Biology subjects

Rossier, O.

Publications and source records attributed to Rossier, O..

8 recordsLinked to original sources

bioRxiv2026

CyranoPS, a novel compact temperate corynephage infecting Corynebacterium glutamicum MB001

Here, we describe CyranoPS, a temperate phage isolated from the river Yvette in France, using the prophage-free strain Corynebacterium glutamicum MB001. Negative-stain electron microscopy revealed virions with siphovirus morphology. Its 14,910-bp genome has a GC content of 53.0% and 14-nt 3' overhangs at both ends. It carries 26 predicted genes, 10 without predicted functions, and no tRNAs. CyranoPS belongs to the newly established actinophage cluster ER, whose members have the smallest known genomes among Corynebacterium-infecting phages. Deletion of genes encoding the porin PorAH and the protein mycoloyltransferase MytC abolished infection, while genes encoding the mycomembrane proteins PorBC and ProtX were dispensable. CyranoPS established lysogeny in C. glutamicum through chromosomal integration probably mediated by its predicted tyrosine integrase. Integration truncated the immunity repressor gene Cyr_18, resulting in a protein devoid of its 15 C-terminal residues. The truncated repressor conferred stronger protection against superinfection than the full-length repressor. CyranoPS lysogens exhibited high levels of spontaneous prophage induction, reaching 109 PFU/mL under standard conditions, with no further increase upon treatment with mitomycin C. These findings suggest a potentially SOS-independent lytic-lysogenic switch and establish CyranoPS as a compact model to study infection and lysogeny regulation in Mycobacteriales.

microbiology↗

bioRxiv2026

Dissecting the genetic determinants of bacterial DNA degradation by bacteriophage T5

Upon infection of Escherichia coli, the virulent bacteriophage T5 employs a unique two-step mechanism to transfer its 122-kb genome: only 8% of the DNA are initially transferred, allowing expression of pre-early genes that alter host functions before the remaining DNA is delivered. Early infection triggers rapid host DNA degradation and nucleotide catabolism, processes partly controlled by the pre-early genes encoding the predicted metallo-phosphatase A1 and the dNMP phosphatase Dmp. However, the functions of most of the 17 proteins encoded by the first-step transfer DNA (FST-DNA) remain unknown. Using reverse genetics, we engineered several T5 mutants carrying deletions in the FST-DNA. One of them carries only four pre-early genes (A1, A2, hdi and 009), demonstrating that thirteen of the 17 predicted genes are dispensable under laboratory conditions. Mutant characterization showed that only A1 and the predicted DNA-binding protein gene A2 are essential for productive infection, while dmp enhances phage virulence. Notably, seven pre-early genes (dmp, A1, hdi, hegG, 011, 013 and 015) proved toxic when expressed in E. coli without other viral factors, causing severe morphological changes or nucleoid disorganization, while one gene compromised membrane integrity. The essential gene A1, which is conserved among all viruses in the Demerecviridae family, emerged as the primary driver of host genome degradation, both essential and sufficient for chromosomal DNA digest in vivo. These findings advance our understanding of how T5 manipulates its host during the critical early stages of infection, offering new insights into phage-host interactions and the molecular strategies viruses use to subvert bacterial cells.

microbiology↗

bioRxiv2024

Amyloid signaling in antiphage defense

Immune regulated cell death (RCD) is a defense strategy common to different domains of life involving purposeful sacrifice of infected cells. In animals and fungi, functional amyloids play a role in the control of RCD as molecular switches activating key cell death effectors, rather than causing direct toxicity as seen with pathological amyloids. Here, we describe a novel amyloid-based signal transduction mechanism in an antiphage defense system in Escherichia coli. This antiphage abortive infection (Abi) system is mediated by two proteins, Bab and Agp which share a common amyloid motif and are encoded by adjacent genes. Upon phage infection, Agp activates Bab through amyloid signaling, leading to cell death. We determined the structure of the Bab cell death execution domain, which is distantly related to pore-forming domains present in fungi, animals and plants. We show that Bab and the fungal HET-S amyloid-controlled cell death execution protein are functionally interchangeable in their respective roles in antiphage defense and allorecognition. These findings add antiphage defense to the functional repertoire of amyloids.

microbiology↗

bioRxiv2023

Actin-Driven Nanotopography Enhances Integrin Molecular Clutch in Developing Tissue

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerisation-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MAS). Super-resolution microscopy revealed that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking showed that, during MAS development, integrins became immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also displayed restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscles cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerisation driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

cell biology↗

bioRxiv2022

Talin and kindlin cooperate to control the density of integrin clusters.

Focal adhesions are important mechanosensitive structures, composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Cellular adhesion to the extracellular matrix depends on the activation of integrins by intracellular mechanisms. Talin and kindlin are major activators of integrins that are recruited to the inner membrane and bind to {beta}-integrin cytoplasmic tails. Many studies showed the importance of integrin activation and clustering and how the organization of extracellular ligands guides the nanoscale organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we performed experiments using a biomimetic in vitro system, made of Giant Unilamellar Vesicles, containing transmembrane integrins, on which purified talin, kindlin, and actomyosin assemble. Here we first show that talin and kindlin individually have the ability to cluster integrins. When added together, talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density in the talin-integrin, kindlin-integrin, and talin-kindlin-integrin clusters reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin significantly enhances the segregation of talin-integrin clusters induced by actomyosin contractility, suggesting that it increases the coupling of these clusters to the actin cytoskeleton. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.

biochemistry↗

bioRxiv2022

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

cell biology↗

bioRxiv2020

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

AO_SCPLOWBSTRACTC_SCPLOWFocal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

cell biology↗

bioRxiv2017

Fast Activation Cycles Of Rac1 At The Lamellipodium Tip Trigger Membrane Protrusion

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE complex, effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Yet, correlation of Rho GTPases activation with cycles of membrane protrusions, suggested that Rac1 activation is not synchronized with membrane protrusion and occurs behind the lamellipodium. However, RhoA activation is maximal at the cell edge and synchronized with edge progression. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function of Rho GTPases mutants, we demonstrate that Rac1 immobilizations at the lamellipodium tip are correlated with Rac1 activation, on the contrary to RhoA. We show that Rac1 effector WAVE and Rac1 regulator IRSp53 accumulate at the lamellipodium tip by membrane free-diffusion and trapping. Nevertheless, wild-type Rac1, which directly interacts with WAVE and IRSp53, only displays slower diffusion at the lamellipodium tip, suggesting fast local activation/inactivation cycles. Local optogenetic activation of Rac1, triggered by Tiam1 membrane recruitment, proves that Rac1 activation must occur at the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. Furthermore, coupling tracking with optogenetic activation of Rac1 demonstrates that Rac1-WT diffusive properties are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model where Rac1 is rapidly switching between activation and inhibition at the lamellipodium tip, ensuring a local and fast control of Rac1 actions on its targets.\n\nSignificanceRac1 and RhoA GTPases are molecular switches controlling the actin cytoskeletal during cell migration. WAVE, Rac1 effector during cell protrusion, is concentrated at the lamellipodium tip. But, recent biosensor imaging studies suggested that Rac1 activation occurs behind the lamellipodium, while RhoA activation is maximal at the cell edge. Using single-molecule imaging and optogentics Rac1 activation we solved this apparent contradiction. We revealed a strong correlation between Rac1 activation and transient immobilizations at the lamellipodium tip, unlike RhoA. Furthermore, we demonstrated that Rac1 must be activated at the lamellipodium tip and not away from it to stimulate protrusion. Thus, fast cycling between activation and inhibition at the proximity of Rac1 targets ensures a local and fast control over Rac1 actions.\n\nAbbreviations

cell biology↗