scholarly journals Two populations of cytoplasmic dynein contribute to spindle positioning in C. elegans embryos

2017 ◽  
Vol 216 (9) ◽  
pp. 2777-2793 ◽  
Author(s):  
Ruben Schmidt ◽  
Lars-Eric Fielmich ◽  
Ilya Grigoriev ◽  
Eugene A. Katrukha ◽  
Anna Akhmanova ◽  
...  
Keyword(s):  
Cytoplasmic Dynein ◽  
Force Generation ◽  
Cell Cortex ◽  
Animal Cells ◽  
Spindle Positioning ◽  
C Elegans ◽  
Knockout Mutants ◽  
Pulling Forces ◽  
Endogenous Proteins ◽  
Pulling Force

The position of the mitotic spindle is tightly controlled in animal cells as it determines the plane and orientation of cell division. Contacts between cytoplasmic dynein and astral microtubules (MTs) at the cell cortex generate pulling forces that position the spindle. An evolutionarily conserved Gα-GPR-1/2Pins/LGN–LIN-5Mud/NuMA cortical complex interacts with dynein and is required for pulling force generation, but the dynamics of this process remain unclear. In this study, by fluorescently labeling endogenous proteins in Caenorhabditis elegans embryos, we show that dynein exists in two distinct cortical populations. One population directly depends on LIN-5, whereas the other is concentrated at MT plus ends and depends on end-binding (EB) proteins. Knockout mutants lacking all EBs are viable and fertile and display normal pulling forces and spindle positioning. However, EB protein–dependent dynein plus end tracking was found to contribute to force generation in embryos with a partially perturbed dynein function, indicating the existence of two mechanisms that together create a highly robust force-generating system.

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 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 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 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 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 Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo

2010 ◽  
Vol 108 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Kenji Kimura ◽  
Akatsuki Kimura
Keyword(s):  
Caenorhabditis Elegans ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Early Embryo ◽  
Organelle Transport ◽  
Animal Cells ◽  
C Elegans ◽  
Cell Functions ◽  
Intracellular Organelles ◽  
Pulling Force

The centrosome is generally maintained at the center of the cell. In animal cells, centrosome centration is powered by the pulling force of microtubules, which is dependent on cytoplasmic dynein. However, it is unclear how dynein brings the centrosome to the cell center, i.e., which structure inside the cell functions as a substrate to anchor dynein. Here, we provide evidence that a population of dynein, which is located on intracellular organelles and is responsible for organelle transport toward the centrosome, generates the force required for centrosome centration in Caenorhabditis elegans embryos. By using the database of full-genome RNAi in C. elegans, we identified dyrb-1, a dynein light chain subunit, as a potential subunit involved in dynein anchoring for centrosome centration. DYRB-1 is required for organelle movement toward the minus end of the microtubules. The temporal correlation between centrosome centration and the net movement of organelle transport was found to be significant. Centrosome centration was impaired when Rab7 and RILP, which mediate the association between organelles and dynein in mammalian cells, were knocked down. These results indicate that minus end-directed transport of intracellular organelles along the microtubules is required for centrosome centration in C. elegans embryos. On the basis of this finding, we propose a model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first model, to our knowledge, providing a mechanical basis for cytoplasmic pulling force for centrosome centration.

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

2018 ◽  
Vol 115 (5) ◽  
pp. E954-E963 ◽  
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.

scholarly journals Visualization of dynein-dependent microtubule gliding at the cell cortex: implications for spindle positioning

2011 ◽  
Vol 194 (3) ◽  
pp. 377-386 ◽  
Author(s):  
Eva M. Gusnowski ◽  
Martin Srayko
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Cortex ◽  
Early Prophase ◽  
Experimental Conditions ◽  
Spindle Positioning ◽  
Level Of Activity ◽  
Pulling Forces ◽  
Cell Embryo ◽  
Pulling Force ◽  
The One

Dynein motors move along the microtubule (MT) lattice in a processive “walking” manner. In the one-cell Caenorhabditis elegans embryo, dynein is required for spindle-pulling forces during mitosis. Posteriorly directed spindle-pulling forces are higher than anteriorly directed forces, and this imbalance results in posterior spindle displacement during anaphase and an asymmetric division. To address how dynein could be asymmetrically activated to achieve posterior spindle displacement, we developed an assay to measure dynein’s activity on individual MTs at the embryo cortex. Our study reveals that cortical dynein motors maintain a basal level of activity that propels MTs along the cortex, even under experimental conditions that drastically reduce anaphase spindle forces. This suggests that dynein-based MT gliding is not sufficient for anaphase spindle-pulling force. Instead, we find that this form of dynein activity is most prominent during spindle centering in early prophase. We propose a model whereby different dynein–MT interactions are used for specific spindle-positioning tasks in the one-cell embryo.

scholarly journals The tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division

2017 ◽  
Author(s):  
Kenji Sugioka ◽  
Lars-Eric Fielmich ◽  
Kota Mizumoto ◽  
Bruce Bowerman ◽  
Sander van den Heuvel ◽  
...  
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Asymmetric Cell Division ◽  
Cell Cortex ◽  
Colorectal Cancers ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
C Elegans ◽  
Pulling Forces ◽  
Pulling Force

AbstractThe adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/ß-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 C. 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 spindle-pulling force attenuation, which may be generally used in asymmetric cell division, and when disrupted potentially contributes to division defects in cancer.Significance StatementAPC (adenomatous polyposis coli) is a Wnt signaling component as well as a microtubule-associated protein, and its mutations are frequently associated with colorectal cancers in humans. Although APC stabilizes microtubules (MTs), its mechanical role during cell division is largely unknown. Here we show that APC is an attenuator of forces acting on the mitotic spindle during asymmetric cell division of the C. elegans zygote. We performed live-imaging, laser-microsurgery, and numerical simulation to show how APC suppresses spindle pulling force generation by stabilizing microtubule plus-ends and reducing microtubule catastrophe frequency at the cell cortex. Our study is the first to document a mechanical role for the APC protein, and provides a physical basis for spindle-pulling force attenuation.

scholarly journals Mechanisms of spindle positioning

2013 ◽  
Vol 200 (2) ◽  
pp. 131-140 ◽  
Author(s):  
Francis J. McNally
Keyword(s):  
Oocyte Maturation ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Meiotic Cell ◽  
Spindle Positioning ◽  
Animal Development ◽  
Cell Divisions ◽  
Spindle Poles ◽  
Pulling Force

Accurate positioning of spindles is essential for asymmetric mitotic and meiotic cell divisions that are crucial for animal development and oocyte maturation, respectively. The predominant model for spindle positioning, termed “cortical pulling,” involves attachment of the microtubule-based motor cytoplasmic dynein to the cortex, where it exerts a pulling force on microtubules that extend from the spindle poles to the cell cortex, thereby displacing the spindle. Recent studies have addressed important details of the cortical pulling mechanism and have revealed alternative mechanisms that may be used when microtubules do not extend from the spindle to the cortex.

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