Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay

A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse. We have used this system to examine the perturbation of elements of the mitotic spindle, which is a microtubule-based bipolar structure involved in the segregation of the replicated genome to daughter cells during cell division. First, we used the small molecule inhibitor monastrol to target kinesin-5, a molecular motor that maintains the microtubule spindle bipolarity. Next, we explored the dynamics of the mitotic spindle by monitoring the exchange between unpolymerized and polymerized tubulin within microtubule fibers. These results show that a simple diffusion-based system can generate biochemical perturbations directly within a cell-free cytoplasm based on Xenopus egg extracts at the time scale of minutes. Our assay is therefore suitable for monitoring the dynamics of supramolecular assemblies within cell-free extracts in response to perturbations. This strategy opens up broad perspectives including phenotype screening or mechanistic studies of biological assembly processes and could be applied to other cell-free extracts such as those derived from mammalian or bacterial cells

A rhenium tris-carbonyl derivative as a single core multimodal probe for imaging (SCoMPI) combining infrared and luminescent properties.

International audienceA rhenium tris-carbonyl derivative has been designed to couple infrared and luminescent detection in cells. Both spectroscopies are consistent with one another; they point out the reliability of the present SCoMPI (for Single Core Multimodal Probe for Imaging) for bimodal imaging and unambiguously indicate a localization at the Golgi apparatus in MDA-MB-231 breast cancer cells

Observation par microscopie de fluorescence de la transcription de l'aon

LYON-ENS Sciences (693872304) / SudocSudocFranceF

Effets du confinement sur l auto-organisation du cytosquelette

Le but de cette thèse a été d étudier quantitativement l effet d un confinement physique, mimant les conditions de compartimentations cellulaires, sur des processus auto-organisationnels de fibres du cytosquelette. Nous avons développé une méthode permettant l encapsulation au sein de gouttelettes et de vésicules d un extrait cellulaire de Xénope, formant un aster à partir des interactions entre microtubules et moteurs moléculaires. Nous avons en particulier montré que cette auto-organisation dépendait fortement de la dimension du confinement. Trois structures distinctes de microtubules, chacune associée à une gamme de taille ont été ainsi observées : asters, asters asymétriques et faisceaux corticaux. Les transitions pour lesquelles les brisures de symétries apparaissent, se produisent pour des diamètres bien définis. Ces observations ont été expliquées qualitativement par un modèle physique simple et reproduites par des simulations numériques prenant en compte les propriétés physiques intrinsèques des objets biologiques (élasticité des microtubules, moteurs moléculaires ). Une seconde étape, consistant à encapsuler l extrait cellulaire, au sein de vésicules a permis d observer de nouvelles structures en forme de protrusions, mettant en évidence le rôle de la rigidité du confinement. L utilisation de cette méthode a permis de réaliser des expériences préliminaires sur l organisation d un réseau d actine au sein de gouttelettes et a mis en évidence la présence d un flux directionnel des filaments d actine pouvant aboutir à la formation d un anneau contractile, reproduisant ainsi une des étapes essentielles de la cytokinèse.We studied the effect of a confined physical environment on cytoskeleton self-organized processes. In order to reproduce the cell s confinement, we developed an efficient bottom-up approach to encapsulate a Xenopus cell extract within droplets and vesicles. The qualitative and quantitative studies on a microtubule aster encapsulated within droplets showed three different patterns: asters, asymmetrical asters, and cortical bundles. Monitoring of the morphologies adopted by microtubule arrays showed that symmetry breaking in the organization of microtubules and formation of an aster in a spherical environment is dependent on the size of the droplet. Computer simulations based on physical considerations reproduced the various structures observed, and a simple physical model identified the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structure, such as protrusions, highlighting the importance of the rigidity of the confinement. We also used this system and this approach to study the re-organization of the actin network within droplets. Preliminary results demonstrate the existence of an actin directional flow around a contractile ring, reproducing in vivo processes present during cytokinesis.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Self-Assembly of Magnetically-Functionalized Molecular Motors and Microtubules into Active Gels

International audienceThe diversity of functions achieved by living cells result from the collective behavior of biological components that interact through multiple scales in time and space. The cytoskeleton constitutes one canonical system forming dynamic organizations when interacting with molecular motors. These materials constitute a state of active matter that exhibit out-of-equilibrium behavior with oriented order in the presence of energy. However, such active materials are highly dependent on the intrinsic properties of their constituents (fibers, molecular motors, and energy), which makes it difficult to control their behavior. Being able to manipulate directly the constitutive elements of the active gel could provide additional control parameters. Here, we report a strategy to functionalize and manipulate active microtubule-based structures upon magnetic actuation. We engineered protein nanocage ferritins as magnetic labels targeting molecular motors (Eg5 kinesin motors). We first mixed these magnetic motors with individual microtubules, allowing for their manipulation. In order to generate a magnetic-responsive gel, we then mixed the magnetic motors with active microtubule-based structures and characterized their dynamic behavior. We found that the magnetic forces applied on magnetic motors slowed down the dynamics of the microtubule structures as well as constrained their rotation. Our results highlight how genetically encoded magnetic elements, behaving as magnetic actuators, could perturb active gels

Actin-Network Architecture Regulates Microtubule Dynamics

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Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function

International audienceThe spatial regulation of messenger RNA (mRNA) translation is central to cellular functions and relies on numerous complex processes. Biomimetic approaches could bypass these endogenous complex processes, improve our comprehension of the regulation, and allow for controlling local translation regulations and functions. However, the causality between local translation and nascent protein function remains elusive. Here, we developed a nanoparticle (NP)-based strategy to magnetically control mRNA spatial patterns in mammalian cell extracts and investigate how local translation impacts nascent protein localization and function. By monitoring the translation of the magnetically localized mRNAs, we show that mRNA–NP complexes operate as a source for the continuous production of proteins from defined positions. By applying this approach to actin-binding proteins, we triggered the local formation of actin cytoskeletons and identified the minimal requirements for spatial control of the actin filament network. In addition, our bottom-up approach identified a role for mRNA as a translation-coupled scaffold for the function of nascent N-terminal protein domains. Our approach will serve as a platform for regulating mRNA localization and investigating the function of nascent protein domains during translation

Towards high throughput production of artificial egg oocytes using microfluidics.

International audienceThe production of micron-size droplets using microfluidic tools offers new opportunities to carry out biological assays in a controlled environment. We apply these strategies by using a flow-focusing microfluidic device to encapsulate Xenopus egg extracts, a biological system recapitulating key events of eukaryotic cell functions in vitro. We present a method to generate monodisperse egg extract-in-oil droplets and use high-speed imaging to characterize the droplet pinch-off dynamics leading to the production of trains of droplets. We use fluorescence microscopy to show that our method does not affect the biological activity of the encapsulated egg extract by observing the self-organization of microtubules and actin filaments, two main biopolymers of the cell cytoskeleton, encapsulated in the produced droplets. We anticipate that this assay might be useful for quantitative studies of biological systems in a confined environment as well as high throughput screenings for drug discovery

α-Synuclein liquid condensates fuel fibrillar α-synuclein growth

International audienceα-Synuclein (α-Syn) aggregation into fibrils with prion-like features is intimately associated with Lewy pathology and various synucleinopathies. Emerging studies suggest that α-Syn could form liquid condensates through phase separation. The role of these condensates in aggregation and disease remains elusive and the interplay between α-Syn fibrils and α-Syn condensates remains unexplored, possibly due to difficulties in triggering the formation of α-Syn condensates in cells. To address this gap, we developed an assay allowing the controlled assembly/disassembly of α-Syn condensates in cells and studied them upon exposure to preformed α-Syn fibrillar polymorphs. Fibrils triggered the evolution of liquid α-Syn condensates into solid-like structures displaying growing needle-like extensions and exhibiting pathological amyloid hallmarks. No such changes were elicited on α-Syn that did not undergo phase separation. We, therefore, propose a model where α-Syn within condensates fuels exogenous fibrillar seeds growth, thus speeding up the prion-like propagation of pathogenic aggregates