Jean-Bernard Fiche

The spatiotemporal organization of proteins and lipids within membranes is crucial for ensuring proper cellular signaling. While the segregation of proteins and lipids into membrane nanodomains is well established, it remains unclear whether nanodomains can generate gradients of small diffusible molecules. In plants, reactive oxygen species (ROS), especially hydrogen peroxide (H ₂ O ₂ ), act as key signaling molecules in response to environmental stimuli such as osmotic stress. However, how extracellular H ₂ O ₂ affects intracellular signaling has remained unknown. Here, we show that osmotic stimulation induces the formation of localized, H ₂ O ₂ -rich nanoenvironments at the cytoplasmic face of the plasma membrane (PM) in Arabidopsis root cells. Using a PM-tethered H ₂ O ₂ biosensor, we found that these oxidized nanodomains arise from the clustering of RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs) and RHO OF PLANTS 6 (ROP6), in coordination with aquaporin-mediated H ₂ O ₂ transport via the PLASMA MEMBRANE INTRINSIC PROTEIN2;7 (PIP2;7). These local redox hotspots at the PM create a feedforward loop in which H ₂ O ₂ enhances ROP6 nanoclustering, thereby amplifying ROS signaling. Disruption of H ₂ O ₂ production or transport dampens both ROP6 clustering and anisotropic cell expansion, indicating a crucial role for spatially confined redox signaling in regulating plant growth under osmotic stress. Our findings propose a model in which ROP6/RBOHD-F/PIP2;7 nanodomains function as discrete redox signaling units, redefining ROS signaling at the PM as a structured, signal-specific, and compartmentalized process.

During their growth, plants encounter and respond to a variety of environmental signals. However, the mechanisms underlying the integration and specificity of signals remain poorly understood. Rho of Plant (ROP) signaling plays a central role in various processes, including polar cell growth and responses to different stimuli, and relies on stimuli-dependent membrane nanodomains. The effector composition of ROP6 nanodomains varies depending on the signal and may be involved in downstream signal specificity. In this study, we explore how ROP6 signaling is regulated by Guanine nucleotide Exchange Factor (GEF) during osmotic stress. We find that GEF14 is required for osmotically induced ROS accumulation. This isoform acts specifically in response to osmotic stimulation, since it is dispensable for other stimuli. We demonstrate that GEF14 activates ROP6 and controls its clustering in a signal-specific manner. Furthermore, we find that GEF14 relocates from the cytoplasm to clusters at the plasma membrane after osmotic stimulation. Together, our results suggest that a single GEF isoform can encode for signal specificity controlling ROP6 activation, clustering and downstream cellular responses.

A bstract: Integrins are crucial for eukaryotic cell attachment and motility within the extracellular matrix (ECM) via focal-adhesion formation, with their evolutionary emergence important for the development of multicellularity. Intriguingly, single gliding cells of the predatory deltaproteobacterium Myxococcus xanthus form bacterial focal-adhesion (bFA) sites; therein, helically-trafficked motors become immobilized at anchored locations through Glt apparatus association with cell-surface integrin αI-domain-like adhesin CglB. Using traction-force, bead-force, and total internal reflection-fluorescence microscopies combined with biochemical approaches, we herein identify the von Willebrand A domain-containing cell-surface lipoprotein CglD to be a β-integrin-like outer-membrane lipoprotein that functionally associates with and anchors the trans-envelope Glt–CglB gliding apparatus, stabilizing and efficiently anchoring this assembly at bFAs. Calcium dependence governs CglD importance, consistent with its integrated ECM eukaryotic cartilage oligomeric matrix protein domains. CglD thus confers mechanosensory and mechanotransductory capabilities to the gliding apparatus, helping explain bFA-mediated trans-envelope force transduction, from inner-membrane-embedded motors to the cell surface.

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall–sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.

The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal-adhesion (bFA) sites to power gliding motility. Using total internal reflection fluorescence and force microscopies, we identify the von Willebrand A domain-containing outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling adhesin of the gliding transducer (Glt) machinery at bFAs. Biochemical and genetic analyses reveal that CglB localizes to the cell surface independently of the Glt apparatus; once there, it is recruited by the OM module of the gliding machinery, a heteroligomeric complex containing the integral OM β barrels GltA, GltB, and GltH, as well as the OM protein GltC and OM lipoprotein GltK. This Glt OM platform mediates the cell-surface accessibility and retention of CglB by the Glt apparatus. Together, these data suggest that the gliding complex promotes regulated surface exposure of CglB at bFAs, thus explaining the manner by which contractile forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum.

Many species, such as fish schools or bird flocks, rely on collective motion to forage, prey, or escape predators. Likewise, Myxococcus xanthus forages and moves collectively to prey and feed on other bacterial species. These activities require two distinct motility machines enabling adventurous (A) and social (S) gliding, however when and how these mechanisms are used has remained elusive. Here, we address this long-standing question by applying multiscale semantic cell tracking during predation. We show that: (1) foragers and swarms can comprise A- and S-motile cells, with single cells exchanging frequently between these groups; (2) A-motility is critical to ensure the directional movement of both foragers and swarms; (3) the combined action of A- and S-motile cells within swarms leads to increased predation efficiencies. These results challenge the notion that A- and S-motilities are exclusive to foragers and swarms, and show that these machines act synergistically to enhance predation efficiency.

In mammals, insulators contribute to the regulation of loop extrusion to organize chromatin into topologically associating domains. In Drosophila the role of insulators in 3D genome organization is, however, under current debate. Here, we addressed this question by combining bioinformatics analysis and multiplexed chromatin imaging. We describe a class of Drosophila insulators enriched at regions forming preferential chromatin interactions genome-wide. Notably, most of these 3D interactions do not involve TAD borders. Multiplexed imaging shows that these interactions occur infrequently, and only rarely involve multiple genomic regions coalescing together in space in single cells. Finally, we show that non-border preferential 3D interactions enriched in this class of insulators arise before TADs and transcription during Drosophila development. Our results are inconsistent with insulators forming stable hubs in single cells, and instead suggest that they fine-tune existing 3D chromatin interactions, providing an additional regulatory layer for transcriptional regulation.

A bstract: The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesion (bFA) sites to power gliding motility. Using TIRF and force microscopy, we herein identify the integrin αI-domain-like outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling protein of the gliding motility complex. Similar to most known OM lipoproteins, CglB is anchored on the periplasmic side of the OM and thus a mechanism must exist to secrete it to the cell surface in order for it to interact with the underlying substratum. We reveal this process to be mediated by a predicted OM β-barrel structure of the gliding complex. This OM platform was found to regulate the conformational activation and secretion of CglB across the OM. These data suggest that the gliding complex promotes surface exposure of CglB at bFAs, thus explaining the manner by which forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum; they also uncover a novel protein secretion mechanism, highlighting the ubiquitous connection between secretion and bacterial motility.

During development, naïve cells gradually acquire distinct cell fates, through sophisticated mechanisms of precise spatio-temporal gene regulation. Acquisition of cell fate is thought to rely on the specific interaction of remote cis -regulatory modules (e.g. enhancers, silencers) (CRM) and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly in single cells within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to systematically detect CRM-promoter looping interactions within topological associating domains (TADs) during Drosophila development. By comparing cis -regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses revealed the existence of local interactions between multiple remote CRMs to form hubs. We found that loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach offers a new perspective on the role of CRM-promoter interactions in defining transcriptional activation and repression states, as well as distinct cell types.

In the course of their growth and development plants have to constantly perceive and react to their environment. This is achieved in cells, by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependant nanodomains within the PM. These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS),that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, ROP6 activation triggers the nanoclustering of two NADPH oxidases that subsequently generates ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically-driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensure signal specificity downstream of independent stimuli.