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Biology subjects

Gueroui, Z.

Publications and source records attributed to Gueroui, Z..

9 recordsLinked to original sources

Coupled nucleoplasmic flows and envelope expansion govern the mechanical resistance of the nucleus

In recent year, it became clear that the cell nucleus can undergo large deformations, during immune cell migration and tumor growth. These deformations generate signals that allow cells to sense their environment and adapt to it. How cells cope with and respond to large deformations thus strongly depends on the nuclear mechanics, but our understanding of the physical properties of the nucleus remains incomplete. In particular, it is not clear how the nuclear volume responds to deformation. Here we combine controlled confinement assays, high-resolution imaging and atomic force microscopy with theoretical modelling to propose a physical model of the cell nucleus that accounts for its surface and bulk properties and addresses both steady-state and transient regimes. Our results establish the nucleus as a poroelastic body in which mechanics are dominated by the envelope and dynamics by the chromatin, and suggest that regulating water permeability may be as important as softening the envelope for cells migrating rapidly through dense tissues.

biophysics↗

Controlling lipid droplet dynamics via tether condensates

Lipid droplets (LDs) exhibit remarkable diversity and functionality within cells, depending on the metabolic needs of cells and the maintenance of lipid homeostasis. Such versatility is acquired through dynamic spatial and temporal positioning, enabling tight communication with other organelles. However, this complexity poses challenges in understanding LD biology. Controlled sequestration and release of LDs within their intracellular environment could offer a method to synchronize their behavior and better understand their function. Here, to advance in this direction, we developed ControLD (Controlled Trapping of Lipid Droplets), a novel approach designed to manipulate LDs and influence their dynamics and life cycle. By orchestrating the assembly/disassembly of engineered condensates, ControLD allows precise sequestration and release of LDs in cells. This technique effectively isolates LDs from the intracellular environment, drastically reducing interactions with other organelles. Notably, our experiments demonstrate that physically isolating LDs impairs their dynamics and remobilization during metabolic needs. ControLD represents a versatile tool for reversible LD trapping, with potential applications in controlling various cellular organelles.

cell biology↗

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

biophysics↗

Condensate functionalization with motors directs their nucleation in space and allows manipulating RNA localization

The localization of RNAs in cells is critical for many cellular processes. Whereas motor-driven transport of RNP condensates plays a prominent role in RNA localization in cells, their studies remain limited by the scarcity of available tools allowing to manipulate condensates in a spatial manner. To fill this gap, we reconstitute in cellula a minimal RNP transport system based on bioengineered condensates which were functionalized with kinesins and dynein-like motors, allowing for their positioning at either the cell periphery or centrosomes. This targeting mostly occurs through the active transport of the condensate scaffolds, which leads to localized nucleation of phase-separated condensates. Then, programming the condensates to recruit specific mRNAs is able to shift the localization of these mRNAs towards the cell periphery or the centrosomes. Our method opens novel perspectives to examine the role of RNA localization as a driver of cellular functions.

cell biology↗

RNA at the surface of phase-separated condensates impacts their size and number

Membrane-less organelles, by localizing and regulating complex biochemical reactions, are ubiquitous functional subunits of intracellular organization. They include a variety of nuclear and cytoplasmic ribonucleoprotein (RNP) condensates, such as nucleoli, P-bodies, germ granules and stress granules. While is it now recognized that specific RNA and protein families are critical for the biogenesis of RNP condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells with controlled RNA and protein composition. Our bioengineered condensates are made of ArtiGranule scaffolds undergoing liquid-liquid phase separation in cells and programmed to specifically recruit a unique RNA species. We found that RNAs localized on condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. This simplified system allowed us to demonstrate that the size of the condensates scales with RNA surface density, the higher the RNA density is, the smaller and more frequent the condensates are. Our observations suggest a mechanism based on physical constraints, provided by RNAs localized on condensate surface, that limit condensate growth and coalescence.

cell biology↗

Engineering E. coli for magnetic control and the spatial localization of functions

The fast-developing field of synthetic biology enables broad applications of programmed microorganisms including the development of whole-cell biosensors, delivery vehicles for therapeutics, or diagnostic agents. However, the lack of spatial control required for localizing microbial functions could limit their use and induce their dilution leading to ineffective action or dissemination. To overcome this limitation, the integration of magnetic properties into living systems enables a contact-less and orthogonal method for spatiotemporal control. Here, we generated a magnetic-sensing Escherichia coli by driving the formation of iron-rich bodies into bacteria. We found that these bacteria could be spatially controlled by magnetic forces and sustained cell growth and division, by transmitting asymmetrically their magnetic properties to one daughter cell. We combined the spatial control of bacteria with genetically encoded-adhesion properties to achieve the magnetic capture of specific target bacteria as well as the spatial modulation of human cell invasions.

synthetic biology↗

Centering based on active diffusion in mouse oocytes is non-specific

The mechanism for nucleus centering in mouse oocytes results from a gradient of actin-positive vesicles. By microinjecting oil droplets and fluorescent beads, we analyze the consequences of the gradient of activity on transport of exogenous tracer particles of different sizes. We also use optical tweezers to probe rheological properties of the cytoplasm. We find that the gradient activity induces a general centering force, akin to an effective pressure gradient, leading to centering of oil droplets with velocities comparable to nuclear ones. High temporal resolution measurements reveal that passive particles, larger than 1{micro}m, experience the activity gradient by a biased diffusion towards the cell center. Unexpectedly, this general and size dependent mechanism is maintained in Meiosis I but contrasted by a further process that specifically off-centers the spindle. These antagonizing processes depend on myosin activity, thus we reconcile how the same molecular actors can have two opposite functions (centering versus off-centering).

cell biology↗

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

Spatial regulations of mRNA translation are central to cellular functions and relies on numerous complex processes. Biomimetic approaches could bypass the endogenous complex processes, improve our comprehension, and allow for controlling local translation regulations and functions. However, the causality between localizing translation and nascent protein function remains elusive. Here, we develop a novel nanoparticle-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 translation on magnetically localized mRNAs, we show that mRNA-nanoparticle operates as a source for the continuous production of proteins from defined positions. By applying magnetic localization of mRNAs coding for Actin Binding Proteins, we trigger the local formation of actin cytoskeleton and identify minimal requirements for spatial control of actin filament network. In addition, our bottom-up approach identifies a novel role of mRNA as translation-coupled scaffold for nascent N-terminal protein domain functions. Our approach will serve as a novel platform for regulating mRNA localization and investigating a functional role of nascent protein domains during translation.

molecular biology↗

RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates

Liquid-liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Here we report the versatile scaffold \"ArtiG\" to generate concentration-dependent RNA-protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high order RNP assemblies. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.

cell biology↗