scholarly journals The coordination of spindle-positioning forces during the asymmetric division of the C. elegans zygote is revealed by distinct microtubule dynamics at the cortex

2019 ◽  
Author(s):  
H. Bouvrais ◽  
L. Chesneau ◽  
Y. Le Cunff ◽  
D. Fairbrass ◽  
N. Soler ◽  
...  
Keyword(s):  
Direct Proof ◽  
Cell Periphery ◽  
Mitotic Progression ◽  
Spindle Positioning ◽  
Anteroposterior Axis ◽  
C Elegans ◽  
Pulling Force ◽  
The Stability ◽  
Force Generator ◽  
Spindle Position

ABSTRACTIn the Caenorhabditis elegans zygote, astral microtubules generate forces, pushing against and pulling from the cell periphery. They are essential to position the mitotic spindle. By measuring the dynamics of astral microtubules at the cortex, we revealed the presence of two populations, residing there for 0.4 s and 1.8 s, which correspond to the pulling and pushing events, respectively. Such an experiment offers a unique opportunity to monitor both forces that position the spindle under physiological conditions and study their variations along the anteroposterior axis (space) and the mitotic progression (time). By investigating pulling-force-generating events at the microscopic level, we showed that an anteroposterior asymmetry in dynein on-rate – encoding pulling-force imbalance – is sufficient to cause posterior spindle displacement. The regulation by spindle position – reflecting the number of microtubule contacts in the posterior-most region – reinforces this imbalance only in late-anaphase. Furthermore, we exhibited the first direct proof that the force-generator increasing persistence to pull (processivity) accounts for the temporal control of pulling force throughout mitosis. We thus propose a three-fold control of pulling force, by the polarity, spindle position and mitotic progression. Focusing on pushing force, we discovered a correlation between its density and the stability of the spindle position during metaphase, which strongly suggests that the pushing force contributes to maintaining the spindle at the cell centre. This force remains constant and symmetric along the anteroposterior axis during the division. The pulling one increases in intensity and becomes dominant at anaphase. In conclusion, the two-population study enabled us to decipher the complex regulation of the spindle positioning during cell division.

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

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.

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 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 EIPR1 controls dense-core vesicle cargo retention and EARP complex localization in insulin-secreting cells

2018 ◽  
Author(s):  
Irini Topalidou ◽  
Jérôme Cattin-Ortolá ◽  
Blake Hummer ◽  
Cedric S. Asensio ◽  
Michael Ailion
Keyword(s):  
Dense Core ◽  
Dense Core Vesicle ◽  
Cell Periphery ◽  
Interacting Protein ◽  
C Elegans ◽  
Insulin Secreting Cells ◽  
The Stability ◽  
Insulinoma Cells ◽  
Golgi Network

AbstractDense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargos including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting protein EIPR-1 as proteins important for controlling levels of DCV cargos in C. elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargos such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues inC. elegansembryos

Development ◽  
2002 ◽  
Vol 129 (19) ◽  
pp. 4469-4481 ◽  
Author(s):  
Meng-Fu Bryan Tsou ◽  
Adam Hayashi ◽  
Leah R. DeBella ◽  
Garth McGrath ◽  
Lesilee S. Rose
Keyword(s):  
Asymmetric Cell Division ◽  
Spindle Pole ◽  
Cellular Polarity ◽  
Wild Type ◽  
Spindle Positioning ◽  
C Elegans ◽  
Spindle Poles ◽  
Nuclear Rotation ◽  
Spindle Position ◽  
Asymmetric Regulation

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.

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 Human Microcephaly Protein RTTN Is Required for Proper Mitotic Progression and Correct Spindle Position

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1441
Author(s):  
En-Ju Chou ◽  
Tang K. Tang
Keyword(s):  
Brain Size ◽  
Neurodevelopmental Disorder ◽  
Mitotic Progression ◽  
Centriole Duplication ◽  
Spindle Poles ◽  
Multipolar Spindles ◽  
Mitotic Abnormalities ◽  
Cortical Localization ◽  
Spindle Position ◽  
Autosomal Recessive Primary Microcephaly

Autosomal recessive primary microcephaly (MCPH) is a complex neurodevelopmental disorder characterized by a small brain size with mild to moderate intellectual disability. We previously demonstrated that human microcephaly RTTN played an important role in regulating centriole duplication during interphase, but the role of RTTN in mitosis is not fully understood. Here, we show that RTTN is required for normal mitotic progression and correct spindle position. The depletion of RTTN induces the dispersion of the pericentriolar protein γ-tubulin and multiple mitotic abnormalities, including monopolar, abnormal bipolar, and multipolar spindles. Importantly, the loss of RTTN altered NuMA/p150Glued congression to the spindle poles, perturbed NuMA cortical localization, and reduced the number and the length of astral microtubules. Together, our results provide a new insight into how RTTN functions in mitosis.

scholarly journals The crosstalk between microtubules, actin and membranes shapes cell division

Open Biology ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 190314 ◽  
Author(s):  
Francesca Rizzelli ◽  
Maria Grazia Malabarba ◽  
Sara Sigismund ◽  
Marina Mapelli
Keyword(s):  
Membrane Trafficking ◽  
Mitotic Progression ◽  
Isolated Cells ◽  
Spindle Positioning ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Epithelial Sheets ◽  
Cell Substrate Adhesion ◽  
Extracellular Signalling

Mitotic progression is orchestrated by morphological and mechanical changes promoted by the coordinated activities of the microtubule (MT) cytoskeleton, the actin cytoskeleton and the plasma membrane (PM). MTs assemble the mitotic spindle, which assists sister chromatid separation, and contact the rigid and tensile actomyosin cortex rounded-up underneath the PM. Here, we highlight the dynamic crosstalk between MTs, actin and cell membranes during mitosis, and discuss the molecular connections between them. We also summarize recent views on how MT traction forces, the actomyosin cortex and membrane trafficking contribute to spindle positioning in isolated cells in culture and in epithelial sheets. Finally, we describe the emerging role of membrane trafficking in synchronizing actomyosin tension and cell shape changes with cell–substrate adhesion, cell–cell contacts and extracellular signalling events regulating proliferation.

scholarly journals Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

2006 ◽  
Vol 17 (8) ◽  
pp. 3557-3568 ◽  
Author(s):  
James C. Warren ◽  
Adam Rutkowski ◽  
Lynne Cassimeris
Keyword(s):  
Host Cell ◽  
Dynamic Instability ◽  
Infection Process ◽  
Microtubule Dynamics ◽  
Cell Periphery ◽  
Adenovirus Infection ◽  
Drug Induced ◽  
Infected Cells ◽  
The Stability ◽  
Induced Changes

Adenovirus translocation to the nucleus occurs through a well characterized minus end-directed transport along microtubules. Here, we show that the adenovirus infection process has a significant impact on the stability and dynamic behavior of host cell microtubules. Adenovirus-infected cells had elevated levels of acetylated and detyrosinated microtubules compared with uninfected cells. The accumulation of modified microtubules within adenovirus-infected cells required active RhoA. Adenovirus-induced changes in microtubule dynamics were characterized at the centrosome and at the cell periphery in living cells. Adenovirus infection resulted in a transient enhancement of centrosomal microtubule nucleation frequency. At the periphery of adenovirus-infected cells, the dynamic instability of microtubules plus ends shifted toward net growth, compared with the nearly balanced growth and shortening observed in uninfected cells. In infected cells, microtubules spent more time in growth, less time in shortening, and underwent catastrophes less frequently compared with those in uninfected cells. Drug-induced inhibition of Rac1 prevented most of these virus-induced shifts in microtubule dynamic instability. These results demonstrate that adenovirus infection induces a significant stabilizing effect on host cell microtubule dynamics, which involve, but are not limited to, the activation of the RhoGTPases RhoA and Rac1.

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