Biology subjects

Guerin, C.

Publications and source records attributed to Guerin, C..

35 records · Page 2Linked to original sources

bioRxiv2023

The enigma of the SARS-CoV-2 microcirculation dysfunction: evidence for modified endothelial junctions.

Published evidence indicates that Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) infection causes endothelial cell (EC) injury in the Coronavirus Disease 2019 (COVID-19). Endothelial junctions (EJ) are crucial to maintain EC integrity and normal microvascular functions due to the adhesive properties of Vascular endothelial (VE)-cadherin to glue EC together. Here we report studies in vitro and in vivo that indicate VE-cadherin to be a target for cleavage by ACE2. We have identified that the extracellular domain of VE-cadherin contains these two amino acid sequences at the positions 256P-F257 and 321PMKP-325L for ACE2 substrate recognition. Incubation of purified sVE with ACE2 revealed a dose-dependent loss of immunoreactivity detected with an antibody directed against the Extracellular domain 1 (EC1) domain of sVE. We confirmed the presence of ACE2 on ECs using immunofluorescence studies, and by western blotting on ECs extracts. We also present evidence from patients with severe COVID-19 disease for a circulating form of ACE2. Its apparent molecular weight of 70 kDa is in agreement with a previously described extracellular form of ACE2 bearing the catalytic site of the ectopeptidase. Consistent with the experimental evidence for our hypothesis, the level of circulating soluble VE-cadherin fragments was increased in the blood of patients with severe COVID-19 disease. Further studies are needed to determine if increased circulating fragments of ACE2 and VE-cadherin may contribute to the future development of post-acute COVID-19 syndrome characterized by vascular endothelial injury, hypoxia, and inflammatory state. Impact StatementSARS-CoV-2 infection promotes vascular dysfunction but the processes are not completely understood. The vascular endothelium is composed of a monolayer of endothelial cells (ECs) that exclusively express VE-cadherin at adherens junctions (AJs). The published structure of VE-cadherin has revealed crucial residues in the domains EC1-2 for ECs adhesiveness. In this report, we demonstrate for the first time that VE-cadherin is a target for ACE2 ectoenzyme in the domains EC2-3. In addition, in COVID-19 patients blood, we identify truncated forms of ACE2 and VE-cadherin that are correlated with severe SARS-CoV-2 infection. Because the turnover rate of ECs is very low, this could provide part of the explanation for Long CoVID-19 disease. These exciting results highlight the role of proteases and AJs, and the need for continuing efforts to elucidate whether these circulating proteins might be of prime significance for clinicians to facilitate personalized medicine.

biochemistry↗

bioRxiv2023

Comprehensive and Comparative Transcriptional Profiling of the Cell Wall Stress Response in Bacillus subtilis

The bacterial cell wall (CW) is an essential protective barrier and the frontline of cellular interactions with the environment and also a target for numerous antimicrobial agents. Accordingly, its integrity and homeostasis are closely monitored and rapid adaptive responses by transcriptional reprogramming induce appropriate counter-measures against perturbations. Here, we report a comprehensive and comparative transcriptional profiling of the primary cell envelope stress responses (CESR), based on combining RNAseq and high-resolution tiling array studies of the Gram-positive model bacterium Bacillus subtilis exposed to a range of antimicrobial compounds that interfere with cytoplasmic, membrane-coupled or extracellular steps of peptidoglycan (PG) biosynthesis. It revealed the complexity of the CESR of B. subtilis and unraveled the contribution of extracytoplasmic function sigma factors (ECFs) and two-component signal transduction systems (TCSs) to protect the cell envelope. While membrane-anchored steps are tightly controlled, early cytoplasmic and late extracellular steps of PG biosynthesis are hardly monitored at all. The ECF {sigma} factors {sigma}W and particularly {sigma}M provide a general CESR, while {sigma}V is almost exclusively induced by lysozyme, against which it provides specific resistance. Remarkably, {sigma}X was slightly repressed by most antibiotics, pointing towards a role in envelope homeostasis rather than CESR. It shares this role with the WalRK TCS, which balances CW growth with controlled autolysis. In contrast, all remaining TCSs are envelope stress-inducible systems. LiaRS is induced by a wide range of PG synthesis inhibitors, while the three paralogous systems BceAB, PsdRS and ApeRS are more compound-specific detoxification modules. Induction of the CssRS TCS by all antibiotics interfering with membrane-anchored steps of PG biosynthesis points towards a physiological link between CESR and secretion stress. Based on the expression signatures, a suite of CESR-specific B. subtilis whole cell biosensors were developed and carefully evaluated. This is the first comprehensive transcriptomic study focusing exclusively on the primary effects of envelope perturbances that shall provide a reference point for future studies on Gram-positive CESR.

microbiology↗

bioRxiv2023

Detection of tumor-derived extracellular vesicles interactions with immune cells is dependent on EV-labelling methods

Cell-cell communication within the complex tumor microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Understanding the interactions between TD-EVs and immune cells is essential for understanding immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs), analyzed by imaging flow cytometry and multicolor spectral flow cytometry. EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labeled either with the lipophilic dye MemGlow-488 (MG-488), with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), or through expression of a MyrPalm-superFolder GFP (sfGFP) that incorporates into EVs during their biogenesis using a genetically engineered cell line. Our results showed that these different labeling strategies, although analyzed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labeling method. Moreover, MG-488-labeled liposomes behaved similarly to MG-488 EVs, highlighting the predominant role of the labelling strategy on the visualization and analysis of TD-EVs uptake by immune cell types. Consequently, the use of different EV labeling methods has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

cell biology↗

bioRxiv2022

Friction patterns guide actin network contraction

The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile acto-myosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we micropatterned actin nucleation promoting factors on glass or on a lipid bilayer. We adapted their concentrations on each surface to induce the assembly of actin networks of similar densities, and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction both the active and resistive forces are essential to direct the actin network deformation. Significance statementCell shape changes are controlled by complex sets of mechanical forces of various origins. Numerous studies have been dedicated to the role of active forces, originating from molecular motors and filament polymerization, but much less is known about the guiding role of resistive forces. Here we show that a non-uniform distribution of friction forces between a contracting acto-myosin network and its underlying substrate can direct its deformation as it contracts. Our results suggest that the contribution of resistive forces, such as anchoring forces but also less specific viscous forces along cell surface, can be as significant as those of active forces in driving network deformation and should be considered in mechanical models describing the regulation of cell shape and movements.

biophysics↗

bioRxiv2022

Recycling limits the lifetime of actin turnover

Intracellular organization is largely mediated by the actin turnover. Cellular actin networks consume matter and energy to sustain their dynamics, while maintaining their appearance. This behavior, called dynamic steady state, enables cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system using actin bead motility in a compartment with a limited amount of monomer. We used the speed and the size of the actin comet tails to evaluate the systems monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated proteins is the key step in allowing the reuse of monomers for multiple assembly cycles. Energy supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

cell biology↗

bioRxiv2022

Actin architecture steers microtubules in active cytoskeletal composite

Cytoskeletal motility assays use surface-immobilised molecular motors to propel cytoskeletal filaments. These assays have been widely employed to characterise the motor properties and interactions of cytoskeletal elements with themselves or with external factors. Moreover, the motility assays are a promising class of bio-inspired active tools for nanotechnological applications. While effective utilisation of these assays involves controlling the filament direction and speed, either as a sensory readout or a functional feature, designing a subtle control embedded in the assay is an ongoing challenge. Here we investigate the interaction between motor-propelled microtubules and networks of actin filaments. We demonstrate that the microtubules respond to a network of actin filaments and that this response depends on the networks architecture. Both linear actin filaments and a network of actin branched by the Arp2/3 complex decelerate microtubule gliding; however, an unbranched actin network provides additional guidance and effectively steers the microtubules. This effect, which resembles the recognition of cortical actin architecture by microtubules, is a conceptually new means of controlling the filament gliding in the motility assay with potential application in the design of active materials and cytoskeletal nano-devices.

biophysics↗

bioRxiv2022

Identification and functional characterization of two heme uptake systems required for virulence of the fish pathogen Flavobacterium psychrophilum

Bacterial pathogens have a critical impact on aquaculture, a sector that accounts for half of the human fish consumption. Flavobacterium psychrophilum (phylum Bacteroidetes) is responsible for bacterial cold-water disease in salmonids worldwide. The molecular factors involved in host invasion, colonization and hemorrhagic septicaemia are mostly unknown. In this study, we identified two new TonB-dependent receptors, HfpR and BfpR, that are required for adaptation to iron conditions encountered during infection and for virulence in rainbow trout. Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of deletion mutants showed that they act without redundancy: BfpR is required for optimal growth in the presence of high hemoglobin level, while HfpR confers the capacity to acquire nutrient iron from heme or hemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein related to the HmuY family. We demonstrated that HfpY binds heme and contributes significantly to host colonization and disease severity. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote heme uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of pathogenicity of a serious pathogen belonging to the understudied family Flavobacteriaceae and point to the newly identified heme receptors as promising targets for antibacterial development.

microbiology↗

bioRxiv2022

The use of pigs vocalisation structure to assess the quality of human-pig relationship

Studying human-animal interactions in domestic species and how they affect the establishment of a positive Human-Animal Relationship (HAR) may help us improve animal welfare and better understand the evolution of interspecific interactions associated with the domestication process. Understanding and describing the quality of an HAR requires information on several aspects of the animal biology and emotional states (social, spatial and postural behaviours, physiological and cognitive states). Growing evidence shows that acoustic features of animal vocalisations may be indicators of emotional states. Here, we tested the hypothesis that vocal structure may indicate the quality of HAR. At weaning, 30 piglets were positively handled by an experimenter who talked to and physically interacted with them three times a day, while 30 other piglets only received the contact necessary for proper husbandry. After two weeks, we recorded the behaviours and vocalisations produced in the presence of the static experimenter for 5 min. We repeated this test two weeks later, after a conditioning period during which human presence with additional positive contacts was used as a reward for all piglets. We hypothesized this conditioning period would lead to a positive human-piglet relationship for all piglets. As expected, piglets that were positively handled at weaning expressed a higher attraction toward the experimenter, and, after the conditioning, piglets that were not positively handled at weaning expressed a similar level of attraction than the positively handled ones. Piglets positively handled at weaning produced shorter grunts than the other ones, regardless of the context of recording, which may indicate a more positive affect. During reunions with the static experimenter, a more positive HAR was associated with a decrease in vocal reactivity to human proximity. However, during reunions with the experimenter providing additional positive contacts and over the conditioning, proximity to the human systematically triggered shorter and higher pitched grunts, indicator of positive a emotional state. Results first show that changes in vocal structure are consistent with indicators of positive states in the presence of a human. Second, these changes are stronger when the human positively interact with the piglets, supposedly emphasizing a higher positive arousal state during these interactions. We show that vocalisation structure may be a promising indicator of the quality of human-pig relationship.

animal behavior and cognition↗

bioRxiv2022

Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase.

Reprogramming of gene expression during transition from exponential growth to stationary phase is crucial for bacterial survival. In the model Gram-positive bacterium Bacillus subtilis, this process is mainly governed by the activity of the global transcription regulators AbrB, CodY and Spo0A. We recently showed that the transcription termination factor Rho, known for its ubiquitous role in the inhibition of antisense transcription, is involved in Spo0A-mediated regulation of differentiation programs specific to the stationary phase in B. subtilis. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain that expresses rho at a relatively stable high level in order to circumvent its decrease occurring in the wild-type cells entering the stationary phase. We show that B. subtilis cells stably expressing Rho fail to sporulate and to develop genetic competence, which is largely, but not exclusively, due to abnormally low expression of the master regulator Spo0A. Moreover, in addition to a global decrease of antisense transcription, these cells exhibit genome-wide alterations of sense transcription. A significant part of these alterations affects genes from global regulatory networks of cellular adaptation to the stationary phase and reflects the attenuated de-repression of the AbrB and CodY regulons and the weakened stringent response. Accordingly, stabilization of Rho level reprograms stationary phase-specific physiology of B. subtilis cells, negatively affects cellular adaptation to nutrient limitations and alters cell-fate decision-making to such an extent that it blocks development of genetic competence and sporulation. Taken together, these results indicate that the activity of termination factor Rho constitutes a previously unknown layer of control over the stationary phase and post-exponential adaptive strategies in B. subtilis, from the adjustment of cellular metabolism to the activation of survival programs.

microbiology↗

bioRxiv2022

The architecture of the actin network can balance the pushing forces produced by growing microtubules.

The position of centrosome, the main microtubule-organizing center (MTOC), is instrumental in the definition of cell polarity. It is defined by the balance of tension and pressure forces in the network of microtubules (MTs). As MTs polymerize against the cell periphery, pressure increases and produces pushing forces on the MTOC. How the mechanical interplay between MTs and the actin network is involved in the regulation of these forces remains poorly understood, in particular because its investigation is technically limited by the structural and biochemical complexity of the cell cytoplasm. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of an aster of dynamic MTs with actin networks of various compositions and architectures in cell-sized microwells. In the absence of actin filaments, the positioning of the MTOC was highly sensitive to variations in MT length. The presence of a bulk actin filament network limited MTs deformation and displacement, and MTOCs were hold in place. In contrast, the assembly of a dense and branched actin network along the edges of the wells centered the MTOCs by preventing MT slippage and thus maintaining an isotropic balance of pushing forces. In agreement with this, an asymmetric peripheral actin network caused the MTOC to decenter by creating an asymmetry in the pushing forces. Numerical simulations demonstrated that steric hindrance by actin networks, at the tip or along the entire length of MTs, can modulate MTOC positioning, as observed in the experiments. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to MT length and enforce robust MTOC centering or decentering depending on its architecture.

cell biology↗

bioRxiv2022

Actin-microtubule dynamic composite forms responsive active matter with memory

Active cytoskeletal materials in vitro demonstrate self-organising properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in the living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possessing adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite, where self-assembling microtubules and actin filaments collectively self-organise due to the activity of microtubules-percolating molecular motors. We demonstrate that microtubules spatially organise actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli suggesting possible autoregulatory behaviour in changing mechanochemical environment. We thus establish artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.

biophysics↗

bioRxiv2020

MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

The Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

cell biology↗

bioRxiv2020

Manipulable object and human contact: preferences and modulation of emotional states in weaned piglets

1Enriching the life of farm animals is an obligation in intensive farming conditions. In pigs, manipulable materials are mandatory when no bedding is available. Like manipulable objects, positive human interactions might be considered as enrichment, as they provide the animals occasions to interact, increase their activity and lead to positive emotional states. In this study, we investigated how weaned piglets perceived a manipulable object, and a familiar human. After a similar familiarization to both stimuli, twenty-four weaned piglets were tested for a potential preference for one of the stimuli and submitted to isolation/reunion tests to evaluate the emotional value of the stimuli. We hypothesized that being reunited with a stimulus would attenuate the stress of social isolation and promote positive behaviors, and even more that the stimulus has a positive emotional value for piglets. Although our behavioural data did not allow to show a preference for one of the stimuli, piglets approached more often the human and were observed laying down only near the human. Using behavioural and bioacoustic data, we showed that reunion with the human decreased more the time spent in an attentive state and mobility of piglets than reunion with the object, and isolation. Vocalizations differed between reunions with the object and the human, and were different from vocalizations during isolation. The human presence led to higher frequency range, more noisy and shorter grunts. Finally, both stimuli decreased the isolation stress of piglets, and piglets seemed to be in a more positive emotional state with the human compared to the object. It confirms the potential need for positive human interactions to be used as pseudo-social enrichment in pigs.

animal behavior and cognition↗

bioRxiv2020

Piglets vocally express the anticipation of (pseudo)-social contexts in their grunts

Emotions not only arise in reaction to an eliciting event but also while anticipating it, making this context a way to assess the emotional value of events. Anticipatory studies have poorly considered vocalisations whereas they carry information about the emotional state. We studied the grunts of piglets that anticipated two (pseudo)social events known to elicit positive emotions more or less intense: arrival of a familiar conspecifics and arrival of a familiar human. Both time and spectral features of the vocal expression of piglets differed according to the emotional context. Piglets produced low-frequency grunts at a higher rate when anticipating conspecifics compared to human. Spectral noise increased when piglets expected conspecifics, whereas the duration and frequency range increased when expecting a human. When the arrival of conspecifics was delayed grunts duration increased, while when the arrival of the human was delayed spectral parameters were comparable to those during isolation. This shows that vocal expressions in piglets during anticipation are specific to the expected reward and to the time duration between signal and the delivery of the reward. Vocal expression (time and spectral features) is thus a good way to explore emotional state of piglets during anticipation of challenging events.

animal behavior and cognition↗

bioRxiv2019

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that PARK7/DJ-1, a key familial Parkinsons disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, which is dysregulated in many complex diseases.

biochemistry↗

bioRxiv2018

Reconstitution of the equilibrium state of dynamic actin networks

Principles of regulation of actin network dimensions, fundamentally important for cell functions, remain unclear. We studied in vitro and in silico the effect of key parameters, actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became globally treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic equilibrium state of actin network emerges from biochemical and structural feedbacks.

cell biology↗

bioRxiv2018

Actin filaments regulate microtubule growth at the centrosome.

The centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here we show that increasing densities of actin filaments at the centrosome of lymphocytes were correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in centrosomal-actin disassembly and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed in vitro reconstitution assays based on (i) purified centrosomes and (ii) on the co-micropatterning of microtubule seeds and actin filaments. The two assays demonstrated that actin filaments perturb microtubule growth by steric hindrance. Finally, we showed that cell adhesion and spreading leads to lower densities of centrosomal actin thus resulting in higher microtubule growth. Hence we propose a novel mechanism by which the number of centrosomal microtubules is regulated by cell adhesion and actin-network architecture.

cell biology↗