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[
International C. elegans Meeting,
2001]
Cell divisions creating daughters of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates into asymmetric spindle positioning remain poorly understood. We examined the nature of the forces governing asymmetric spindle positioning in the single cell stage C. elegans embryo. In order to reveal the forces acting on each spindle pole, we removed the central spindle in living embryos either physically with a UV laserbeam, or genetically by RNA mediated interference (RNAi) of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thus explaining the overall posterior displacement observed in wild-type embryos. Importantly, we further show that the net force acting on each spindle pole is under control of the par genes required for cell polarity along the anterior-posterior (AP) embryonic axis. In addition, we demonstrate that inactivation of heterotrimeric G protein alpha subunits results in a loss of external forces (see abstract by Colombo et al.), thus explaining the symmetric phenotype observed in these cases. In summary, our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force acting on each spindle pole.
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[
International Worm Meeting,
2003]
During unequal cell division the mitotic spindle is eccentrically positioned prior to cell cleavage. In one-cell C. elegans embryos this off-center location is determined by an imbalance in polarity-controlled forces acting on the spindle poles. The mechanistic basis for this difference in net force is unknown. By disintegrating centrosomes with a UV laser and by performing a fluctuation analysis on the movement of centrosomal fragments, we find that this imbalance results from an increase in the number of force generators available to pull upon astral microtubules of the posterior compared to the anterior aster. Furthermore, a functional G signaling pathway is required to generate astral force, and is therefore a critical link between cortical polarity and the dynamic behavior of the mitotic spindle.
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[
International Worm Meeting,
2011]
Establishment of polarity is essential for conferring different developmental fates to the dividing cells of an embryo. In Caenorhabditis elegans one cell embryos, anteroposterior polarization is facilitated by long-ranged flow of the actomyosin cortex. Even though the flowing cortex contains many actin binding proteins (ABPs) that contribute to its structure and dynamics, there are only a limited number of mechanical properties that are important at large length and time scales relevant for polarization, for example contractility and cortical viscosity (Mayer, Bois, Depken, Julicher, Grill, 2010). Importantly, this suggests that there is only a reduced spectrum of cortical flow phenotypes that one might expect to obtain by modulating these few mechanical properties through different molecular mechanisms. To bridge the gap between molecular and cellular scales, we here sought to investigate which cell-scale mechanical properties are controlled by which ABPs. We devised a candidate RNAi screen of ABPs and found that several ABPs affect cortical flow. This was achieved by analyzing myosin foci size and density and several flow characteristics, such as peak velocities and spatio-temporal velocity-velocity correlations, for each ABP knockdown. The velocity-velocity correlations provided us with an estimation of the characteristic hydrodynamic length of cortical flow, which describes the extent to which flows are long-ranged. Interestingly, all those ABPs that displayed a detectable cortical flow phenotype did so through affecting this hydrodynamic length. RNAi either resulted in short-ranged flows, indicative of a less viscous cortex, or it resulted in flows that were longer-ranged than wild type, indicative of a cortex that is more viscous that under wild-type conditions. Our results suggest that the characteristic hydrodynamic length is a central physical property subject to precise regulation. They also point towards a type of "mechanical redundancy" in animal development, with many molecular mechanisms affecting the same cell-scale physical property.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Polarization of the one-cell embryo proceeds through the establishment of two mutually exclusive cortical domains. These domains define the anterior and posterior axis and specify the unequal inheritance of cell fate determinants during the first asymmetric cell division. The formation of these domains is generally thought to rely on mutually antagonistic interactions between two sets of conserved polarity proteins: PAR-3, PAR-6 and aPKC which define the anterior domain, and PAR-1 and PAR-2 which define the posterior. A key outstanding question in polarity establishment is how these molecular interactions between individual PAR complexes combine to generate a stable and properly posit ioned domain boundary, particularly one that stably separates two PAR domains that span several hundred square microns. We have analyzed the distribution and dynamics of PAR proteins in live embryos, focusing on the interface region between the two PAR domains during maintenance phase. Our data suggests that this interface consists of overlapping diffusive gradients that arise as PAR proteins diffuse laterally out of their respective domains and into the opposing domains where they are subject to displacement by mutual antagonism. We conclude that the stably positioned PAR boundary likely reflects a non-equilibrium steady-state maintained through continuous cycles of membrane association, lateral diffusion of PAR proteins down their respective concentration gradients spanning the PAR interface, and ultimately displacement back into the cytoplasm as PAR proteins encounter one another within the boundary region.
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[
International Worm Meeting,
2021]
Establishment of the major body axes is essential for embryonic development. The C. elegans dorsoventral axis is established during the two-cell stage, and its orientation is defined by the division of the anterior AB blastomere. Earlier work in various holoblastic animals has shown that embryo geometry strongly affects early embryonic division patterns. However, whether and how embryo geometry affects the orientation of the dividing AB cell, and thereby the dorsoventral axis is unknown. To study this, we performed quantitative 3D live imaging of the cytoskeletal machinery in dividing AB blastomeres of embryos that are compressed perpendicular to the anteroposterior axis. In agreement with previous studies, we find that the AB cell division, and therefore the future dorsoventral axis, aligns parallel to the long cellular axis (which is perpendicular to the compression direction). Moreover, we show that, when viewed down the anteroposterior axis, the mitotic spindle of the AB blastomere is initially oriented randomly with respect to the long axis. During spindle elongation, it undergoes a rapid, large scale rotation to finally align parallel with the long axis. This mitotic spindle rotation is accompanied by a rotation of the cytokinetic furrow, ultimately resulting in the cell division axis to be aligned with the long cellular axis. Although the molecular mechanisms are still topic of debate, the mitotic spindle in many contexts is known to find the long axis of the cell. However, by performing conditional genetic perturbations, we find that force generation in the actomyosin layer is required and governs the kinetics of alignment of the mitotic spindle along the future DV axis. We speculate that the cooperative action of spindle and cortex promotes timely alignment of the cytokinetic machinery in fast-dividing cells.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Asymmetric cell divisions are essential for the development of multicellular organisms. During the first division of the C. elegans zygote, antero-posterior polarization is established by a large-scale flow of the actomyosin cortex, which segregates cortical and cytoplasmic fate determinants. A gradient in cortical tension, thought to arise from an uneven myosin distribution, has been proposed as the driving force for cortical flow. However, the simple picture of an elastic relaxation fails to explain how the cortex undergoes continuous flows over several minutes. To investigate the forces driving flow, we sought to understand the relationship between cortical flows, contractility and tension. Using UV laser ablation, we measure tension in the embryo cortex in a location-and direction-dependent manner. We find that orthogonal to flow, cortical tension differs between the anterior and posterior domain, and is regulated by the Rho-GTP cycle. We furthermore reveal an anterior anisotropy in tension, with higher tension across than along the AP axis. In contrast, tension is isotropically high throughout the cortex when polarization is impaired, demonstrating that anisotropy is a direct consequence of flow. Notably, cortical tension along the AP axis does not differ between anterior and posterior, i.e. flows are not associated with tension gradients. A quantification of myosin density and flow profiles together with a theoretical analysis reveal that the cortex needs to be sufficiently viscous to support long-range cortical rearrangements and robust polarization. Our results demonstrate that contractile gradients, but not tension gradients, constitute the driving force of cortical flow, and that a highly viscous cortex enables flows to be long-ranged. Cortical flows play an important role in other developmental stages, both during C. elegans embryogenesis and other biological systems, and we anticipate that our findings are fundamental to various flow-dependent processes.
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[
International Worm Meeting,
2015]
During cytokinesis' onset the recruitment of multiple proteins is orchestrated by specific signaling events while reproducible cortical actin flows have been observed. This brings up the question of the contribution of the mechanics during this initial phase of ring assembly. In this study we investigate how myosin induced cortical flows impact actin network organization and filament alignment, thereby directly initiating stable ingression by mechanically remodeling the architecture.We perform our analysis in the early C. elegans zygote. In this organism, a pseudocleavage furrow is formed during the phase of polarizing cortical flows, at a time when the biochemical triggers involved in cytokinesis are not yet present. In this system, we can thus uncouple the mechanical contributions to furrow formation from the biochemical effects involved in the later stages of cytokinesis. We observed that actin filaments align in converging and compressive flow. Hence, alignment and order arises in a disordered network in response to compression by flow and orthogonally to its direction. We show that this partially ingressing furrow is a direct consequence of actin filament alignment induced by the initial flow phase. Quantitative analysis of the dynamics of flows, filaments orientation and cell shape changes, together with theoretical modeling in the framework of a thin film of active and nematic fluid, allowed for a precise characterization of how deformation and shear in the cortical flow gives rise to ordering of actin filaments. Notably, such cortical dynamics and pattern formation arise in a very similar manner both during pseudocleavage and cytokinesis phases.Taken together, our work paints a simple picture of how furrow ingression arises: the presence of a contractility gradient induces the formation of convergent flows, which in turn result in the local alignment of filaments. Filament alignment by compressive flow in turn drives asymmetric stress generation and furrow ingression. We thus identify the key physical principles that lead to the generation of an ingression for cytokinesis.
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[
European Worm Meeting,
2006]
Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.
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Chowdury, Debanjan, Nicola, Ernesto, Grill, Stephan W., Khuc-Trong, Philip, Hyman, Anthony A, Goehring, Nathan W.
[
International Worm Meeting,
2009]
A conserved network of PAR polarity proteins is required for establishment of cellular polarity in a wide variety of systems. These proteins are typically segregated into mutually exclusive, functionally distinct membrane domains which define the axis of polarity. In the one-celled C. elegans embryo, discrete sets of PAR proteins are partitioned into roughly equal sized anterior and posterior domains, which are essential for the proper inheritance of cell fate determinants during the asymmetric first cell division. The formation of PAR domains has been shown to depend on a highly contractile cortical actomyosin network that is itself polarized and is required for establishing PAR polarity. PAR domains also depend on mutual antagonistic interactions between PAR proteins, which are essential for maintaining their stable, mutually exclusive distribution within discrete domains. However, despite progress in understanding the molecular activities involved in these processes, we lack a basic physical mechanism for explaining how the activities of the actomyosin cortex and the PAR proteins are coupled in order to give rise to the stable, reproducibly sized PAR domains that are observed in the embryo. In order to provide insight into the types of mechanisms that could underlie the formation of PAR domains, we undertook a quantitative description of the dynamics of PAR proteins in C. elegans embryos. The results of this analysis indicate that PAR proteins diffuse extensively on the membrane of embryos, exchange between membrane-associated and cytoplasmic states, and are subject to advection by actomyosin-dependent cortical flow. We find that these observed behaviors, when coupled to the documented antagonism between PAR proteins, are sufficient to generate a reaction-diffusion driven system for establishing PAR polarity. The theoretical model we propose provides a single, rather simple physical mechanism that explains the actomyosin-dependent polarization of the embryo, the maintenance of mutually exclusive PAR domains, and the reproducible determination of domain size. Importantly, in this model the stable partitioning of the embryo into domains is a function of intrinsic properties of the PAR proteins and does not appear to depend on an underlying scaffold function of the actomyosin cortex. Rather, this work suggests that the actomyosin cortex acts primarily through the generation of cortical flow which provides a robust trigger to ensure that polarization of the PAR system occurs with the proper timing and geometry.
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Myers, Gene, YANG, Xinyi, MAGHELLI, Nicola, ROYER, Loic, Grill, Stephan, FERRARO, Teresa, Labouesse, Michel, PONTABRY, Julien
[
International Worm Meeting,
2017]
The process of morphogenesis in C. elegans embryos is largely driven by epidermal cells. Unlike Drosophila and zebrafish, no cell division or cell rearrangement is involved in C. elegans morphogenesis. Epidermis shape changes, which are characterized by junction lengthening along the anterior/posterior (A/P) direction, play a key role in this process. The nature of junction lengthening and planar polarity establishment, as well as the cellular mechanisms involved in these processes during C. elegans embryonic elongation are the main objectives of this project. Our lab observed that junction elongation along the A/P direction increases after muscle becomes active, and fails in muscle defective embryos. To better understand which role muscles play in polarized junction lengthening, we examined the global and local movement patterns of embryo using Single Plane Illumination Microscopy, focusing on epidermal adherens junctions and muscle nuclei. We found that wild-type embryos rotated strongly soon after muscle became active, and equally frequently to an outward or inward direction. However, muscle defective and Rho-kinase mutant embryos, which stop elongation at the 2-fold stage, scarcely rotated, suggesting that rotations are important for embryo elongation. By comparing the changes of seam cell aspect ratio, we observed that the head, body and tail mechanically behaved as partially independent entities. We next sought to understand how such movements could account for the polarized junction lengthening, keeping in mind that C. elegans embryos are radially symmetric. By measuring the distance between two dorsal or ventral muscle nuclei, respectively, we found that dorsal and ventral muscles mostly contract alternatively, accounting for embryo rotations. Intriguingly, analysis of junction roughness showed that during embryo rotations, junctions along the A/P direction were stretched when seam cells were positioned outwards. Laser ablation targeting DLG-1::GFP results further discovered that these junctions were under higher tension when stretched. These results suggest that asymmetric muscle activity defines the source of polarity in C. elegans embryo and provides the local driving force for epidermis stretching. To study how this tension impacts polarized junction lengthening, we are investigating the insertion of new E-cad molecule into junctions during embryo rotations by single molecule imaging. Altogether, our results suggest that C. elegans embryos extend in a ratchet mode due to the alternating muscle contractions.