Biology subjects

Schaeffer, A.

Publications and source records attributed to Schaeffer, A..

13 recordsLinked to original sources

bioRxiv2026

Uncoupling microtubule lifetime, stability and post-translational modifications.

Microtubules (MTs) undergo continuous cycles of growth and disassembly. Because the transitions between these states are stochastic, MT age varies widely within a population. As MTs age, they are thought to accumulate post-translational modifications (PTMs) that, directly or indirectly, enhance their stability and thereby extend their lifetime. The rare MTs that withstand prolonged exposure to destabilizing drugs such as nocodazole (NZ) are indeed enriched in PTMs; yet the relationships between MT age, PTMs and stability remain unclear. Using microinjection of labelled tubulin, we measured microtubule network turnover in immortalized mouse embryonic fibroblasts. Half of the network was renewed within 3 minutes and 80% within 10 minutes, while approximately 5% of microtubules persisted for more than 20 minutes. These dynamics were comparable in quiescent and senescent cells, although the fraction of slowly renewing or non-renewing microtubules rose to 20% in senescent cells. Unexpectedly, neither the amount of PTMs (acetylation and detyrosination) nor resistance to NZ increased with MT age, and resistance to NZ was independent of these PTMs. Degrees of acetylation and detyrosination should therefore not be taken as readouts of MT age or stability. Because these PTMs do not accumulate on MTs over time, the chemical modification of polymerized tubulin is likely more reversible and dynamic than previously assumed.

cell biology↗

bioRxiv2026

Microglial TLR4 Mediates Post-UTI Chronic Pelvic Pain

Urinary tract infections (UTIs) are a major risk factor for interstitial cystitis/bladder pain syndrome (IC/BPS), yet the mechanisms driving chronic pelvic pain and associated symptoms remain poorly understood. Here, we investigated the role of microglia and Toll-like receptor 4 (TLR4) in a mouse model of post-UTI chronic pelvic pain (PUPP). Infection with E. coli induced persistent pelvic allodynia that was significantly attenuated by microglial depletion (PLX5622) or inhibition (minocycline), indicating a key role for microglia in pain maintenance. In contrast, microglial depletion did not improve urinary dysfunction or anxiety- and depression-like behaviors. Prefrontal cortex microglia of PUPP mice exhibited reduced microglial branching complexity and a less ramified phenotype, indicative of an activated microglial state. Transcriptomic profiling of brain CD11b+ cells revealed a reactive microglial signature enriched for chemokines, NFKB-related genes, and immediate early response genes, alongside pathways involved in immune regulation and leukocyte recruitment. Both general and microglia-specific TLR4 deletion reduced pelvic allodynia and reduced microglial morphological features of activation. Consistent with this, pharmacological TLR4 inhibition in vitro suppressed LPS-induced NFKB activation, cytokine secretion, and CD68 expression. Together, these findings identify microglial TLR4 as a critical mediator of post-UTI chronic pelvic pain.

neuroscience↗

bioRxiv2024

DNMT/G9a Complex Inhibition Uncovers Epigenetic Vulnerabilities and Induces IFN-Response in Acute Myeloid Leukemia

Epigenetic dysregulation is a hallmark of Acute Myeloid Leukemia (AML), with mutations in DNA Methyltransferases (e.g., DNMT3A) being frequent and promising therapeutic targets. DNMTs form complexes with Histone Methyltransferases (HMTs), driving gene silencing loop via chromatin methylation crosstalk. However, potential connections between this DNMTs/HMTs cooperative activity and oncogenic requirements across the AML mutational spectrum remain poorly understood. Here, we demonstrate that AMLs carrying DNMT3A and Nucleophosmin (NPM1) mutations exhibit a specific epigenetic vulnerability toward a complex formed by DNMTs and G9a, a specific histone H3 Lysine 9 Methyltransferase (H3K9-HMT). Dual inhibition of DNMT/G9a restores differentiation, reduces tumor growth, and spares healthy progenitors compared to standard hypomethylating agents. Mechanistically, DNMT/G9a regulates NPM1 stability, inhibits HOXA9/MEIS1 activity, and triggers interferons (IFN) response via viral mimicry pathways by modulating hypermethylated retrotransposons. Collectively, our data unravel specific epigenetic vulnerabilities within the complex AML mutational landscape and provide a compelling rationale for the design of personalized epigenetic therapies with enhanced efficacy and safer clinical outcomes.

cancer biology↗

bioRxiv2024

Grasping the constraints of pure bacterial strains for the complete catabolism of micropollutants: a proteomic and kinetic study

Research into the microbial degradation of organic micropollutants (OMP) often involves monitoring depletion of the parent compound and analyzing the biotransformation pathways that can lead to the production of metabolites, some being toxic, and/or to their mineralization. For the antibiotic sulfamethoxazole (SMX), previous studies testing a range of SMX concentration (mg down to ng L-1), have shown incomplete biotransformation of the parent SMX. This occurred both during wastewater treatment with mixed microbial communities and in studies with pure bacterial strains acclimated to SMX. This study explores the mechanism of SMX biotransformation and relationships with the proteome profile as possible reasons for the incomplete degradation of the parent SMX. Microbacterium sp BR1 served as an acclimated bacterial degrader of SMX in the range of {micro}g L-1 to ng L-1. Depletion of the SMX was incomplete whereas the metabolite 3-amino-5-methylisoxazole (3A5MI) accumulated. The activity of the enzymes for the initial transformation of the parent SMX (SadA) was higher than that of further biotransformation steps (SadB). These results showcase that even a highly sensitive and metabolically active strain at very low SMX concentrations may require complementary enzymatic machineries to degrade metabolites that have an inhibitory impact in the biodegradation and persistence of this antibiotic. SynopsisA complete removal of organic micropollutants from water is challenging. This article delves into the bacterial degradation of the antibiotic SMX and proteome analysis to clarify underlying causes of its incomplete elimination

microbiology↗

bioRxiv2024

Contractile forces direct the chiral swirling of minimal cell collectives

Chirality is a conserved biological feature with critical implications in tissue morphogenesis and embryonic development. In culture, large multicellular groups exhibit spontaneous chiral symmetry break when moving collectively on micropatterned surfaces. Although several studies have shown that actin network integrity and acto-myosin network contractility participate to the establishment of the chirality of the movement, the exact contribution of contractile forces to the directionality of the chiral bias in collectives remains to be elucidated. Here we studied the contractile forces produced by a minimal collective constituted of a pair of endothelial cells. We first show that cell doublets confined on disk-shaped micropatterns undergo spontaneous and persistent chiral swirling, displaying a mild but robust clockwise (CW) bias, as the one observed in bigger collectives. This bias could be amplified or reversed by modulating contractile forces. Traction force measurements revealed that large forces tend to drive counter-clockwise (CCW) rotation whereas low forces rather favor a CW rotation. Furthermore, the study of heterotypic doublets indicates that the speed and direction of the rotation is determined by the more contractile cells within the doublets. These results thus revealed that contractile leader cells could drive the chiral motion of minimal collectives. Significance StatementChirality, which represents a fundamental property of living systems, manifests in cell collectives by their persistent biased directional swirling. Despite the clear identification of the implication of actomyosin cytoskeleton in driving the internal chiral symmetry break occurring in cells, little is known about the actual role of cellular forces produced by this network in the development of handedness in collectives. Our findings establish that the level of mechanical energy developed by pairs of confined endothelial cells regulates the strength and direction of their rotation. Our results also identify the more contractile cell of the doublet as the cell driving the direction and speed of rotation of the pair. This study thus sheds new light on the importance of the generation and integration of mechanical forces within a small collective in the determination of its chiral rotation.

cell biology↗

bioRxiv2024

Global genomics of the man-o'-war (Physalia) reveal biodiversity at the ocean surface

The open ocean is a vast, highly connected environment, and the organisms found there have been hypothesized to represent massive, well-mixed populations. Of these, the Portuguese man-o-war (Physalia) is uniquely suited to dispersal, sailing the ocean surface with a muscular crest. We tested the hypothesis of a single, panmictic Physalia population by sequencing 133 genomes, and found five distinct lineages, with multiple lines of evidence showing strong reproductive isolation despite range overlap. We then scored thousands of citizen-science photos and identified four recognizable morphologies linked to these lineages. Within lineages, we detected regionally endemic subpopulations, connected by winds and currents, and identified individual long-distance dispersal events. We find that, even in these sailing species, genetic variation is highly partitioned geographically across the open ocean. SummaryThe open ocean is a vast and highly connected environment. The organisms that live there have a significant capacity for dispersal and few geographic boundaries to separate populations. Of these, the Portuguese man-o-war or bluebottle (genus Physalia) is uniquely suited to long-distance travel, using its gas-filled float and muscular crest to catch the wind and sail the sea surface. Physalia are distributed across the globe, and like many pelagic organisms, have been hypothesized to represent a massive, well-mixed population that extends across ocean basins. We tested this hypothesis by sequencing whole genomes of 133 samples collected from waters of over a dozen countries around the globe. Our results revealed five distinct lineages, with multiple lines of evidence indicating strong reproductive isolation, despite regions of range overlap. We combined these data with an independent dataset of thousands of images of Physalia uploaded to the citizen-science website inaturalist.org, which we scored for morphological characters including sail size, tentacle arrangement, and color. From these images, we identified four recognizable morphologies, described their geographical distribution, and linked them to four of the lineages identified with genomic data. We conclude there are at least four species, three of which correspond to species proposed by scientists in the 18th and 19th centuries: P. physalis, P utriculus, and P. megalista, along with one as yet unnamed species Physalia sp. from the Tasman Sea. Within each species, we observe significant population structure, with evidence of persistent subpopulations at a regional scale, as well as evidence for individual long-distance dispersal events. Our findings indicate that, instead of one well-mixed, cosmopolitan species, there are in fact multiple Physalia species with distinct but overlapping ranges, each made up of regionally endemic subpopulations that are connected by major ocean currents and wind patterns.

zoology↗

bioRxiv2024

Centrosome positioning independently of microtubule-based forces

The regulation of centrosome position is central to the establishment of polarized cell functions. This process is thought to rely on the generation of mechanical forces along microtubules, the balance of which determines centrosome position. By studying these forces in adherent cells in culture, we found that neither pushing nor pulling forces propagate along microtubules to the centrosome. Inhibiting dyneins or disassembling microtubules did not disrupt the maintenance of the centrosome at the center of the cell. In contrast, the actomyosin contractile network appears to be responsible for the generation of a centripetal flow that drives the centrosome towards the center of the cell independently of the microtubules. Furthermore, we found that centering of the centrosome depends more on the reorganization of cell shape around the centrosome than on an effective centrosome displacement throughout the cytoplasm. Interestingly, despite their lack of mechanical role, microtubules appear to direct this remodeling of cell shape. This revised view of centrosome positioning offers a new perspective for understanding the establishment of cell polarity.

cell biology↗

bioRxiv2024

Microtubule-dependent orchestration of centriole amplification in brain multiciliated cells

Multiciliated cell (MCC) differentiation is a calibrated version of the canonical cell cycle. The MCC cell cycle variant sustains amplification of centrioles for the nucleation of dozens of motile cilia, while avoiding cell division. In this study, we show that the MCC cell cycle variant is an accelerated version of the canonical cell cycle, which superposes two cycles of centriole biogenesis, in order to obtain multiple mature centrioles within a single -instead of double- cell cycle iteration. We further show that the precocious maturation of amplified procentrioles is even determinant for their spatial self-organization, disengagement and apical migration for cilia nucleation. Our findings collectively suggest that the decomposition of centriole biogenesis over two cycle iterations in dividing cells may have been adopted to ensure the growth of a solitary primary cilium, and exemplify how minimal deviations of the canonical cell cycle allow MCC progenitors to both amplify, and accelerate, centriole biogenesis for vital motile ciliogenesis.

cell biology↗

bioRxiv2023

Microtubules under mechanical pressure can breach dense actin networks

The crosstalk between actin network and microtubules is key to the establishment of cell polarity. It ensures that the asymmetry of actin architec ture along cell periphery directs the organization of microtubules in cell interior. In particular, the way the two networks are physically inter-twined regulates the spatial organization and the distribution of forces in the microtubule network. While their biochemical crosstalk is getting uncovered, their mechanical crosstalk is still poorly understood. Here we designed an in vitro reconstitution assay to study the physical interaction between dynamic microtubules with various structures made of actin fil aments. We found that microtubules can align and move by their polymerization force along linear bundles of actin filaments. But they cannot enter dense and branched actin meshworks, such as those found in the lamellipodium along cell periphery. However, when microtubules are immobilized, by their crosslinking with actin structures or others means, the force of polymerization builds up pressure in the microtubules that is sufficient to allow them to breach and penetrate these dense actin meshworks. This mechanism may explain the final progression of microtubules up to cell periphery through the denser parts of the actin network.

biophysics↗

bioRxiv2022

Evaluating microbial contaminations of alternative heating oils

Since 2008, legislative initiatives for climate protection and reduced dependency on fossil resource imports led to the introduction of biofuels as CO2-reduced alternatives in the heating oil sector. In the case of biodiesel, the oil industry or its customers were confronted with accelerated and escalating microbial contaminations during heating oil storage. Since then, other fuel alternatives, like hydrogenated vegetable oils, gas-to-liquid products (GtL), or Oxymethylenether (OME) have been or will be developed and potentially introduced to the market. In this study, we use online monitoring of microbial CO2 production and the simulation of onset of microbial contamination to investigate the contamination potential of fuel alternatives during storage. As reference and blends, fossil heating oils of various refineries, in the course of this from various crude oils, and refinery processes reveal considerable variation in potential microbial activity. Oxymethylene ethers have an antimicrobial effect, while various forms of biodiesel confirm the promotion of microbial activity and diversity. The paraffinic Fischer-Tropsch products and biogenic hydrogenation products demonstrate high resistance to microbial contamination despite allowing microbial diversity. Through an array of analytics, including advanced chromatography coupled mass spectrometry, elemental analysis, and microbial sequencing, we can discuss critical fuel properties that promote or inhibit microbial contaminations. In summary, novel, non-fossil heating oils show different strengths and weaknesses for long-term storage. Designing blends for microbial activity reduced long-term storage might be an option. While being niche products, these fuels will contribute to the rapid reduction of fossil resource use.

microbiology↗

bioRxiv2022

Microtubules self-repair in living cells

Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule network has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events taking place in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along pre-existing green microtubule network within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. We then characterized the size and spatial frequency of these incorporations as a function of injected tubulin concentration and post-injection delay. The saturation of these measurements contradicted the hypothesis of nonspecific adsorption along microtubules and suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 micrometers of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations. These results provide evidences that microtubules do self-repair in living cells, and they provide a quantitative characterization of the temporal and spatial dynamics of this process in PtK2 cells.

cell biology↗

bioRxiv2021

Compensatory CSF2-driven macrophage activation promotes adaptive resistance to CSF1R inhibition in breast-to-brain metastasis

Tumor microenvironment-targeted therapies are emerging as promising treatment options for different cancer types. Tumor-associated macrophages and microglia (TAMs) represent an abundant non-malignant cell type in brain metastases and have been proposed to modulate metastatic colonization and outgrowth. We used an inhibitor of colony stimulating factor 1 receptor (CSF1R) to target TAMs at distinct stages of the metastatic cascade in preclinical breast-to-brain metastasis models and found that CSF1R inhibition leads to anti-tumor responses in prevention and intervention trials. However, in established brain metastases, compensatory CSF2Rb-STAT5-mediated pro-inflammatory TAM activation blunted the ultimate efficacy of CSF1R inhibition by inducing neuro-inflammation gene signatures in association with wound repair responses that fostered tumor recurrence. Consequently, combined blockade of CSF1R and STAT5 signaling led to sustained tumor control, a normalization of microglial activation states and amelioration of neuronal damage.

cancer biology↗

bioRxiv2020

Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4 receptors.

Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow, where they regulate the permanent production and renewal of all blood-cell types. HSPC proliferation and differentiation is locally regulated by their interaction with cells forming specific microenvironments close to the bone matrix or close to blood vessels. However, the cellular mechanisms underlying HSPCs interaction with these cells and their potential impact on HSPC polarity is still poorly understood. Here we modelled the bone-marrow niche using microfluidic technologies in a bone-marrow on a chip device, and evaluated long-duration cell-cell contacts between single HSPCs and stromal cells or endothelial cells in a custom-designed microwell cell-culture system. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of their cytoskeleton architectures. As in the case with immune synapses formed by lymphocytes, the centrosome was located in proximity of the cell-cell contact. The entire microtubule network emanated from the centrosome, and the nucleus was confined to the side opposite of the cell-cell contact. The capacity of the HSPC to polarize appeared specific as it was not observed in contact with skin fibroblasts. The receptors ICAM, VCAM and CXCR4 were identified in the polarizing contact, and were all independently capable of inducing morphological polarization. However, only CXCR4 was independently capable of inducing the polarization of the centrosome-microtubule network. Altogether these results revealed a novel mechanism of HSPC polarization associated with its anchorage to specific cells in the bone-marrow, which might be instrumental in the regulation of their fate.

cell biology↗