Object Segmentation and Ground Truth in 3D Embryonic Imaging

Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets

Stem cell differentiation increases membrane-actin adhesion regulating cell blebability, migration and mechanics

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/K. S. is funded by an EPSRC PhD studentship. S.T. is funded by an EU Marie Curie Intra European Fellowship (GENOMICDIFF)

Pléchâtel – Les Landes de Lanserva

À environ 200 m au sud du site néolithique de La Hersonnais, plusieurs tertres à faible élévation et de forme oblongue avaient été signalés dans les Landes de Lanserva et l’un d’eux a fait l’objet d’une opération préventive en 1981 (rapport de Maurice Gautier, 1981), en prévision de la réalisation du nouvel axe routier Rennes-Nantes. En l’absence de structures et de mobilier, les sondages de reconnaissance s’étaient révélés négatifs sur ce premier tertre aujourd’hui disparu. Parallèlement à l..

Mouvements actifs, regules par le calcium, de la touffe ciliaire des cellules ciliées mecano-sensorielles de l'oreille interne. .

The dynamical behaviour of a hair bundle – the mechanosensitive organelle of the hair cells found in the inner ear – is rich. A hair bundle can oscillate spontaneously, “twitch” or simply relax in response to a force step.Using iontophoresis to affect the Ca2+ concentration near a hair bundle from the bullfrog’s sacculus and displacement-clamp measurements of the bundle’s force-displacement relations, we were able to reconcile these contrasting manifestations of active hair-bundle motility. We used Ca2+ and offsets of the bundle’s mean position to control the fraction of open transduction channels at steady state and thus the bundle’s operating point. In the case of non oscillatory hair bundles, we found that the polarity and kinetics of active hair-bundle movement evoked by a step stimulus depended on the bundle’s operating point in the nonlinear force- displacement relation. When the force-displacement relation displayed a region of negative stiffness, spontaneous hair-bundle oscillations arose when the hair bundle was required to operate within this unstable region.Only two ingredients are necessary to account for the various incarnations of active hair-bundle motility: non-linear gating compliance of the transduction apparatus and the Ca2+-regulated activity of the myosin-based adaptation motor. Numerical simulations successfully reproduced a wide range of observations from different experimental situations and animal species, thereby suggesting that only one force-generating mechanism is needed to describe the seemingly opposite movements that the hair-bundle can produce.Le comportement dynamique d’une touffe ciliaire (l’organelle mécano-sensible des cellules ciliées de l’oreille interne) est très varié. Une touffe ciliaire peut osciller spontanément, avoir une réponse de type « excitable » ou simplement relaxer en réponse à un échelon de force.En utilisant la iontophorèse pour modifier la concentration en calcium à proximité d’une touffe ciliaire du saccule de la grenouille taureau, et en mesurant les relations force/déplacements de cette organelle, nous sommes parvenus à réconcilier ces manifestations contrastées de sa motilité active. Nous utilisons le calcium et/ou des biais statiques appliqués à la position de la touffe ciliaire pour contrôler la probabilité d’ouverture au repos et par là, son point de fonctionnement. Dans le cas de touffes ciliaires non oscillantes, nous montrons que la polarité et la cinétique des mouvements ciliaires actifs observés en réponse à un échelon de stimulation dépendent du point de fonctionnement de la cellule. Lorsque la relation force/déplacement de la touffe ciliaire possède une région de raideur négative, des oscillations spontanées peuvent être déclenchées lorsque le point de fonctionnement est placé au sein de cette région instable.Seuls deux ingrédients sont nécessaires pour interpréter ces manifestations de la motilité ciliaire active : l’existence d’une relation force/déplacement non linéaire conséquence de l’activation mécanique directe des canaux de transduction, et l’activité du moteur d’adaptation, fondé sur les myosines et dépendant du calcium. Des simulations numériques reproduisent avec succès un grand nombre d’observations expérimentales, dans des conditions différentes et sur des animaux variés, suggérant qu’un seul mécanisme actif est nécessaire pour décrire toute la gamme des mouvements actifs observés sur les touffes ciliaires des cellules ciliées

Synthetic asters as elastic and radial skeletons

The radial geometry with rays radiated from a common core occurs ubiquitously in nature for its symmetry and functions. Herein, we report a class of synthetic asters with well-defined core-ray geometry that can function as elastic and radial skeletons to harbor nano- and microparticles. We fabricate the asters in a single, facile, and high-yield step that can be readily scaled up; specifically, amphiphilic gemini molecules self-assemble in water into asters with an amorphous core and divergently growing, twisted crystalline ribbons. The asters can spontaneously position microparticles in the cores, along the radial ribbons, or by the outer rims depending on particle sizes and surface chemistry. Their mechanical properties are determined on single- and multiple-aster levels. We further maneuver the synthetic asters as building blocks to form higher-order structures in virtue of aster-aster adhesion induced by ribbon intertwining. We envision the astral structures to act as rudimentary spatial organizers in nanoscience for coordinated multicomponent systems, possibly leading to emergent, synergistic functions

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin-driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase

Objective comparison of particle tracking methods

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers

Villevêque – Les Pâtures

Cette intervention a fait suite à des prospections systématiques le long du tracé de l’autoroute A11 Le Mans-Angers. À la confluence de deux petits ruisseaux, sur une légère pente exposée au nord, est apparue une zone de plus forte concentration de silex (avec nucléus à débitage lamellaire). Après décapage mécanique de la terre labourée, environ 48 m2 ont été fouillés. La nature générale des sédiments est un sable alluvionnaire reposant sur des marnes à ostracées. Dans la couche supérieure, d..

RhoD Inhibits RhoC-ROCK-Dependent Cell Contraction via PAK6.

RhoA-mediated regulation of myosin-II activity in the actin cortex controls the ability of cells to contract and bleb during a variety of cellular processes, including cell migration and division. Cell contraction and blebbing also frequently occur as part of the cytopathic effect seen during many different viral infections. We now demonstrate that the vaccinia virus protein F11, which localizes to the plasma membrane, is required for ROCK-mediated cell contraction from 2 hr post infection. Curiously, F11-induced cell contraction is dependent on RhoC and not RhoA signaling to ROCK. Moreover, RhoC-driven cell contraction depends on the upstream inhibition of RhoD signaling by F11. This inhibition prevents RhoD from regulating its downstream effector Pak6, alleviating the suppression of RhoC by the kinase. Our observations with vaccinia have now demonstrated that RhoD recruits Pak6 to the plasma membrane to antagonize RhoC signaling during cell contraction and blebbing

Automated cell tracking using StarDist and TrackMate [version 1; peer review: awaiting peer review]

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images