LET-99 inhibits lateral posterior pulling forces during asymmetric spindle elongation in C. elegans embryos

Cortical pulling on astral microtubules positions the mitotic spindle in response to PAR polarity cues and G protein signaling in many systems. In Caenorhabditis elegans single-cell embryos, posterior spindle displacement depends on Gα and its regulators GPR-1/2 and LIN-5. GPR-1/2 and LIN-5 are necessary for cortical pulling forces and become enriched at the posterior cortex, which suggests that higher forces act on the posterior spindle pole compared with the anterior pole. However, the precise distribution of cortical forces and how they are regulated remains to be determined. Using spindle severing, single centrosome assays, and centrosome fragmentation, we show that both the anterior and posterior cortices generate more pulling force than the lateral–posterior region. Lateral inhibition depends on LET-99, which inhibits GPR-1/2 localization to produce a bipolar GPR-1/2 pattern. Thus, rather than two domains of cortical force, there are three. We propose that the attenuation of lateral forces prevents counterproductive pulling, resulting in a higher net force toward the posterior that contributes to spindle elongation and displacement.

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Evidence for anaphase pulling forces during C. elegans meiosis

The Journal of Cell Biology ◽

10.1083/jcb.202005179 ◽

2020 ◽

Vol 219 (12) ◽

Cited By ~ 2

Author(s):

Brennan M. Danlasky ◽

Michelle T. Panzica ◽

Karen P. McNally ◽

Elizabeth Vargas ◽

Cynthia Bailey ◽

...

Keyword(s):

Spindle Pole ◽

Outer Face ◽

Chromosome Movement ◽

Female Meiosis ◽

Homologous Chromosomes ◽

C Elegans ◽

Spindle Poles ◽

Pulling Forces ◽

Spindle Elongation ◽

Anaphase B

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

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Faculty Opinions recommendation of Heterotrimeric G protein signaling functions with dynein to promote spindle positioning in C. elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092303.546718 ◽

2007 ◽

Author(s):

Bob Goldstein

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Protein Signaling ◽

Heterotrimeric G Protein ◽

Spindle Positioning ◽

C Elegans ◽

Heterotrimeric G Protein Signaling

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Heterotrimeric G protein signaling functions with dynein to promote spindle positioning in C. elegans

The Journal of Cell Biology ◽

10.1083/jcb.200707085 ◽

2007 ◽

Vol 179 (1) ◽

pp. 15-22 ◽

Cited By ~ 90

Author(s):

Claudia Couwenbergs ◽

Jean-Claude Labbé ◽

Morgan Goulding ◽

Thomas Marty ◽

Bruce Bowerman ◽

...

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Protein Signaling ◽

Dynein Light Chain ◽

Loss Of Function ◽

Functional Link ◽

Heterotrimeric G Protein ◽

Spindle Positioning ◽

C Elegans ◽

Heterotrimeric G Protein Signaling

Proper orientation and positioning of the mitotic spindle is essential for the correct segregation of fate determinants during asymmetric cell division. Although heterotrimeric G proteins and their regulators are essential for spindle positioning in many cell types, their mechanism of action remains unclear. In this study, we show that dyrb-1, which encodes a dynein light chain, provides a functional link between heterotrimeric G protein signaling and dynein activity during spindle positioning in Caenorhabditis elegans. Embryos depleted of dyrb-1 display phenotypes similar to a weak loss of function of dynein activity, indicating that DYRB-1 is a positive regulator of dynein. We find that the depletion of dyrb-1 enhances the spindle positioning defect of weak loss of function alleles of two regulators of G protein signaling, LIN-5 and GPR-1/2, and that DYRB-1 physically associates with these two proteins. These results indicate that dynein activity functions with regulators of G protein signaling to regulate common downstream effectors during spindle positioning in the early C. elegans embryo.

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Cortical microtubule pulling forces contribute to the union of the parental genomes in the C. elegans zygote

10.1101/2021.11.19.469242 ◽

2021 ◽

Author(s):

Griselda VELEZ-AGUILERA ◽

Batool OSSAREH-NAZARI ◽

Lucie VAN HOVE ◽

Nicolas Joly ◽

Lionel Pintard

Keyword(s):

Mitotic Spindle ◽

Cortical Microtubule ◽

Temporal Coordination ◽

C Elegans ◽

Astral Microtubule ◽

Pulling Forces ◽

Single Nucleus ◽

Spindle Elongation

Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single C. elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required for the formation of a pronuclear envelopes scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera, 2020). Here we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation. We also demonstrate that weakening of the pronuclear envelopes, via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

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Faculty Opinions recommendation of Heterotrimeric G protein signaling functions with dynein to promote spindle positioning in C. elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092303.549957 ◽

2007 ◽

Author(s):

Michel Labouesse

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Protein Signaling ◽

Heterotrimeric G Protein ◽

Spindle Positioning ◽

C Elegans ◽

Heterotrimeric G Protein Signaling

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Multiple RGS proteins alter neural G protein signaling to allow C. elegans to rapidly change behavior when fed

Genes & Development ◽

10.1101/gad.14.16.2003 ◽

2000 ◽

Vol 14 (16) ◽

pp. 2003-2014 ◽

Cited By ~ 2

Author(s):

Meng-Qiu Dong ◽

Daniel Chase ◽

Georgia A. Patikoglou ◽

Michael R. Koelle

Keyword(s):

G Protein ◽

Egg Laying ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

C Elegans ◽

Change Behavior ◽

Rgs Protein ◽

Heterotrimeric G Protein Signaling

Regulators of G protein signaling (RGS proteins) inhibit heterotrimeric G protein signaling by activating G protein GTPase activity. Many mammalian RGS proteins are expressed in the brain and can act in vitro on the neural G protein Go, but the biological purpose of this multiplicity of regulators is not clear. We have analyzed all 13 RGS genes in Caenorhabditis elegans and found that three of them influence the aspect of egg-laying behavior controlled by Go signaling. A previously studied RGS protein, EGL-10, affects egg laying under all conditions tested. The other two RGS proteins, RGS-1 and RGS-2, act as Go GTPase activators in vitro but, unlike EGL-10, they do not strongly affect egg laying when worms are allowed to feed constantly. However, rgs-1; rgs-2double mutants fail to rapidly induce egg-laying behavior when refed after starvation. Thus EGL-10 sets baseline levels of signaling, while RGS-1 and RGS-2 appear to redundantly alter signaling to cause appropriate behavioral responses to food.

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Tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712052115 ◽

2018 ◽

Vol 115 (5) ◽

pp. E954-E963 ◽

Cited By ~ 9

Author(s):

Kenji Sugioka ◽

Lars-Eric Fielmich ◽

Kota Mizumoto ◽

Bruce Bowerman ◽

Sander van den Heuvel ◽

...

Keyword(s):

Cell Division ◽

Tumor Suppressor ◽

Mitotic Spindle ◽

Asymmetric Cell Division ◽

Cell Cortex ◽

Dependent Manner ◽

C Elegans ◽

Pulling Forces ◽

Underlying Mechanisms ◽

Pulling Force

The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/β-catenin signaling and accurate chromosome segregation and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that Caenorhabditis elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par–aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus-ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle-pulling forces. Our results document a physical basis for the attenuation of spindle-pulling force, which may be generally used in asymmetric cell division and, when disrupted, potentially contributes to division defects in cancer.

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LET-99-dependent spatial restriction of active force generators makes spindle’s position robust

10.1101/103937 ◽

2017 ◽

Author(s):

H. Bouvrais ◽

L. Chesneau ◽

S. Pastezeur ◽

M. Delattre ◽

J. Pécréaux

Keyword(s):

Biochemical Networks ◽

Active Force ◽

Mitotic Progression ◽

Contact Density ◽

Posterior Displacement ◽

C Elegans ◽

Anaphase Onset ◽

Pulling Forces ◽

Pulling Force ◽

Force Generator

AbstractDuring the asymmetric division of the Caenorhabditis elegans nematode zygote, the polarity cues distribution and daughter cell fates depend on the correct positioning of the mitotic spindle, which results from both centering and cortical pulling forces. Revealed by anaphase spindle rocking, these pulling forces are regulated by the force generator dynamics, which are in turn consequent of mitotic progression. We found a novel, additional, regulation of these forces by the spindle position. It controls astral microtubule availability at the cortex, on which the active force generators can pull. Importantly, this positional control relies on the polarity dependent LET-99 cortical band, which restricts or concentrates generators to a posterior crescent. After delaying anaphase onset, we detected this positional pulling force regulation in C. elegans as a precocious spindle rocking with respect to anaphase onset. We ascribed this control to the microtubule dynamics at the cortex. Indeed, in mapping the cortical contacts, we found a correlation between the centrosome–cortex distance and the microtubule contact density. In turn, it modulates pulling force generator activity. We modelled this control, predicting and experimentally validating that the posterior crescent extent controlled where the anaphase oscillations started, in addition to mitotic progression. We found in particular that the oscillation onset position resists changes in cellular geometry and moderate variations of active force generator count. Finally, we propose that spatially restricting force generator to a posterior crescent sets the spindle’s final position, reflecting polarity through the LET-99 dependent restriction of force generators to a posterior crescent. This regulation superimposes that of force generator processivity. This novel control confers a low dependence on microtubule and active force generator exact numbers or dynamics, provided that they exceed the threshold needed for posterior displacement. Interestingly, this robustness originates in cell mechanics rather than biochemical networks.

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A cytoplasmic dynein required for mitotic aster formation in vivo

Journal of Cell Science ◽

10.1242/jcs.111.17.2607 ◽

1998 ◽

Vol 111 (17) ◽

pp. 2607-2614 ◽

Cited By ~ 2

Author(s):

S. Inoue ◽

O.C. Yoder ◽

B.G. Turgeon ◽

J.R. Aist

Keyword(s):

Spindle Pole Body ◽

Cytoplasmic Dynein ◽

Spindle Pole ◽

Mitotic Spindles ◽

Laser Microbeam ◽

Filamentous Ascomycete ◽

Spindle Elongation ◽

Pulling Force ◽

Anaphase B

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

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