scholarly journals Tipping the spindle into the right position

2016 ◽  
Vol 213 (3) ◽  
pp. 293-295 ◽  
Author(s):  
Anna Akhmanova ◽  
Sander van den Heuvel
Keyword(s):  
Mitotic Spindle ◽  
Cleavage Plane ◽  
Animal Cells ◽  
Spindle Positioning ◽  
Cell Biol ◽  
Pulling Forces ◽  
The Right

The position of the mitotic spindle determines the cleavage plane in animal cells, but what controls spindle positioning? Kern et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510117) demonstrate that the microtubule plus end–associated SKAP/Astrin complex participates in this process, possibly by affecting dynein-dependent pulling forces exerted on the tips of astral microtubules.

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 Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport

2017 ◽  
Author(s):  
Daniel José Barbosa ◽  
Joana Duro ◽  
Dhanya K. Cheerambathur ◽  
Bram Prevo ◽  
Ana Xavier Carvalho ◽  
...  
Keyword(s):  
Binding Activity ◽  
Organelle Transport ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Animal Cells ◽  
Spindle Positioning ◽  
C Elegans ◽  
Pulling Forces

ABSTRACTThe microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin’s microtubule binding activity, located in p150’s CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Using precise mutants engineered by genome editing, we show that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating cortical pulling forces. Instead, p150 CAP-Gly mutants inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of the C-terminal tyrosine of α-tubulin, and both p150 CAP-Gly and tubulin tyrosination mutants decrease the frequency of organelle transport from the cell periphery towards centrosomes during centration. In light of recent work on dynein-dynactin motility in vitro, our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing embryo, and that this function of dynactin is important for generating robust cytoplasmic pulling forces for centrosome centration.

scholarly journals Cytokinetic astralogy

2009 ◽  
Vol 187 (6) ◽  
pp. 757-759 ◽  
Author(s):  
Julie C. Canman
Keyword(s):  
Mitotic Spindle ◽  
Direct Contact ◽  
Cell Cortex ◽  
Animal Cells ◽  
Division Plane ◽  
Cell Biol ◽  
Spindle Microtubules

Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.

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 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 Stoichiometric interactions explain spindle dynamics and scaling across 100 million years of nematode evolution

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Reza Farhadifar ◽  
Che-Hang Yu ◽  
Gunar Fabig ◽  
Hai-Yin Wu ◽  
David B Stein ◽  
...  
Keyword(s):  
Quantitative Genetics ◽  
Mitotic Spindle ◽  
Mechanistic Explanation ◽  
Nematode Species ◽  
Spindle Positioning ◽  
Spindle Dynamics ◽  
Pulling Forces ◽  
Final Length ◽  
Force Generator ◽  
Rule Out

The spindle shows remarkable diversity, and changes in an integrated fashion, as cells vary over evolution. Here, we provide a mechanistic explanation for variations in the first mitotic spindle in nematodes. We used a combination of quantitative genetics and biophysics to rule out broad classes of models of the regulation of spindle length and dynamics, and to establish the importance of a balance of cortical pulling forces acting in different directions. These experiments led us to construct a model of cortical pulling forces in which the stoichiometric interactions of microtubules and force generators (each force generator can bind only one microtubule), is key to explaining the dynamics of spindle positioning and elongation, and spindle final length and scaling with cell size. This model accounts for variations in all the spindle traits we studied here, both within species and across nematode species spanning over 100 million years of evolution.

scholarly journals Optogenetic dissection of mitotic spindle positioning in vivo

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Lars-Eric Fielmich ◽  
Ruben Schmidt ◽  
Daniel J Dickinson ◽  
Bob Goldstein ◽  
Anna Akhmanova ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Membrane Anchor ◽  
Spindle Positioning ◽  
C Elegans ◽  
Cell Cleavage ◽  
Evolutionarily Conserved ◽  
Pulling Forces ◽  
Endogenous Proteins ◽  
Pulling Force

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP–GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

scholarly journals Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

2018 ◽  
Author(s):  
Masako Okumura ◽  
Toyoaki Natsume ◽  
Masato T. Kanemaki ◽  
Tomomi Kiyomitsu
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Human Cells ◽  
Microtubule Binding ◽  
Spindle Positioning ◽  
The Core ◽  
Pulling Forces ◽  
Focal Structures

AbstractTo position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment/activation by NuMA’s N-terminal long arm, and NuMA’s direct microtubule-binding activities to achieve a multiplicity of microtubule interactions. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

scholarly journals A novel patch assembly domain in Num1 mediates dynein anchoring at the cortex during spindle positioning

2012 ◽  
Vol 196 (6) ◽  
pp. 743-756 ◽  
Author(s):  
Xianying Tang ◽  
Bryan St. Germain ◽  
Wei-Lih Lee
Keyword(s):  
Mitotic Spindle ◽  
Distinct Point ◽  
Point Mutations ◽  
Pleckstrin Homology Domain ◽  
Spindle Positioning ◽  
Bud Neck ◽  
Pulling Forces ◽  
Necessary And Sufficient ◽  
Caax Motif ◽  
Pa Domain

During mitosis in budding yeast, cortically anchored dynein generates pulling forces on astral microtubules to position the mitotic spindle across the mother–bud neck. The attachment molecule Num1 is required for dynein anchoring at the cell membrane, but how Num1 assembles into stationary cortical patches and interacts with dynein is unknown. We show that an N-terminal Bin/Amphiphysin/Rvs (BAR)–like domain in Num1 mediates the assembly of morphologically distinct patches and its interaction with dynein for spindle translocation into the bud. We name this domain patch assembly domain (PA; residues 1–303), as it was both necessary and sufficient for the formation of functional dynein-anchoring patches when it was attached to a pleckstrin homology domain or a CAAX motif. Distinct point mutations targeting the predicted BAR-like PA domain differentially disrupted patch assembly, dynein anchoring, and mitochondrial attachment functions of Num1. We also show that the PA domain is an elongated dimer and discuss the mechanism by which it drives patch assembly.

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