scholarly journals Optogenetic dissection of mitotic spindle positioning in vivo

2018 ◽  
Author(s):  
Lars-Eric Fielmich ◽  
Ruben Schmidt ◽  
Daniel J. Dickinson ◽  
Bob Goldstein ◽  
Anna A. Akhmanova ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Membrane Anchor ◽  
Spindle Positioning ◽  
Membrane Recruitment ◽  
Cell Cleavage ◽  
Pulling Forces ◽  
Germline Expression ◽  
Endogenous Proteins ◽  
The Individual

ABSTRACTThe position of the mitotic spindle determines the plane of cell cleavage, and thereby the location, size, and content of daughter cells. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules. This process requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. It remains unknown whether this complex merely forms a membrane anchor for dynein, or whether the individual components have additional functions, for instance through Gα-GTP or dynein activation. To functionally dissect this system, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. Controlled germline expression and membrane recruitment of the Gα regulators RIC-8Ric-8A and RGS-7Loco/RGS3, and replacement of Gα with a light-inducible membrane anchor demonstrated that Gα-GTP signaling is dispensable for pulling force generation. In the absence of Gα, cortical recruitment of GPR-1/2 or LIN-5, but not dynein itself, induced high pulling forces. Local recruitment 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. This study also highlights the possibilities for optogenetic control of endogenous proteins within an animal system.

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 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 Author response: Optogenetic dissection of mitotic spindle positioning in vivo

2018 ◽  
Author(s):  
Lars-Eric Fielmich ◽  
Ruben Schmidt ◽  
Daniel J Dickinson ◽  
Bob Goldstein ◽  
Anna Akhmanova ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Author Response ◽  
Spindle Positioning

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 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.

scholarly journals MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression

2013 ◽  
Vol 200 (6) ◽  
pp. 773-787 ◽  
Author(s):  
Mei Zhu ◽  
Florian Settele ◽  
Sachin Kotak ◽  
Luis Sanchez-Pulido ◽  
Lena Ehret ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cell Cortex ◽  
Mitotic Progression ◽  
Uncharacterized Protein ◽  
Spindle Positioning ◽  
Pulling Forces ◽  
A Subunit ◽  
Division Axis ◽  
Dynactin Complex ◽  
Cortical Distribution

Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150glued, a subunit of the dynein–dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.

scholarly journals PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone

2002 ◽  
Vol 157 (7) ◽  
pp. 1175-1186 ◽  
Author(s):  
Cristiana Mollinari ◽  
Jean-Philippe Kleman ◽  
Wei Jiang ◽  
Guy Schoehn ◽  
Tony Hunter ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Complete Suppression ◽  
Null Mutant ◽  
Microtubule Binding ◽  
Cell Cleavage ◽  
Spindle Midzone ◽  
Cdk Phosphorylation

Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

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