scholarly journals RanGTP and CLASP1 cooperate to position the mitotic spindle

2013 ◽  
Vol 24 (16) ◽  
pp. 2506-2514 ◽  
Author(s):  
Stephen L. Bird ◽  
Rebecca Heald ◽  
Karsten Weis
Keyword(s):  
Mitotic Spindle ◽  
Nucleocytoplasmic Transport ◽  
Cytoplasmic Dynein ◽  
Small Gtpase ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Molecule Inhibitor ◽  
Pulling Forces ◽  
Cortical Localization ◽  
Β Function

Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-β function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.

scholarly journals Normal spindle positioning in the absence of EBPs and dynein plus-end tracking in C. elegans

2017 ◽  
Author(s):  
Ruben Schmidt ◽  
Anna Akhmanova ◽  
Sander van den Heuvel
Keyword(s):  
Mitotic Spindle ◽  
Cortical Microtubule ◽  
Complete Removal ◽  
Cytoplasmic Dynein ◽  
Animal Cells ◽  
Spindle Positioning ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Pulling Forces ◽  
Cortical Localization

AbstractThe position of the mitotic spindle is tightly controlled in animal cells, as it determines the plane and orientation of cell division. Interactions between cytoplasmic dynein at the cortex and astral microtubules generate pulling forces that position the spindle. In yeast, dynein is actively delivered to the cortex through microtubule plus-end tracking complexes. In animal cells, an evolutionarily conserved Gα-GPR-1/2Pins/LGN–LIN-5NuMA cortical complex interacts with dynein and is required to generate pulling forces, but the mechanism of dynein recruitment to the cortex is unclear. Using CRISPR/Cas9-assisted recombineering, we fluorescently labeled endogenous DHC-1 dynein in C. elegans. We observed strong dynein plus-end tracking, which depended on the end-binding protein EBP-2. Complete removal of the EBP family abolished dynein plus-end tracking but not LIN-5-dependent cortical localization. The ebp-1/2/3 deletion mutant, which was viable and fertile, showed increased cortical microtubule retention; however, pulling forces and spindle positioning were normal. These data indicate that dynein recruited from the cytoplasm creates robust pulling forces.

scholarly journals Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

2013 ◽  
Vol 24 (7) ◽  
pp. 901-913 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Jing Liu ◽  
Huabin Zhu ◽  
Xiaogang Chu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescence Recovery After Photobleaching ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Astral Microtubule ◽  
Mammalian Homologue ◽  
Cortical Localization

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

scholarly journals Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

1997 ◽  
Vol 138 (5) ◽  
pp. 1055-1066 ◽  
Author(s):  
Tirso Gaglio ◽  
Mary A. Dionne ◽  
Duane A. Compton
Keyword(s):  
Mitotic Spindle ◽  
Motor Proteins ◽  
Cultured Cells ◽  
Recent Observation ◽  
Somatic Cells ◽  
Cytoplasmic Dynein ◽  
Common Mechanism ◽  
Mitotic Apparatus ◽  
Free System ◽  
Spindle Poles

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.

scholarly journals Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

2020 ◽  
Author(s):  
Safia Omer ◽  
Katia Brock ◽  
John Beckford ◽  
Wei-Lih Lee
Keyword(s):  
Budding Yeast ◽  
Current Model ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Direct Imaging ◽  
Spindle Positioning ◽  
Sliding Mechanism ◽  
Pulling Forces ◽  
Pulling Force

ABSTRACTCurrent model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing only ∼6 copies of Num1 molecules mediate dynein-dependent spindle pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling force.

scholarly journals Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation

2012 ◽  
Vol 198 (6) ◽  
pp. 1039-1054 ◽  
Author(s):  
Anja K. Dunsch ◽  
Dean Hammond ◽  
Jennifer Lloyd ◽  
Lothar Schermelleh ◽  
Ulrike Gruneberg ◽  
...  
Keyword(s):  
Light Chain ◽  
Mitotic Spindle ◽  
Regulatory System ◽  
Cytoplasmic Dynein ◽  
Spindle Orientation ◽  
Growth Surface ◽  
Cell Cortex ◽  
Dynein Light Chain ◽  
Dynein Motor ◽  
Pulling Forces

The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. Here, we show that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule–associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex.

scholarly journals Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

2020 ◽  
Vol 133 (20) ◽  
pp. jcs246363 ◽  
Author(s):  
Safia Omer ◽  
Katia Brock ◽  
John Beckford ◽  
Wei-Lih Lee
Keyword(s):  
Budding Yeast ◽  
Current Model ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Direct Imaging ◽  
Spindle Positioning ◽  
Sliding Mechanism ◽  
Pulling Forces ◽  
Assembly Factor ◽  
Pulling Force

ABSTRACTThe current model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by its assembly factor Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing around only six Num1 molecules mediate dynein-dependent spindle pulling via a lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but do not interfere with the dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling forces.This article has an associated First Person interview with the first author of the paper.

scholarly journals The Nuclear Mitotic Apparatus (NuMA) Protein: A Key Player for Nuclear Formation, Spindle Assembly, and Spindle Positioning

2021 ◽  
Vol 9 ◽  
Author(s):  
Tomomi Kiyomitsu ◽  
Susan Boerner
Keyword(s):  
Protein A ◽  
Spindle Assembly ◽  
Mitotic Cell ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Nuclear Mitotic Apparatus

The nuclear mitotic apparatus (NuMA) protein is well conserved in vertebrates, and dynamically changes its subcellular localization from the interphase nucleus to the mitotic/meiotic spindle poles and the mitotic cell cortex. At these locations, NuMA acts as a key structural hub in nuclear formation, spindle assembly, and mitotic spindle positioning, respectively. To achieve its variable functions, NuMA interacts with multiple factors, including DNA, microtubules, the plasma membrane, importins, and cytoplasmic dynein. The binding of NuMA to dynein via its N-terminal domain drives spindle pole focusing and spindle positioning, while multiple interactions through its C-terminal region define its subcellular localizations and functions. In addition, NuMA can self-assemble into high-ordered structures which likely contribute to spindle positioning and nuclear formation. In this review, we summarize recent advances in NuMA’s domains, functions and regulations, with a focus on human NuMA, to understand how and why vertebrate NuMA participates in these functions in comparison with invertebrate NuMA-related proteins.

scholarly journals Novel Roles for Saccharomyces cerevisiae Mitotic Spindle Motors

1999 ◽  
Vol 147 (2) ◽  
pp. 335-350 ◽  
Author(s):  
Frank R. Cottingham ◽  
Larisa Gheber ◽  
Dana L. Miller ◽  
M. Andrew Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Cytoplasmic Dynein ◽  
Spindle Positioning ◽  
Bipolar Spindle ◽  
Accessory Factor ◽  
Spindle Function ◽  
Related Proteins ◽  
Spindle Structure

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.

scholarly journals Control of nuclear centration in the C. elegans zygote by receptor-independent Gα signaling and myosin II

2007 ◽  
Vol 178 (7) ◽  
pp. 1177-1191 ◽  
Author(s):  
Morgan B. Goulding ◽  
Julie C. Canman ◽  
Eric N. Senning ◽  
Andrew H. Marcus ◽  
Bruce Bowerman
Keyword(s):  
Mitotic Spindle ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Wild Type ◽  
Spindle Positioning ◽  
C Elegans ◽  
Nonmuscle Myosin Ii ◽  
Pulling Forces ◽  
Actomyosin Contraction

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction.

scholarly journals Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.

1996 ◽  
Vol 135 (2) ◽  
pp. 399-414 ◽  
Author(s):  
T Gaglio ◽  
A Saredi ◽  
J B Bingham ◽  
M J Hasbani ◽  
S R Gill ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Cytoplasmic Dynein ◽  
Dominant Negative ◽  
Polar Regions ◽  
Mitotic Apparatus ◽  
Independent Manner ◽  
Free System ◽  
Cell Free System

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end-directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.

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