Biology subjects

Aulas, A.

Publications and source records attributed to Aulas, A..

8 recordsLinked to original sources

bioRxiv2026

Circulating colorectal tumor cells remodel their surfaceome to increase their viability and metastatic potential in the bloodstream

Circulating tumor cells (CTCs) are the potential seeds of distant metastases; however, little is known about how they survive in the bloodstream. Using a large cohort of colorectal cancer (CRC) patients, we found that the pseudokinase receptor PTK7 is highly expressed in primary tumors and metastatic lesions. Consistent with previous reports, high PTK7 expression is associated with reduced disease-free survival and increased metastatic dissemination. Surprisingly, PTK7 is absent from most CTCs and undergoes a cell-autonomous ONtumor/OFFCTC/ONmetastasis switch that can be recapitulated in a xenografted mouse model, in in vitro systems, and a fluidic platform. PTK7-negative cancer cells exhibit increased expression of YAP1-driven genes, senescence-like features, and enhanced resistance to hemodynamic stress following loss of cell-cell and cell-matrix adhesion. This adaptive phenotype depends on metalloproteases, notably ADAM17, whose cleavage activity remodels the CTCs surfaceome. Functionally, the PTK7 OFFCTC state confers enhanced metastatic potential in vivo, and can be pharmacologically suppressed using metalloprotease inhibitors. Collectively, our findings identify a reversible, cell-autonomous, protease-driven surfaceome remodeling program that enables metastatic adaptation during hematogenous dissemination. Highlights / statement of significanceBy investigating potential markers for circulating colorectal tumor cells with strong metastatic potential, we describe a reversible and cell-autonomous remodeling of the circulating tumor cell surfaceome in patients that confers resistance to anoikis and stress induced by entry into the bloodstream. One Sentence SummaryThe dynamic regulation of PTK7 serves as a surrogate marker for tumor cell plasticity, aggressiveness, survival in the bloodstream, and efficiency in forming metastases. Trial registrationCTC colon Cohort: registered on https://ClinicalTrials.gov identifier NCT03256084; date of registration 2017-07-17 B-Org cohort: registered on https://ClinicalTrials.gov NCT05384184; date of registration 2019-06-06 Ethics statement for animal experimentsStudies on animals were conducted in accordance with the current ethical standards of the European Community (Directive 2010/63/EU), the Ethics Committee for Animal Experimentation (CEEA#14) and the French Ministry of Higher Education and Research, which approved and authorized the entire procedure described in this paper (project number APAFIS #35294).

cancer biology↗

bioRxiv2026

Application of D4 Fluorescent Probes for Quantitative and Spatial Analysis of Cholesterol in Cells

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

biochemistry↗

bioRxiv2026

Ganglioside GM1-enriched rafts regulate the neuronal chloride co-transporter 1 KCC2.

During brain development, dynamic remodeling of membrane lipid composition accompanies the maturation of inhibitory neurotransmission and the progressive establishment of low intracellular chloride levels. Central to this developmental transition is the neuronal K-Cl- cotransporter KCC2, whose stabilization at the plasma membrane enables the emergence of hyperpolarizing GABAergic signaling. Although KCC2 regulation by protein partners has been extensively characterized, whether lipid remodeling actively contributes to its membrane organization and chloride transport remains unclear. Here we identify the ganglioside GM1, a complex lipid abundant in plasma membrane of neurons, as a developmentally regulated lipid determinant of KCC2 membrane localization and function. We show that KCC2 interacts with GM1 within plasma membrane lipid rafts and that this interaction increases during postnatal brain maturation. Molecular modeling identified a conserved ganglioside-binding domain (GBD) in KCC2 centered on tryptophan 318 (W318). Biophysical analyses revealed a specific and saturable interaction between this domain and GM1 that is abolished by the epilepsy-associated W318S mutation. Disruption of KCC2-GM1 interactions, either by W318S mutation or by pharmacological depletion of GM1, excludes KCC2 from lipid rafts, alters its membrane diffusion and clustering, and reduces its surface stability. Functionally, these perturbations impair KCC2-mediated chloride extrusion and disrupt the somato-dendritic chloride gradient in hippocampal neurons. Consistent with these cellular effects, GM1-deficient (St3gal5-/-) mice exhibit selective reduced hippocampal KCC2 expression. Together, these findings reveal a lipid-protein mechanism that links developmental membrane remodeling to KCC2 stabilization and chloride homeostasis, highlighting membrane lipids as active regulators of transporter maturation and inhibitory circuit development.

neuroscience↗

bioRxiv2025

Lipid rafts are the new Stress Granules regulators

Stress granules are cytoplasmic inclusions1 with cyto-protective functions2-6 assembling in response to stress. They are now accepted to be part of the pathological mechanism in several diseases, from cancer to neurodegenerative disorders7-10. However, the field is still struggling to find common regulators of their assembly and function7,11. In this study, we describe an unraveled mechanism involving lipid raft, via gangliosides and cholesterol, in the regulation of SG formation. This is the first report about regulation of SG by the cell membrane composition. This discovery could have a significant impact on the understanding of several disease mechanism. MATERIAL AND METHODESO_ST_ABSCell culture & cell treatmentC_ST_ABSMDA-MB-231 (ATCC) and SH-SY5Y (ATCC) cells were maintained at 37 {degrees}C with 5% CO2 in Gibco Dulbeccos Modified Eagle Medium: Nutrient Mixture F12 (DMEM-F12, GIBCO, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Eurobio, Les Ulis, France), 20 mM HEPES (GIBCO, Waltham, MA, USA), 1X Penicillin streptomycin (GIBCO, Waltham, MA, USA). Cells are treated with methyl-{beta}-cyclodextrine (M{beta}CD) (MDA-MD-231 5mM, SH-SY5Y 1mM) 48h before experimentation, or with d,l-threo-l-Phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) (MDA-MD-231 5M, SH-SY5Y 10M) for 24h. ImmunofluorescenceCells were seeded on coverslips, treated 48h with PPMP or 24h with M{beta}CD before the experiment. After stress treatment, cells are washed quickly with PBS before to be fixed for 15min with 4% Paraformaldehyde (Thermo Scientific, Waltham, MA, USA) in PBS. Cells were then permeabilized and blocked with IF buffer PBS-0.3% TX100 (Euromedex, Souffelweyersheim, France), 1% Glycine (Sigma, Saint-Louis, MO, USA), 5% Normal Horse Serum (Sigma, Saint-Louis, MO, USA), 5% Bovine Serum Albumine (Sigma, Saint-Louis, MO, USA) for 30 min at room temperature. Primary antibodies (Table S1) were diluted in IF buffer and incubated 1 h at room temperature. Coverslips were washed three times for 5 min with 1X PBS between primary and secondary antibody incubations. Subsequently, secondary antibodies (Table S1) were added along with DAPI for 1 h at room temperature in IF buffer. Cells were washed extensively 3 times with 1X PBS and mounted with ProLong Antifade reagent (Invitrogen, Carlsbad, CA, USA). Pictures were taken with confocal microscope LEICA LSM880 Western BlotFollowing drug(s) treatment(s), cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer (150mM NaCl, 50mM Tris pH7.4, 1%TritonX100, 0.1% SDS, 1% Sodiun desoxycholate) with Halt phosphatase and protease inhibitors (Thermo Scientific). Laemmlis sample buffer supplemented was added to samples to 1X final concentration. Samples were boiled, 5min 95{degrees}C before being loaded on a NuPAGE 4-12% Bis-Tris gel (Invitrogen) and transferred to nitrocellulose membrane (GE Healthcare). Membranes were blocked with Tris-buffered saline with 0.1% Tween-20 (TBS-T) with 5% BSA for at least 30 min at room temperature. Antibodies were diluted in 2.5% BSA in TBS-T. Primary antibodies were incubated overnight at 4{degrees}C and secondary antibodies for 1 h at room temperature; mouse anti G3BP1 antibody (Santa Cruz sc-365338), rabbit anti Caprin-1 antibody (ProteinTech Group 15112-1-AP), mouse anti puromycin antibody (Millipore MABE342), mouse anti GAPDH (abcam ab8245). Antibody detection was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Revelation of the blot was made using G:BOX machine (Syngene) via the GeneSys software. Blot analysis and quantification were done using ImageJ software. Statistical AnalysisStatistical analyses were done on 3 independent experiments. Student T-TEST were performed to compare control to PPMP samples or control to M{beta}CD samples.

molecular biology↗

bioRxiv2024

Stress granule formation enables anchorage-independence survival in cancer cells

Stress granules (SGs) are dynamic cytoplasmic structures assembled in response to various stress stimuli that enhance cell survival under adverse environmental conditions. Here we show that SGs contribute to breast cancer progression by enhancing the survival of cells subjected to anoikis stress. SG assembly is triggered by inhibition of Focal Adhesion Kinase (FAK) or loss of adhesion signals. Combined proteomic analysis and functional studies reveal that SG formation enhances cancer cell proliferation, resistance to metabolic stress, anoikis resistance, and migration. Importantly, inhibiting SG formation promotes the sensitivity of cancer cells to FAK inhibitors being developed as cancer therapeutics. Furthermore, we identify the Rho-ROCK- PERK-eIF2 axis as a critical signaling pathway activated by loss of adhesion signals and inhibition of the FAK-mTOR-eIF4F complex in breast cancer cells. By triggering SG assembly and AKT activation in response to anoikis stress, PERK functions as an oncoprotein in breast cancer cells. Overall, our study highlights the significance of SG formation in breast cancer progression and suggests that therapeutic inhibition of SG assembly may reverse anoikis resistance in treatment-resistant cancers such as triple-negative breast cancer (TNBC). HighlightsO_LIEither anoikis stress or loss of adhesion induce stress granule (SG) formation C_LIO_LIThe Rho-ROCK-PERK-eIF2 axis is a crucial signaling pathway triggered by the absence of adhesion signals, leading to the promotion of SG formation along with the inhibition of the FAK- AKT/mTOR-eIF4F complex under anoikis stress. C_LIO_LIPERK functions as an oncogene in breast cancer cells, initiating SG formation and activating AKT under anoikis stress. C_LIO_LIInhibiting SG formation significantly enhances the sensitivity to Focal Adhesion Kinase (FAK) inhibitors, suggesting a potential for combined therapy to improve cancer treatment efficacy. C_LI

cell biology↗

bioRxiv2021

Mechanisms of translation inhibition and suppression of Stress Granule formation by cisplatin

Platinum-based antineoplastic drugs, such as cisplatin, are commonly used to induce tumor cell death. Cisplatin is believed to induce apoptosis as a result of cisplatin-DNA adducts that inhibit DNA and RNA synthesis. Although idea that DNA damage underlines anti-proliferative effects of cisplatin is dominant in cancer research, there is a poor correlation between the degree of the cell sensitivity to cisplatin and the extent of DNA platination. Here, we propose a novel mechanism of cisplatin-mediated cytotoxicity. We show that cisplatin suppresses formation of Stress Granules (SGs), pro-survival RNA granules with multiple roles in cellular metabolism. Mechanistically, cisplatin inhibits cellular translation to promote disassembly of polysomes and aggregation of ribosomal subunits. As SGs are in equilibrium with polysomes, cisplatin-induced shift towards ribosomal aggregation suppresses SG formation and promotes cellular death. Our data also explain nephrotoxic, neurotoxic and ototoxic effects of cisplatin treatment.

cell biology↗

bioRxiv2020

TDP-43 stabilizes transcripts encoding stress granule protein G3BP1: potential relevance to ALS/FTD

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. G3BP1 (Ras-GAP SH3-domain-binding protein 1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

neuroscience↗

bioRxiv2020

A Novel Stress Response Pathway Regulates Ribosome Biogenesis

Production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and, when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but represents an independent stress-response pathway that we term Ribosome Biogenesis Stress Response (RiBiSR). Failure to coordinately regulate ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils a novel stress response pathway that aims at conserving energy, preserving the nucleolus, and prevents further stress by regulation of rRNA processing.

biochemistry↗