRSF1 and its role in mitosis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Magdalena M Maslon ◽  
Sara R Heras ◽  
Nicolas Bellora ◽  
Eduardo Eyras ◽  
Javier F Cáceres
Keyword(s):  
Sequencing Analysis ◽  
Mitotic Progression ◽  
Sr Protein ◽  
Control Of Gene Expression ◽  
Translational Activity ◽  
M Phase ◽  
Splicing Regulator ◽  
Cycle Regulation ◽  
Deep Sequencing Analysis

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.

scholarly journals Mad2-independent Spindle Assembly Checkpoint Activation and Controlled Metaphase–Anaphase Transition inDrosophilaS2 Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 850-863 ◽  
Author(s):  
Bernardo Orr ◽  
Hassan Bousbaa ◽  
Claudio E. Sunkel
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Progression ◽  
Experimental Conditions ◽  
Checkpoint Activation ◽  
Nuclear Envelope Breakdown ◽  
Chromatin Bridges ◽  
Delay Progression

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase–anaphase transition and normal patterns of chromosome segregation.

scholarly journals Direct Interaction of Pericentrin with Cytoplasmic Dynein Light Intermediate Chain Contributes to Mitotic Spindle Organization

1999 ◽  
Vol 147 (3) ◽  
pp. 481-492 ◽  
Author(s):  
Aruna Purohit ◽  
Sharon H. Tynan ◽  
Richard Vallee ◽  
Stephen J. Doxsey
Keyword(s):  
Molecular Motor ◽  
Physiological Role ◽  
Direct Interaction ◽  
Cytoplasmic Dynein ◽  
Mitotic Spindles ◽  
Microtubule Organization ◽  
Cellular Targets ◽  
Spindle Poles ◽  
And Function

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150Glued subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin–dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.

Sign in / Sign up

Export Citation Format

Share Document