Biology subjects

Rivero-Muller, A.

Publications and source records attributed to Rivero-Muller, A..

5 recordsLinked to original sources

bioRxiv2023

TANGO-Light - optogenetic control of transcriptional modulators

Cell signalling pathways, in particular downstream receptor activation, frequently converge in the activation of transcriptional modulators. Yet, cells are able to differentiate the stimulation of each receptor. It has become clear that transcriptional modulators, such as transcription factors, do not work in on or off states but rather in patterns of active/inactivate conformations. Thus, it is the intensity and duration of such fluctuating activity that result in differential cellular and genes expression changes, and this is challenging to replicate using traditional methods such as inhibitors or genetic constructs. Optogenetics, which is based on the use of light-responsive proteins, offers precise control over biological processes, in a spatio-temporal manner, allowing targeted fine-tuned modulation of specific proteins or signalling pathways. Here, we engineered an optogenetic system to control transcriptional modulators, by fusing a photoactivatable receptor and the TANGO system. By this mean we show that we are able to control a plethora of transcriptional modulators by light. And by doing so, changing cells fate - inducing cells to acquire a more mesenchymal or epithelial phenotype. This optogenetic system was also adapted to mimic signalling pathways such as Notch and Wnt in a light-dose dependent manner. Finally, we show that this light-responsive system can be induced by natural light sources upon cell-cell contact/proximity.

cell biology↗

bioRxiv2023

SYNTHETIC CIRCUITS BASED ON SPLIT CAS9 TO DETECT CELLULAR EVENTS

Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviors by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT - under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/533022v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f63cf0org.highwire.dtl.DTLVardef@11ab298org.highwire.dtl.DTLVardef@1c3270eorg.highwire.dtl.DTLVardef@1394488_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Glycoprotein-glycoprotein receptor binding detection using bioluminescence resonance energy transfer (BRET)

The glycoprotein receptors, members of the large G protein-coupled receptors (GPCRs) family, are characterized by a large extracellular domains responsible of binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays most often using radiolabeling but also by thermal shift assays. However, the use of radioisotopes requires appropriate laboratory conditions, and moreover, for this purpose, purified cell membranes are most often used instead of living cells. This in turn poses another challenge due to the altered stability of membrane proteins in detergents used for purification. Here, we overcome such limitations by applying bioluminescence resonance energy transfer (BRET) in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc) luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (yoked human chorionic gonadotropin (yhCG) or luteinizing hormone (LH)) fused to the enhanced green fluorescent protein (eGFP). We first show that the Gluc-LHCGR is expressed on the plasma membrane and is fully functional, as well as the chimeric eGFP-hormones that are properly secreted and able to bind and activate the WT LHCGR. Finally, we applied the method to determine the interactions between clinically relevant mutations in the hormone as well as the receptor and show that this assay is fast and effective, plus safer and cost efficient alternative to radioligand-based assays, to screen for mutations in either the receptor or ligand. It enables kinetic measurements in living cells, detection of biosynthesis of the receptor (membrane expression) and it is compatible with downstream cellular assays - including firefly luciferase-based readouts.

physiology↗

bioRxiv2020

iGIST - a kinetic bioassay for pertussis toxin based on its effect on inhibitory GPCR signaling

Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require annually thousands of mice. Here, we describe iGIST (Interference in Gi-mediated Signal Transduction) - an animal-free kinetic bioassay for detection of PTX by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory -subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells co-expressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high affinity agonist octreotide, and a luminescent 35-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in picogram/ml range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e. a commercial PTX- toxoid containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer-independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.

microbiology↗

bioRxiv2019

Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological processes that also exert important roles in disease progression Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the future in vivo.

bioengineering↗